Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

OECD 422


An OECD 422 Combined Repeated Dose Toxicity Study with Reproductive/Developmental Toxicity Screening Study is available for Tall Oil via the oral route in the diet for Sprague-Dawley rats (Clubb, 2003). The test was carried out according to the requirements of the guideline and in compliance with GLP.


 


Groups of 10 male and 10 female Sprague-Dawley rats were fed a diet containing Tall Oil (CAS No 8002-26-4) at concentrations of 0, 1000, 5000 and 20,000 ppm. Males were treated for at least 4 weeks, starting from 2 weeks prior to mating; females were treated from 2 weeks prior to mating until at least Day 6 of lactation.


 


The only indication of reproductive toxicity was a decrease in mean number of implantation sites at 20,000 ppm (90% of mean control value), with a corresponding decrease in the mean total number of pups born (94% of mean control value) compared to all other dose groups. However, due to the very slight differences compared to the Control group, there is some doubt as to the biological significance of this finding. Litter survival, as indicated by the birth index and viability index, was similar in all groups. There were no histology findings attributed to treatment. The NOAEL for reproductive parameters has been set at greater than or equal to the high dose level of 20000 ppm. Based on mean body weight and food consumption data over the duration of the study, this is equivalent to approximately 1598 mg/kg/day for males and 1972 mg/kg/day for females.


OECD 443


A key guideline OECD 443 Extended One Generation Reproductive Toxicity Study, including Cohorts 1A, 1B (extended to a F2 generation) was conducted with Tall Oil (Meijer, 2022). Test material was administered via the oral route in the diet to Wistar Han rats. The test was carried out according to the requirements of the guideline and in compliance with GLP.


Animals (25 per sex per treatment group) were administered Tall Oil by dietary administration (pellet diet) with diet concentrations of 1500, 5000 and 15000 ppm. During the lactation period when relative food consumption increases significantly, diet concentrations were decreased on three occasions to maintain a stable mean test item intake throughout the different study periods. The rats of the control group received similarly prepared pellets without the test item. Duration of treatment is presented in Table 1 below.


Table 1                Duration of Treatment for Different Subsets of Animals

















































Generation



Subset of Animals



Duration of the Treatment Period



F0



Males



11-12 Weeks (including 10 weeks pre-mating)



Females



16-17 Weeks (including 10 weeks pre-mating)



Females which failed to deliver or female suspected of a total litter loss



14-15 Weeks



F1



Cohort 1A



10-11 Weeks



Cohort 1B Males



12-13 Weeks (including 10 weeks pre-mating)



Cohort 1B Females



16-19 Weeks (including 10 weeks pre-mating)



Cohort 1C



3-4 Weeks



Cohort Surplus



N/A



Females which failed to deliver



15 Weeks



In contrast to the earlier OECD 422 study, no reproductive toxicity was seen in the P0 generation – despite the longer duration of exposure to the test material. The only sign of reproductive toxicity in the P1 generation was a reduction in the mean number of implantation sites at the top dose of 15,000 ppm (82% of mean control value). Consistent with this, there was a decrease in mean live litter size (to 85% of mean control value). However, the number of implantation sites fell within the range of Historical Control Data for most animals. Similarly, litter size for the majority of females fell within the range of Historical Control Data. Therefore, these findings were not considered to be adverse. The NOEL for reproductive parameters was set at ≥15,000 ppm. Based on mean body weight and food consumption data over the duration of the study, this is equivalent to approximately 1197 mg/kg/day in males and 1386 mg/kg/day in females.

Link to relevant study records

Referenceopen allclose all

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21st March 2002 to 26th February 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Remarks:
It was based on the version of the test guideline available at the time (Adopted 22nd March 1996). There are some differences in the information reported compared with stipulations in the current version of OECD 422 (Adopted 29th July 2016). These are: • Functional Observations not performed • Thyroid hormones not measured • Study terminated on PND6, rather than continuing until minimally PND13 • Anogenital distance was not reported There were no deviations from the study plan that impacted upon the integrity of the study.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
no certificate of analysis or details of test substance supplied, however given the nature of the distillation products this deviation was not thought to have affected the integrity of the study.
Principles of method if other than guideline:
N/A
GLP compliance:
yes
Limit test:
no
Justification for study design:
The dose levels were selected and agreed with the Sponsor, following evaluation of existing toxicological data. This included data from a one-week dose range finding study in rats carried out under a separate contract and project number at Inveresk (Inveresk Project No. 493160).
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
At room temperature, in the dark, under nitrogen>

STABILITY UNDER TEST CONDITIONS
Not evaluated during study.

FORM AS APPLIED IN THE TEST
Batches of diet were prepared weekly.
An appropriate quantity of the test item was dissolved in a suitable volume of acetone. This solution was added to a suitable quantity of untreated diet, then mixed for ca one hour with fan assisted venting to remove the ethanol to form a dose premix. A control premix was prepared using the same proportion of acetone and untreated diet. The diets for the Intermediate and High dose groups were prepared by dilution of the dose premix with untreated diet to give the desired concentrations. The Low dose diet was prepared by dilution of the High dose diet with untreated diet. The diet premixes were then placed on a Winkworth mixer for ca 20 min. The Control diet was prepared by dilution of the control premix with untreated diet such that the diet contained the same proportion of premix as the High dose diet.
Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
The rat is a standard rodent species for the reproduction toxicity testing in animals required by regulatory authorities. The normal processes of reproduction in the rat are well documented in the test laboratory.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd
- Age at study initiation: approximately 6 weeks old
- Weight at study initiation: Males: 180-190 g. Females: 113-161 g.
- Fasting period before study: No
- Housing: Initially two per polypropylene cages.
- Diet (e.g. ad libitum): Ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: 12 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ±2
- Humidity (%): 50 ±15
- Air changes (per hr): minimum 15
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 08.04.02 To: 27.05.02
Route of administration:
oral: feed
Vehicle:
acetone
Details on exposure:
The test material was administered orally because this is a possible route of exposure in humans. Administration via the diet is an established experimental routine in rats.

The dose levels were selected and agreed with the Sponsor, following evaluation of existing toxicological data. This included data from a one-week dose range finding study in rats carried out under a separate contract and project number at Inveresk (Inveresk Project No. 493160).
Details on mating procedure:
Pairing was on a one male to one female basis, within the same treatment group. Each female was transferred to the cage of an appropriate co-group male near the end of the working day, and remained there until mating was detected. Vaginal lavages were taken early each morning commencing on the day of pairing, until mating was detected, and the day of observation of a copulatory plug in situ and/or sperm in the lavage was designated Day 0 of gestation. Each female remained with its first designated male for a maximum of 7 consecutive nights.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of the formulated diets was undertaken regarding concentration and homogeneity. Diet prepared for Week 1 and Week 4 of treatment was sampled. Triplicate samples of each formulation, including control, were taken immediately after preparation. The samples were analysed by the Inveresk Product Chemistry Laboratory using a method previously validated in the Inveresk laboratory under a separate protocol and contract (Inveresk Project No. 491836).
Duration of treatment / exposure:
The males were treated for at least four weeks overall, starting from two weeks prior to mating until termination. The females were treated for two weeks prior to mating, then through mating, until termination after Day 4 of lactation.
Frequency of treatment:
Continuous in diet
Details on study schedule:
- Age at mating of the mated animals in the study: approximately 10 weeks
Dose / conc.:
0 ppm (nominal)
Remarks:
Group 1 (control)
Dose / conc.:
1 000 ppm (nominal)
Remarks:
Group 2
Dose / conc.:
5 000 ppm (nominal)
Remarks:
Group 3
Dose / conc.:
20 000 ppm (nominal)
Remarks:
Group 4
No. of animals per sex per dose:
Ten
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected and agreed following evaluation of existing data and a one week dose range-finding study in rats.
- Rationale for animal assignment (if not random): Random
- Rationale for selecting satellite groups: No satellite groups.
Positive control:
No positive control
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily


BODY WEIGHT: Yes
- Time schedule for examinations: Males: once during the week prior to the commencement of dosing and once weekly thereafter. Females: once during the week prior to commencement of treatment, and weekly thereafter until the start of the mating period, then on Day 0 of gestation (the day of detection of a positive mating sign) followed by Days 7, 14 and 20 of gestation, and then Days 1 and 4 of lactation (where Day 0 is the day of parturition).


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes
- Group mean achieved dosages of test substance calculated: Yes


FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No


WATER CONSUMPTION: No


OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: Yes
- Time schedule for collection of blood: During Week 5 of dosing for males and on Day 6 of lactation for females.
- Anaesthetic used for blood collection: No
- Animals fasted: No
- How many animals: Five males and five females
- Parameters checked in table No.1 were examined.


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: During Week 5 of dosing for males and on Day 6 of lactation for females.
- Animals fasted: No
- How many animals: Five males and five females
- Parameters checked in table No.1 were examined.
Oestrous cyclicity (parental animals):
Not examined
Sperm parameters (parental animals):
Parameters examined in male parental generations: testis weight, epididymis weight
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, physical abnormalities.


GROSS EXAMINATION OF DEAD PUPS: yes, for external abnormalities
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals following four weeks of treatment.
- Maternal animals: All surviving animals after four days of lactation.


GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.


HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 2 were prepared for microscopic examination and weighed, respectively.
Postmortem examinations (offspring):
SACRIFICE
- All of the F1 offspring.
- These animals were subjected to postmortem examinations: externally visible abnormalities only.


GROSS NECROPSY: None


HISTOPATHOLOGY / ORGAN WEIGTHS: None
Statistics:
Body weight and food consumption (prior to mating for females), haematology and clinical chemistry data were statistically analysed for homogeneity of variance using the 'F-max' test. If the group variances appeared homogeneous, a parametric ANOVA was used and pairwise comparisons made via Student's t-test using Fisher's F-protected LSD. If the variances were heterogeneous, log or square root transformations were used in an attempt to stabilise the variances. If the variances remained heterogeneous then Kruskal-Wallis ANOVA was used. Organ weights were also analysed likewise, and by analysis of covariance (ANCOVA) using terminal kill body weight as covariate. Histology incidence data were analysed using Fisher's Exact Probability Test. The following pairwise comparisons were performed against the Control group (Group 1): Control group vs Low dose; control group vs intermediate dose; Control group vs high dose. All statistical tests were two-sided and performed at the 5% significance level.
Reproductive indices:
Fertility Index (female) = Number Pregnant/Number Paired
Fertility Index (male) = Number Siring a Litter/Number paired
Gestation Index = Number bearing live pups/Number pregnant
Birth Index = Total number of pups born (live and dead)/number of implantation scars
Live Birth Index = Number of pups live on Day 0 of lactation/Total number born (live and dead)
Offspring viability indices:
Viability Index = Number of pups live on Day 4 of lactation/number live on Day 0
Clinical signs:
no effects observed
Description (incidence and severity):
All clinical observations were considered to be consistent with those normally seen in rats of this age and strain.
Mortality:
no mortality observed
Description (incidence):
N/A
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Please see Table 3-Table 5 for data.

At 20000 ppm, there was a transient decrease in weight gain in both sexes. In males, decreased weight gain was most notable for over the first week, although absolute weights were significantly lower over the first 3 weeks of treatment. In females, there was a notable decrease throughout the pre-mating phase. The resulting deficit in body weight was never regained in either sex. In pregnant females, reduced weight gain was evident over Day 7- 20 of gestation, compared to the Control animals.

There were no obvious effects of treatment at 5000 or 1000 ppm.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
FOOD CONSUMPTION

Please see Table 6 to Table 8 for data.

At 20000 ppm, food consumption in males was reduced for the first 2 weeks of treatment (attaining significance during Week 1) and in Week 4 (not recorded Week 3 as paired for mating). In females, food consumption was significantly decreased during the pre-mating period. Consumption was also reduced during the first half of the gestation period, compared to the Control animals.

There were no obvious effects of treatment at 5000 or 1000 ppm.

COMPOUND INTAKE

Please see Table 9 for data.
Food efficiency:
not examined
Description (incidence and severity):
N/A
Water consumption and compound intake (if drinking water study):
not examined
Description (incidence and severity):
N/A
Ophthalmological findings:
not examined
Description (incidence and severity):
N/A
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Please see Table 10 & Table 11 for data.

At 20000 ppm, there was a non-significant decrease In white blood cells in females. Any other intergroup differences were not considered to reflect an effect of treatment.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Please see Table 12 & Table 13 for data.

Alkaline phosphatase levels were significantly Increased in females at 5000 and 20000 ppm, and in males at 20000 ppm. In males, there was a non-significant increase in levels at 5000 ppm, and in females at 1000 ppm there was an equivocal increase, but, given the small group sjze, it was considered that the difference was too small to reflect an effect of treatment.

Total bilirubin was increased in both sexes at 20000 ppm.

In addition, at 20000 ppm, cholesterol levels were Increased in males; albumin (and consequently total protein) were reduced in females.
Endocrine findings:
not examined
Description (incidence and severity):
N/A
Urinalysis findings:
not examined
Description (incidence and severity):
N/A
Behaviour (functional findings):
not examined
Description (incidence and severity):
N/A
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
N/A
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
All histology findings were typical of spontaneously arising background findings in rats of this strain and age.
Other effects:
not examined
Description (incidence and severity):
N/A
Reproductive function: oestrous cycle:
not examined
Description (incidence and severity):
N/A
Reproductive function: sperm measures:
not examined
Description (incidence and severity):
N/A
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
Please see Table 16 to Table 18 for data.

Mating performance was not affected by treatment. There were no obvious effects on the duration of gestation at any of the dose levels applied.

At 20000 ppm mean number of implant sites per pregnancy was marginally decreased and hence mean total number of pups born was lower than that of all other dose groups. However, due to the very slight differences compared to the Control group, there is some doubt as to the reproducibility of this finding.
N/A
Key result
Dose descriptor:
NOEL
Effect level:
ca. 1 000 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Decreased weight gain and food consumption in both sexes at 20000 ppm. Changes in liver function in both sexes at 20000 ppm. At 5000 ppm there was increased liver weight and alkaline phosphatase in both sexes.
Clinical signs:
not specified
Description (incidence and severity):
N/A
Mortality / viability:
not specified
Description (incidence and severity):
N/A
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Please see Table 19 for data.

At 20000 ppm, mean litter weights were slightly reduced compared to the Controls, reflecting the decrease In litter size.
Food consumption and compound intake (if feeding study):
not examined
Description (incidence and severity):
N/A
Food efficiency:
not examined
Description (incidence and severity):
N/A
Water consumption and compound intake (if drinking water study):
not examined
Description (incidence and severity):
N/A
Ophthalmological findings:
not examined
Description (incidence and severity):
N/A
Haematological findings:
not examined
Description (incidence and severity):
N/A
Clinical biochemistry findings:
not examined
Description (incidence and severity):
N/A
Urinalysis findings:
not examined
Description (incidence and severity):
N/A
Sexual maturation:
not examined
Description (incidence and severity):
N/A
Anogenital distance (AGD):
not examined
Description (incidence and severity):
N/A
Nipple retention in male pups:
not examined
Description (incidence and severity):
N/A
Organ weight findings including organ / body weight ratios:
not examined
Description (incidence and severity):
N/A
Gross pathological findings:
no effects observed
Description (incidence and severity):
N/A
Histopathological findings:
not examined
Description (incidence and severity):
N/A
Other effects:
not examined
Description (incidence and severity):
N/A
Behaviour (functional findings):
not examined
Description (incidence and severity):
N/A
Developmental immunotoxicity:
not examined
Description (incidence and severity):
N/A
N/A
Key result
Dose descriptor:
NOEL
Generation:
F1
Effect level:
>= 20 000 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other:
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Reproductive effects observed:
no

Table 3                Body Weights & Body Weight Gain (g). Male



































































 



GROUP 1


CONTROL



GROUP 2


1000 PPM



GROUP 3


5000 PPM



GROUP 4


20000 PPM



BODY WEIGHT



DAY 0



MEAN


SD


N



320


17


10



313


9


10



316


11


10



313


10


10



DAY 7



MEAN


SD


N



374


26


10



369


16


10



373


16


10



351**


13


10



DAY 14



MEAN


SD


N



414


34


10



408


17


10



415


20


10



386*


18


10



DAY 21



MEAN


SD


N



447


37


10



437


19


10



444


26


10



413**


22


10



DAY 28



MEAN


SD


N



468


45


10



460


23


10



471


28


10



434


29


10



BODY WEIGHT GAIN



DAY 0 to DAY 28



MEAN


SD


N



149


30


10



147


16


10



154


20


10



121*


24


10



Significantly different from the Control: * P<0.05, ** P<0.01, *** P<0.001


 


Table 4                Body Weights & Body Weight Gain (g). Female – Pre-mating



















































 



GROUP 1


CONTROL



GROUP 2


1000 PPM



GROUP 3


5000 PPM



GROUP 4


20000 PPM



BODY WEIGHT



DAY 0



MEAN


SD


N



194


9


10



199


15


10



197


8


10



188


9


10



DAY 7



MEAN


SD


N



217


13


10



221


18


10



219


10


10



200**


9


10



DAY 14



MEAN


SD


N



237


16


10



240


22


10



237


10


10



214**


15


10



BODY WEIGHT GAIN



DAY 0 to DAY 14



MEAN


SD


N



43


9


10



41


12


10



40


4


10



25**


12


10



Significantly different from the Control: * P<0.05, ** P<0.01, *** P<0.001


 


Table 5                Mean Body Weights & Mean Body Weight Gain (g). Female – Gestation and Lactation














































































 



GROUP 1


CONTROL



GROUP 2


1000 PPM



GROUP 3


5000 PPM



GROUP 4


20000 PPM



BODY WEIGHT



DAY 0


Of gestation



MEAN



245



247



240



218



DAY 7


Of gestation



MEAN



286



285



275



256



DAY 14


Of gestation



MEAN



329



320



317



288



DAY 20


Of gestation



MEAN


 



410



407



388



345



BODY WEIGHT GAIN



DAY 0 to DAY 20



MEAN


(% of Control)



165


-



160


97



148


90



127


77



BODY WEIGHT



DAY 1


Of lactation



MEAN



298



286



273



261



DAY 4


Of Lactation



MEAN



319



313



300



281



 


 


Table 6                Food Consumption (g/animal/day). Male





































 



GROUP 1


CONTROL



GROUP 2


1000 PPM



GROUP 3


5000 PPM



GROUP 4


20000 PPM



DAYS 0-7



MEAN


SD


N (CAGE)



31.5


2.2


5



30.9


2.0


5



32.6


1.6


5



27.2**


2.2


5



DAYS 7-14



MEAN


SD


N (CAGE)



35.5


3.9


5



34.1


1.9


5



34.9


1.3


5



32.1


2.4


5



DAYS 21-28



MEAN


SD


N (CAGE)



32.0


2.8


5



31.5


1.5


5



32.4


2.5


5



29.9


1.9


5



Significantly different from the Control: * P<0.05, ** P<0.01, *** P<0.001


 


Table 7                Food Consumption (g/animal/day). Female – pre-mating





























 



GROUP 1


CONTROL



GROUP 2


1000 PPM



GROUP 3


5000 PPM



GROUP 4


20000 PPM



DAYS 0-7



MEAN


SD


N (CAGE)



21.1


1.0


5



21.7


1.2


5



20.5


2.0


5



17.2***


1.3


5



DAYS 7-14



MEAN


SD


N (CAGE)



22.9


1.3


5



22.6


1.1


5



21.6


1.5


5



20.3**


1.5


5



Significantly different from the Control: * P<0.05, ** P<0.01, *** P<0.001


 


Table 8                Mean Food Consumption (g/animal/day). Female – Gestation and Lactation













































 



GROUP 1


CONTROL



GROUP 2


1000 PPM



GROUP 3


5000 PPM



GROUP 4


20000 PPM



DAYS 0-7


Of gestation



MEAN



26.9



26.5



26.2



23.9



DAYS 7-14


Of gestation



MEAN



29.9



30.0



27.9



26.7



DAYS 14-20


Of gestation



MEAN



31.8



32.8



32.6



30.1



DAYS 0-4


Of lactation



MEAN



33.9



31.9



31.6



30.1



 


Table 9                Group Mean Achieved Dosage of Test Item (mg/kg/day)








































































 



GROUP 2


1000 PPM



GROUP 3


5000 PPM



GROUP 4


20000 PPM



MALES



WEEK 1



91



473



1639



WEEK 2



88



443



1742



WEEK 4



70



354



1412



FEMALES



WEEK 1



103



493



1773



WEEK 2



98



474



1961



DAYS 0-7


(OF GESTATION)



100



509



2017



DAYS 7-14


(OF GESTATION)



99



471



1963



DAYS 14-20


(OF GESTATION



90



462



1902



DAYS 1-4


(OF LACTATION)



107



551



2221



 


Table 10             Haematology, Week 5. Males






































































































































 



 



GROUP 1


CONTROL



GROUP 2


1000 PPM



GROUP 3


5000 PPM



GROUP 4


20000 PPM



Hb



MEAN


SD


N



16.1


0.6


5



16.1


0.4


5



15.5


0.1


5



15.7


0.5


5



RBC



MEAN


SD


N



8.28


0.32


5



8.34


0.23


5



7.95


0.27


5



8.10


0.25


5



Hct



MEAN


SD


N



0.453


0.019


5



0.452


0.014


5



0.442


0.004


5



0.440


0.015


5



MCH



MEAN


SD


N



19.5


0.4


5



19.3


0.5


5



19.5


0.7


5



19.4


0.3


5



MCV



MEAN


SD


N



54.7


1.5


5



54.2


1.4


5



55.5


1.7


5



54.3


0.9


5



MCHC



MEAN


SD


N



35.6


0.3


5



35.7


0.4


5



35.1*


0.2


5



35.7


0.4


5



WBC



MEAN


SD


N



14.50


2.21


5



14.33


2.85


5



15.82


4.05


5



12.97


3.93


5



Neut



MEAN


SD


N



1.54


0.37


5



2.30


1.46


5



1.57


0.49


5



1.49


0.74


5



Lymp



MEAN


SD


N



12.39


1.96


5



11.41


3.59


5



13.71


4.23


5



10.91


3.02


5



Mono



MEAN


SD


N



0.25


0.07


5



0.28


0.10


5



0.20


0.02


5



0.22


0.11


5



Eos



MEAN


SD


N



0.19


0.05


5



0.17


0.07


5



0.16


0.07


5



0.15


0.10


5



Baso



MEAN


SD


N



0.04


0.02


5



0.04


0.02


5



0.06


0.03


5



0.04


0.02


5



LUC



MEAN


SD


N



0.09


0.03


5



0.12


0.03


5



0.12


0.05


5



0.16


0.10


5



Plat



MEAN


SD


N



1023


102


5



975


122


5



967


119


5



994


195


5



PT



MEAN


SD


N



13


1


5



13


1


5



13


1


5



12*


1


5



Significantly different from the Control: * P<0.05, ** P<0.01, *** P<0.001


 


 


Table 11             Haematology, Week 7. Females






































































































































 



 



GROUP 1


CONTROL



GROUP 2


1000 PPM



GROUP 3


5000 PPM



GROUP 4


20000 PPM



Hb



MEAN


SD


N



14.0


1.0


5



13.9


0.6


5



13.2


0.4


5



13.0


1.7


5



RBC



MEAN


SD


N



7.30


0.46


5



7.13


0.31


5



6.88


0.29


5



6.87


0.85


5



Hct



MEAN


SD


N



0.394


0.029


5



0.390


0.021


5



0.370


0.014


5



0.365


0.048


5



MCH



MEAN


SD


N



19.1


0.6


5



19.5


0.2


5



19.2


0.4


5



19.0


0.4


5



MCV



MEAN


SD


N



54.0


1.4


5



54.7


1.3


5



53.8


1.3


5



53.2


1.6


5



MCHC



MEAN


SD


N



35.4


0.6


5



35.7


0.7


5



35.7


0.4


5



35.7


0.3


5



WBC



MEAN


SD


N



12.99


3.46


5



12.79


3.85


5



12.15


2.84


5


 



8.89


2.28


5



Neut



MEAN


SD


N



3.84


1.68


5



3.30


1.19


5



3.62


1.09


5



2.44


0.98


5



Lymp



MEAN


SD


N



8.55


2.89


5



8.78


2.85


5



7.99


2.05


5



6.04


1.83


5



Mono



MEAN


SD


N



0.28


0.13


5



0.34


0.22


5



0.26


0.04


5



0.15*


0.04


5



Eos



MEAN


SD


N



0.18


0.07


5



0.15


0.07


5



0.14


0.03


5



0.17


0.17


5



Baso



MEAN


SD


N



0.03


0.03


5



0.04


0.03


5



0.03


0.01


5



0.02


0.01


5



LUC



MEAN


SD


N



0.11


0.06


5



0.17


0.10


5



0.12


0.07


5



0.08


0.02


5



Plat



MEAN


SD


N



1073


133


5



1061


145


5



1110


170


5



906


478


5



PT



MEAN


SD


N



14


1


5



14


1


4



14


1


4



13


2


3



Significantly different from the Control: * P<0.05, ** P<0.01, *** P<0.001


 


Table 12             Clinical Chemistry, Week 5. Male














































































































































 



 



GROUP 1


CONTROL



GROUP 2


1000 PPM



GROUP 3


5000 PPM



GROUP 4


20000 PPM



Urea



MEAN


SD


N



7.3


0.5


5



7.4


1.1


5



6.5


0.5


5



6.4


0.6


5



Glu



MEAN


SD


N



7.92


0.42


5



7.95


0.76


5



8.12


0.88


5



7.90


0.29


5



AST



MEAN


SD


N



86


8


5



92


15


5



92


6


5



83


5


5



ALT



MEAN


SD


N



59


2


5



71


8


5



70


11


5



65


9


5



AP



MEAN


SD


N



683


39


5



699


137


5



842


183


5



1891**


905


5



Na



MEAN


SD


N



144


1


5



144


2


5



143


2


5



142


3


5



k



MEAN


SD


N



4.9


0.2


5



5.0


0.1


5



4.9


0.3


5



5.0


0.2


5



Cl



MEAN


SD


N



101


1


5



102


2


5



101


2


5



101


4


5



TP



MEAN


SD


N



68


2


5



70


2


5



66


2


5



66


2


5



Alb



MEAN


SD


N



41


1


5



43


1


5



41


1


5



41


2


5



AG-R



MEAN


SD


N



1.6


0.2


5



1.6


0.1


5



1.6


0.3


5



1.7


0.1


5



Chol



MEAN


SD


N



2.0


0.4


5



1.9


0.3


5



2.0


0.3


5



2.4*


0.1


5



Crea



MEAN


SD


N



51


2


5



53


2


5



52


3


5



56


4


5



Ca



MEAN


SD


N



2.91


0.06


5



2.95


0.07


5



2.94


0.07


5



2.94


0.07


5



Phos



MEAN


SD


N



2.01


1.13


5



2.58


0.09


5



2.60


0.14


5



2.08


1.17


5



T. Bi



MEAN


SD


N



0.6


0.2


5



1.0


0.6


5



0.9


0.2


5



1.7***


0.4


5



Significantly different from the Control: * P<0.05, ** P<0.01, *** P<0.001


 


Table 13             Clinical Chemistry, Week 7. Female














































































































































 



 



GROUP 1


CONTROL



GROUP 2


1000 PPM



GROUP 3


5000 PPM



GROUP 4


20000 PPM



Urea



MEAN


SD


N



10.7


1.2


5



11.1


2.7


5



11.8


1.2


5



10.5


0.7


5



Glu



MEAN


SD


N



6.68


0.90


5



6.65


0.70


5



6.00


0.35


5



6.44


0.77


5



AST



MEAN


SD


N



96


17


5



88


17


5



98


8


5



84


14


5



ALT



MEAN


SD


N



105


23


5



114


31


5



128


28


5



113


21


5



AP



MEAN


SD


N



509


203


5



638


152


5



980*


378


5



1649***


929


5



Na



MEAN


SD


N



145


3


5



141


3


5



144


1


5



142


3


5



k



MEAN


SD


N



5.3


0.7


5



5.6


0.3


5



5.9


0.4


5



5.9


0.5


5



Cl



MEAN


SD


N



106


4


5



103


4


5



105


2


5



104


1


5



TP



MEAN


SD


N



64


1


5



65


4


5



62


2


5



59*


4


5



Alb



MEAN


SD


N



40


1


5



39


2


5



39


2


5



35***


1


5



AG-R



MEAN


SD


N



1.6


0.1


5



1.5


0.1


5



1.7


0.2


5



1.4*


0.1


5



Chol



MEAN


SD


N



2.4


0.5


5



2.2


0.5


5



1.9*


0.1


5



2.7


0.3


5



Crea



MEAN


SD


N



59


4


5



58


2


5



62


4


5



63


3


5



Ca



MEAN


SD


N



2.84


0.12


5



2.90


0.06


5



2.79


0.08


5



2.77


0.09


5



Phos



MEAN


SD


N



1.89


0.27


5



1.93


0.31


5



1.58


0.21


5



1.86


0.24


5



T. Bi



MEAN


SD


N



0.9


0.2


5



0.9


0.6


5



1.4


0.6


5



1.9**


0.5


5



Significantly different from the Control: * P<0.05, ** P<0.01, *** P<0.001


 


Table 14             Absolute Organ Weights (Following Covariance Analysis). Males





































































































































 



GROUP 1


CONTROL



GROUP 2


1000 PPM



GROUP 3


5000 PPM



GROUP 4


20000 PPM



BODY WEIGHT



MEAN


SE


N



458


11


10



458


11


10



458


11


10



458


11


10



ADRENAL GLANDS



MEAN


SE


N



0.0757


0.0036


10



0.0803


0.0036


10



0.0674


0.0036


10



0.0634*


0.0038


10



BRAIN



MEAN


SE


N



2.10


0.03


10



2.12


0.03


10



2.12


0.03


10



2.08


0.03


10



EPIDIDYMIDES



MEAN


SE


N



1.2041


0.0292


10



1.2032


0.0287


10



1.2048


0.0291


10



1.2047


0.0311


10



HEART



MEAN


SE


N



1.72


0.06


10



1.82


0.06


10



1.73


0.06


10



1.72


0.06


10



KIDNEYS



MEAN


SE


N



3.77


0.10


10



3.85


0.10


10



4.01


0.10


10



4.07


0.11


10



LIVER



MEAN


SE


N



17.88


0.53


10



18.88


0.52


10



19.46*


0.53


10



20.12**


0.56


10



LUNG



MEAN


SE


N



1.82


0.06


9



1.86


0.05


10



1.76


0.05


10



1.75


0.06


10



PITUITARY GLAND



MEAN


SE


N



0.0013


0.0001


10



0.012


0.001


10



0.013


0.001


10



0.011


0.001


10



PROSTATE



MEAN


SE


N



0.771


0.046


10



0.777


0.045


10



0.704


0.046


10



0.761


0.049


10



SPLEEN



MEAN


SE


N



0.85


0.03


10



0.83


0.03


10



0.85


0.03


10



1.04***


0.03


10



SALIVARY GLANDS



MEAN


SE


N



0.7770


0.0240


10



0.7572


0.0236


10



0.7501


0.0239


10



0.7978


0.0255


10



TESTES



MEAN


SE


N



3.55


0.08


10



3.51


0.07


10



3.69


0.08


10



3.74


0.08


10



THYMUS



MEAN


SE


N



0.491


0.032


10



0.415


0.031


10



0.435


0.032


10



0.385


0.034


10



THYROID GLANDS



MEAN


SE


N



0.0219


0.0010


10



0.0233


0.0009


10



0.0225


0.0010


9



0.0230


0.0010


10



Significantly different from the Control: * P<0.05, ** P<0.01, *** P<0.001


 


Table 15             Absolute Organ Weights (Following Covariance Analysis). Females





























































































































 



GROUP 1


CONTROL



GROUP 2


1000 PPM



GROUP 3


5000 PPM



GROUP 4


20000 PPM



BODY WEIGHT



MEAN


SE


N



310


7


10



310


7


10



310


7


10



310


8


8



ADRENAL GLANDS



MEAN


SE


N



0.0911


0.0037


10



0.0843


0.0032


10



0.0791


0.0032


10



0.0774


0.0042


8



BRAIN



MEAN


SE


N



1.87


0.02


10



1.87


0.02


10



1.90


0.02


10



1.96


0.03


8



HEART



MEAN


SE


N



1.12


0.04


10



1.16


0.04


10



1.25


0.04


10



1.18


0.05


8



KIDNEYS



MEAN


SE


N



2.59


0.05


10



2.53


0.05


10



2.47


0.05


10



2.53


0.06


8



LIVER



MEAN


SE


N



16.69


0.48


10



16.16


0.43


10



17.77


0.43


10



17.87


0.55


8



LUNG



MEAN


SE


N



1.32


0.04


9



1.38


0.03


10



1.29


0.03


10



1.37


0.04


8



OVARIES



MEAN


SE


N



0.114


0.005


10



0.106


0.004


10



0.104


0.004


10



0.097


0.006


8



PITUITARY GLAND



MEAN


SE


N



0.014


0.001


10



0.016


0.001


10



0.015


0.001


10



0.013


0.001


8



SPLEEN



MEAN


SE


N



0.59


0.03


10



0.63


0.03


10



0.68


0.03


10



0.72


0.03


8



SALIVARY GLANDS



MEAN


SE


N



0.6038


0.0208


10



0.6027


0.0184


10



0.6121


0.0185


10



0.5755


0.0238


8



THYMUS



MEAN


SE


N



0.198


0.024


10



0.233


0.021


10



0.201


0.021


10



0.242


0.028


8



THYROID GLANDS



MEAN


SE


N



0.0166


0.0010


9



0.0165


0.0009


10



0.0152


0.0009


10



0.0171


0.0011


8



UTERUS



MEAN


SE


N



0.53


0.02


10



0.53


0.02


10



0.51


0.02


10



0.50


0.03


8



Significantly different from the Control: * P<0.05, ** P<0.01, *** P<0.001


 


Table 16             Mating performance and fertility indices





































































 



GROUP 1


CONTROL



GROUP 2


1000 PPM



GROUP 3


5000 PPM



GROUP 4


20000 PPM



Number of males paired



10



10



10



10



Number of siring males



10



10



10



8



Number of females paired



10



10



10



10



Pregnant females



10



10



10



8



Number of females producing a liver litter



10



10



10



8



Fertility Index, males (%)



100



100



100



80



Fertility Index, females (%)



100



100



100



80



Gestation Index (%)



100



100



100



100



 


 


Table 17             Implantation sites summary





























 



GROUP 1


CONTROL



GROUP 2


1000 PPM



GROUP 3


5000 PPM



GROUP 4


20000 PPM



IMPLANTATIONS



MEAN


SD


N



13.4


3.6


10



15.4


2.5


10



14.2


3.8


10



12.1


1.5


10


      

 


 


Table 18             Developmental data























































 



GROUP 1


CONTROL



GROUP 2


1000 PPM



GROUP 3


5000 PPM



GROUP 4


20000 PPM



Mean number of live pups per litter ± SD, Day 0



12.4 ± 3.5



14.7 ± 2.5



13.1 ± 3.4



11.4 ± 1.7



Mean number of live pups per litter ± SD, Day 1



12.3 ± 3.3



14.0 ± 3.9



13.0 ± 3.4



11.4 ± 1.7



Mean number of live pups per litter ± SD, Day 4



12.3 ± 3.3



13.1 ± 4.7



13.0 ± 3.4



11.4 ± 1.7



Birth index (%)



90



96



93



97



Live birth index (%)



99



100



100



97



Viability index (%)



99



88



99



100



 


 


Table 19             Group Mean Litter and Pup Weight (g) ± SD


 



































































 



GROUP 1


CONTROL



GROUP 2


1000 PPM



GROUP 3


5000 PPM



GROUP 4


20000 PPM



LITTER



Day 1



84 ± 17



85 ± 21



82 ± 16



79 ± 9



Day 4



126 ± 23



124 ± 50



121 ± 22



117 ± 12



Mean of Litter Mean Pup Weight



MALES



Day 1



7.2 ± 0.9



6.4 ± 0.9



6.7 ± 1.2



7.2 ± 0.8



Day 4



10.8 ± 2.0



9.7 ± 1.9



10.1 ± 2.2



10.7 ± 1.4



FEMALES



Day 1



6.9 ± 1.2



5.9 ± 0.8



6.2 ± 1.0



6.8 ± 0.6



Day 4



10.5 ± 2.2



9.0 ± 2.6



9.4 ± 1.7



10.2 ± 1.2



 

Conclusions:
In a good quality Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test conducted to OECD test guideline 422, and GLP, the parental NOAEL, for Tall Oil administered continuously in the diet to rats (Sprague-Dawley), was 1000ppm. The NOAEL for reproductive toxicity was 5000 ppm.
Executive summary:

Four groups of 10 male and 10 female Sprague-Dawley rats received Tall Oil in diets at concentrations of 0, 1000, 5000 and 20000 ppm. The males were dosed for at least four weeks, starting from two weeks prior to mating. The females were dosed from two weeks prior to mating until at least Day 6 of lactation. The animals were monitored for clinical signs, body weight, food consumption, mating and litter performance. Blood samples were taken from five males (during week five) and five females (lactation Day 6) per group for laboratory investigations. All parental animals were subjected to necropsy, which included weighing of major organs. Histopathology was conducted on tissues from five males from Control and High dose, and seven females from the Control and eight females from the High dose.

At 20000 ppm in-life observations included decreased weight gain and food consumption in both sexes. Increased male liver weight, and increases in bilirubin and alkaline phosphatase were noted in both sexes. In addition, small decreases were noted in adrenal gland weight in both sexes, and in albumin, white blood cell count and ovary weight in females; spleen weight and cholesterol were slightly increased in males.

At 5000 ppm liver weight in males and alkaline phosphatase in both sexes were increased. Female adrenal gland weight was reduced.

The only indication of reproductive toxicity was a marginal decrease in implant sites at 20000 ppm with a corresponding decrease in the mean total number of pups born compared to all other dose groups. However, due to the very slight differences compared to the Control group, there is some doubt as to the reproducibility of this finding. In conclusion, under the conditions of this study, toxicity was exhibited at levels of 5000 and 20000 ppm, but there were no clear effects of toxicity at 1000 ppm. Therefore the parental NOAEL was considered to be 1000 ppm. For reproductive parameters the NOEL was considered to be 5000 ppm.

Endpoint:
extended one-generation reproductive toxicity - with F2 generation (Cohorts 1A, and 1B with extension)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 10th June 2020 to (still in draft at pre-finalization stage).
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
N/A
Qualifier:
according to guideline
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
Version / remarks:
Adopted 25th June 2018
Deviations:
no
Remarks:
No deviations that impact the integrity of the study or its conclusions.
Qualifier:
according to guideline
Guideline:
EU Method B.56 (Extended One-Generation Reproductive Toxicity Study)
Principles of method if other than guideline:
N/A
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Justification for study design:
SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS:

- Basis for dose level selection:
The dose levels were selected based on the results of a combined repeated dose and reproduction / developmental toxicity screening test by dietary administration in the rat (Clubb and Daly, 2003), a prenatal developmental toxicity study of Tall Oil by oral gavage in rats (Bressers, 2021), and a repeated dose 90-day study by dietary administration in rats (Lourens, 2020). The intention was to produce graded responses to the test item.

In the combined repeated dose and reproduction / developmental toxicity screening test in the rat, the parental systemic No Observed Adverse Effect Level (NOAEL) was 1000 ppm and the NOAEL for reproductive parameters was 5000 ppm. In the prenatal developmental toxicity study, a NOAEL of 1000 mg/kg/day was established. Finally, in the 90-day study, a NOAEL of 15000 ppm (which equates to approximate achieved doses of 1104 mg/kg/day in males and 1221 mg/kg/day in females) was established.

Therefore, the high dose level of 15000 ppm was selected for this study. Due to the duration of dosing this was anticipated to achieve approximately 1000 mg/kg/day, which is the limit dose for this type of study. This dose level was expected to produce some toxicity, such as a reduction in body weight gain or food consumption, but not excessive lethality that would prevent meaningful evaluation. The mid-dose level of 5000 ppm, which is the approximate geometric mean between the high and low-dose was expected to produce minimal to moderate toxicity. The low-dose level of 1500 ppm should produce no observable indications of toxicity.

- Premating exposure duration for parental (P0) animals:
Parental males (F0) were treated for 10 weeks pre-mating (this period fully covered the time required for epididymal transit of maturing spermatozoa and to allow the detection of post-testicular effects on sperm during the final stages of spermiogenesis and epididymal sperm maturation at mating). Parental females (F0) were treated for 10 weeks pre-mating (this period fully covered several complete oestrous cycles and was sufficient to detect any adverse effects on cyclicity).

- Inclusion/exclusion of extension of F1 Cohort 1B:

Extension included.

- Inclusion/exclusion of developmental neurotoxicity F1 Cohorts 2A and 2B:

Excluded

- Inclusion/exclusion of developmental immunotoxicity F1 Cohort 3:

Excluded

-Additional F1 animals:

Cohort 1C: For each test group, an additional 20 male and 20 female pups (one of each sex per litter) were to be retained to increase the statistical power to investigate sexual maturation in F1.

Cohort Surplus: For each test group, an additional 10 male and 10 female pups (one male or female per litter) were to be retained to increase the statistical power to detect thyroid hormone disruption in F1.

Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
In freezer (≤ -15°C) protected from light, container flushed with nitrogen

STABILITY UNDER TEST CONDITIONS
Stability in the freezer (≤ -15°C) and at room temperature over 3 weeks was confirmed under Test Facility Study No. 20180612 (Analytical Method Development and Validation Study).

FORM AS APPLIED IN THE TEST
The test item was mixed with the use of a vehicle (acetone), at the test item:acetone ratio of 5:3, directly with some powder feed (premix) and subsequently mixed with the bulk of the diet. Standard powder rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was used. Water (approximately 15% in total) was added to aid pelleting. The pellets were dried for approximately 24 hours at 35°C before storage. The control animals received similarly prepared pellets but without the test item.
Diets were prepared freshly for use at room temperature for a maximum of 10 days. Diets were kept in the freezer (≤ -15°C) and/or at room temperature until use, if not used on the day of preparation. Any remaining food left after filling the food hoppers were stored at room temperature for a maximum of 10 days for supplementing food during the respective food consumption measurement interval.
Species:
rat
Strain:
Wistar
Remarks:
(Han)
Details on species / strain selection:
The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive, and toxicants.

The total number of animals used in this study was considered to be the minimum required to properly characterize the effects of the test item. This study has been designed such that it does not require an unnecessary number of animals to accomplish its objectives.

At this time, studies in laboratory animals provide the best available basis for extrapolation to humans and are required to support regulatory submissions. Acceptable models which do not use live animals currently do not exist.

This study plan was reviewed and agreed by the Animal Welfare Body of Charles River Laboratories Den Bosch B.V. within the framework of Appendix 2 of project license AVD2360020172866 approved by the Central Authority for Scientific Procedures on Animals (CCD) as required by the Dutch Act on Animal Experimentation (December 2014).
Sex:
male/female
Details on test animals or test system and environmental conditions:
At initiation of administration, animals were 6 weeks old and weighed between 115 and 180 g (males) and between 101 and 145 g (females).
A health inspection was performed before the initiation of administration. The F0-animals were allowed to acclimate to the Test Facility toxicology accommodation for 12 days before the commencement of administration.
Housing - On arrival, prior to mating and during the post-weaning period, animals were group housed (up to 5 animals of the same sex and same dosing group and cohort together) in polycarbonate cages (Makrolon type IV; height 18 cm).
During the mating phase, males and females were cohabitated on a 1:1 basis in Makrolon plastic cages (type III; height 18 cm).
During the post-mating phase, males were housed in their home cage (Makrolon plastic cages, type IV; height 18 cm) with a maximum of 5 males/cage. Females were individually housed in Makrolon plastic cages (type III, height 18 cm).
During the lactation phase, females were housed in Makrolon plastic cages (type III, height 18 cm). Pups were housed with the dam until termination or weaning (on PND 21).
The cages contained appropriate bedding (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and were equipped with water bottles. The rooms in which the animals were kept were documented in the study records.
Animals were separated during designated procedures/activities.
Each cage was clearly labeled with a color-coded cage card indicating Test Facility Study No., group, animal number(s), and sex.
Environmental Conditions - Target temperatures of 18 to 24°C with a relative target humidity of 40 to 70% were maintained. The actual daily mean temperature during the study period was 21 to 24°C with an actual daily mean relative humidity of 33 to 73%. The values that were outside the targeted range occurred for 7 days with a minimum of 33% and a maximum of 73% and were without a noticeable effect on the clinical condition of the animals or on the outcome of the study. A 12 hour light/12 hour dark cycle was maintained. Ten or greater air changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms.
Food - Prepared diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum throughout the study, except during designated procedures. During the acclimatization period, animals had free access to similarly prepared pellets without the test item (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
The feed was analyzed by the Supplier for nutritional components and environmental contaminants. Results of the analysis were provided by the supplier and are on file at the Test Facility. It is considered that there were no known contaminants in the feed that would interfere with the objectives of the study.
Water - Municipal tap water was freely available to each animal via water bottles.
Periodic analysis of the water was performed, and results of these analyses are on file at the Test Facility. It is considered that there were no known contaminants in the water that would interfere with the objectives of the study.
Animal Enrichment - Animals were socially housed for psychological/environmental enrichment and were provided with items such as devices for hiding in, paper and/or objects for chewing, except when interrupted by study procedures/activities. Results of analysis for contaminants are provided by the Supplier and are on file at the Test Facility. It is considered that there were no known contaminants that would interfere with the objectives of the study.
Veterinary Care - Veterinary care was available throughout the course of the study, and animals were examined by the veterinary staff as warranted by clinical signs or other changes. All veterinary examinations and recommended therapeutic treatments were documented in the study records.
Route of administration:
oral: feed
Vehicle:
acetone
Remarks:
VWR International, Leuven, Belgium
Details on exposure:
The oral route of administration via the diet was selected because this is a possible route of human exposure during manufacture, handling, or use of the test item.

The dose levels were selected based on the results of a combined repeated dose and reproduction / developmental toxicity screening test by dietary administration in the rat (Clubb and Daly, 2003), a prenatal developmental toxicity study of Tall Oil by oral gavage in rats (Bressers, 2021), and a repeated dose 90-day study by dietary administration in rats (Lourens, 2020). The intention was to produce graded responses to the test item.

In the combined repeated dose and reproduction / developmental toxicity screening test in the rat, the parental systemic No Observed Adverse Effect Level (NOAEL) was 1000 ppm and the NOAEL for reproductive parameters was 5000 ppm. In the prenatal developmental toxicity study, a NOAEL of 1000 mg/kg/day was established. Finally, in the 90-day study, a NOAEL of 15000 ppm (which equates to approximate achieved doses of 1104 mg/kg/day in males and 1221 mg/kg/day in females) was established.

Therefore, the high dose level of 15000 ppm was selected for this study. Due to the duration of dosing this was anticipated to achieve approximately 1000 mg/kg/day, which is the limit dose for this type of study. This dose level was expected to produce some toxicity, such as a reduction in body weight gain or food consumption, but not excessive lethality that would prevent meaningful evaluation. The mid-dose level of 5000 ppm, which is the approximate geometric mean between the high- and low-dose was expected to produce minimal to moderate toxicity. The low-dose level of 1500 ppm should produce no observable indications of toxicity.

Dose levels for F0 and F1 animals are summarized in Table1 and Table 2, respectively. Dose levels were adjusted during the weaning period, and these can be found for F0 and F1 female in Table 3 and Table 4, respectively.
Details on mating procedure:
P0

Animals were cohabitated after at least 10 weeks of treatment on a 1:1 basis within the same treatment group, avoiding sibling mating. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated.

A maximum of 14 days was allowed for mating, after which females who had not shown evidence of mating were separated from their males.

For two couples (Male No. 12, Female No. 112 (control) and Male No. 87, Female No. 187 (15000 ppm)), detection of mating was not confirmed in first instance. The actual mating date was determined based on a re-evaluation of the vaginal lavage for presence of sperm cells. Consequently, these couples were separated 3 and 4 days, respectively, after the actual mating date. The actual mating date was designated Day 0 post-coitum.

Detection of mating was not confirmed in first instance for Female Nos. 104 and 166. Evidence of mating was obtained indirectly by delivery of a litter. Apparently, mating was overlooked in the assessment of the vaginal lavage, which explains the continuation of di-estrus during the mating in this female. The mating date of these animals was estimated at 21 days prior to the actual delivery date. This day was designated Day 0 post-coitum.

F1 COHORT 1B

Animals were cohabitated on a 1:1 basis within the same treatment group, avoiding sibling mating after at least 10 weeks of treatment. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated.

A maximum of 14 days was allowed for mating, after which females who have not shown evidence of mating were separated from their males.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Duplicate sets of samples (approximately 5 g) for each sampling time point were used for concentration analysis, the remaining samples were retained at the Test Facility as backup samples. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 20% for diet of target concentration. After acceptance of the analytical results, backup samples were discarded.
Duration of treatment / exposure:
F0-males and females were treated up to and including the day before scheduled necropsy.
Pups were not treated directly but were potentially exposed to the test item in utero, via maternal milk, from exposure to maternal urine/feces, spilled diet from the food hopper from the water bottle or when they commence eating for themselves.
From weaning onwards (PND 21), F1-animals of Cohort 1A received diet containing test item up to and including the day before scheduled necropsy. The F1 animals of Cohort 1B, 1C, Cohort Surplus and Spares (not assigned to one of the cohorts) were administered diet containing test item until the day of necropsy.
The F1-animals of Cohort 1B were treated from weaning onwards, including at least 10 weeks prior to the second mating period, the duration of pregnancy and at least 21 days after delivery up to and including the day of scheduled necropsy.
The first day test diets preparations containing test item were available to the animals was designated as Day 1.
The amount of test item incorporated in the diet was kept at a constant level in terms of ppm, throughout one specified phase of the study period. After termination, the actual test item intake was estimated based on the body weight and food consumption values.
The same diets remained in the food hopper for a maximum of 10 days. On the day of weighing the remaining diet in the food hopper was replaced with new diet retained from the freezer acclimated to room temperature conditions for at least 1 hour prior to opening the diet bag.

See Table 5 for duration of treatment per subset of animals.
Frequency of treatment:
The test item and control item were administered to the appropriate animals by inclusion in the diet ad libitum.
Details on study schedule:
N/A
Dose / conc.:
0 ppm
Remarks:
Group 1 / Control
Dose / conc.:
1 500 ppm
Remarks:
Group 2
Dose / conc.:
5 000 ppm
Remarks:
Group 3
Dose / conc.:
15 000 ppm
Remarks:
Group 4
No. of animals per sex per dose:
P0

25 animals per sex per dose

F1 COHORT 1A

20 animals per sex per dose

F1 COHORT 1B

20 animals per sex per dose

F1 COHORT 1C

20 animals per sex per dose

F1 COHORT SURPLUS

10 animals per sex per dose

Numbers of animals per dose are summarized in Table 1 (F0) and Table 2 (F1).
Control animals:
yes, plain diet
Details on study design:
The diet concentrations in this study were selected to be 0, 1500, 5000 and 15000 ppm, based on the results of a combined repeated dose and reproduction / developmental toxicity screening test by dietary administration in the rat (Clubb and Daly, 2002), a prenatal developmental toxicity study of Tall Oil by oral gavage in rats (Bressers, 2021), and a repeated dose 90-day study by dietary administration in rats (Lourens, 2020). The intention was to produce graded responses to the test item.
F0-animals were randomly assigned to groups at arrival. Males and females were randomized separately. Animals in poor health were not assigned to groups.
Positive control:
None
Parental animals: Observations and examinations:
MORTALITY
Throughout the study, animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day. Animals were not removed from the cage during observation, unless necessary for identification or confirmation of possible findings.

CLINICAL OBSERVATIONS
Clinical observations were performed at least once daily, beginning during the first administration of the test item and lasting throughout the treatment periods up to the day prior to necropsy. The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored. In the data tables, the scored grades were reported, as well as the percentage of animals affected in summary tables.

ARENA OBSERVATIONS
Clinical observations were conducted in a standard arena once before the first administration of the test item and at weekly intervals during the treatment period

BODY WEIGHTS
Animals were weighed individually on the first day of treatment (prior to administration), and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, 14 and 21. In order to monitor the health status, Male No. 76 (Group 4) was also weighed on Day 52. A terminal weight was recorded on the day of scheduled necropsy.

FOOD CONSUMPTION
Food consumption was quantitatively measured weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, 14 and 21.

WATER CONSUMPTION
Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was suspected.

GENERAL REPRODUCTION DATA
From the mating period onwards, the following parameters were recorded for each female: male number paired with, mating date, confirmation of pregnancy and delivery day.
Females were allowed to litter normally. Postnatal day (PND) 1 is defined as the day when a litter is found completed (i.e. membranes and placentas cleaned up, nest built and/or feeding of pups started). The day prior to PND 1 is considered to be the day when the female started to deliver and is defined as PND 0 and used for recording of delivery. Females that were littering were left undisturbed.
Cage debris of pregnant females was examined for evidence of premature delivery and pregnant females were examined to detect signs of difficult or prolonged parturition or deficiencies in maternal care.
CLINICAL PATHOLOGY
Samples for clinical pathology evaluation are shown in Table 6. Blood of 10 selected animals/sex/group (Table 7) was collected on the day of scheduled necropsy. Samples were collected, between 7.00 and 10.30 a.m., from the retro-orbital sinus under anesthesia using isoflurane in the animal facility.
The selected F0-animals animals were fasted overnight with a maximum of approximately 24 hours before blood sampling, but water was available.
Urine was collected into a specimen vial from the 10 selected animals/sex/group of animals housed in individual metabolism cages overnight (approximately 15-20 hrs) with absence of food, but water was available.
Haematology - Blood samples at a target volume of 0.5 mL were collected into tubes containing K3-EDTA as anticoagulant. Samples were analyzed for the parameters specified in Table 8.
Coagulation - Blood samples at a target volume of 0.45 mL were collected into tubes containing Citrate as anticoagulant. Samples were processed for plasma, and plasma was analyzed for Prothrombin Time (PT) and Activated Partial Thromboplastin Time (APTT).
Clinical chemistry - Blood samples at a target volume of 0.5 mL were collected into tubes containing Li-Heparin as anticoagulant. Samples at a target volume of 1.0 mL were collected in tubes without anticoagulant (same sample as for thyroid hormone measurement, see below). Blood samples were processed for plasma or serum (bile acids), which was analyzed for the parameters specified in Table 9.
Thyroid hormones - Blood samples at a target volume of 1.0 were collected into tubes without anticoagulant. Blood samples were processed for serum and used for measurement of T3, T4 and TSH.
Urinalysis - Urine samples were analyzed for the parameters specified in Table 10.
Oestrous cyclicity (parental animals):
Estrous stages were determined by examining the cytology of vaginal lavage samples.
Daily vaginal lavage was performed for all F0-females beginning 14 days prior to mating and during mating until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period.
On the day of scheduled necropsy, a vaginal lavage was also taken. This was done for all females.
Sperm parameters (parental animals):
For all males, the following assessments were performed:
Sperm samples were taken from the proximal part of the vas deferens (right) at necropsy. Sperm motility and progressive motility were assessed from all samples. Sperm smears for morphological evaluation were fixed from all samples and stained with hematoxylin and eosin. Abnormal forms of sperm from a differential count of at least 200 spermatozoa per animal was recorded. Evaluation was performed for all samples.
One epididymis (right) was removed, placed in labeled bags, and kept in the freezer at ≤ 15°C. After thawing, the right epididymis was weighed, homogenized and evaluated for sperm numbers. Evaluation was performed for all samples.
Litter observations:
OBSERVATIONS UNTIL WEANING (PND 21) - F1- AND F2- GENERATION
The in-life procedures, observations, and measurements listed below were performed for the pups until weaning (PND 21).

LITTER STANDARDIZATION
To reduce variability among the litters, on PND 4 eight pups from each litter of equal sex distribution (if possible) were selected. Selective elimination of pups, e.g. based upon body weight or AGD, was not done.
Whenever the number of male or female pups prevented having four of each sex per litter, partial adjustment (for example, five males and three females) was acceptable.

MORTALITY/MORIBUNDITY
Pups were observed twice daily for general health/mortality, simultaneously with the mortality/moribundity check of the dam. The number of live and dead pups was determined on PND 1 and daily thereafter. Pups were not removed from the cage during observation, unless necessary for identification or confirmation of possible findings.

CLINICAL OBSERVATIONS
Clinical observations were performed at least once daily for all pups. Only days on which clinical signs were present between the first and last litter check were given in the respective report tables.

BODY WEIGHTS
Live pups were weighed individually on PND 1, 4, 7, 13 and 21.
For animals of Cohort Surplus and F2-animals of Cohort 1B (10 selected litters/group; one male and one female), a terminal weight was recorded on the day of scheduled necropsy.

SEX
Sex was externally determined for all pups on PND 1, 4, 7 and 13.

ANOGENITAL DISTANCE
Anogenital distance (AGD) was measured for all live pups on PND 1. The AGD was normalized to the cube root of body weight.

AREOLA/NIPPLE RETENTION
All male pups in each litter were examined for the number of areola/nipples on PND 13.


OBSERVATIONS FROM WEANING (PND 21) ONWARDS - F1- AND F2-GENERATION
MORTALITY/MORIBUNDITY (COHORTS 1A, 1B AND 1C)
Throughout the study, animals were observed for general health/mortality and moribundity twice daily. Animals were not removed from cage during observation, unless necessary for identification or confirmation of possible findings. Animals showing pain, distress or discomfort which was considered not transient in nature or is likely to become more severe, were sacrificed for humane reasons based on OECD guidance document on humane endpoints (ENV/JM/MONO/ 2000/7). The circumstances of any death were recorded in detail.

CLINICAL OBSERVATIONS (COHORTS 1A, 1B AND 1C)
Clinical observations were performed at least once daily, beginning during the first administration of the test item and lasting throughout the dosing periods up to the day prior to necropsy. The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored. In the data tables, the scored grades were reported, as well as the percentage of animals affected in summary tables.

ARENA OBSERVATIONS (COHORTS 1A, 1B AND 1C)
Clinical observations were conducted in a standard arena once on day of weaning before dosing and at weekly intervals thereafter.

BODY WEIGHTS (COHORTS 1A, 1B AND 1C)
Animals were weekly weighed individually. This started on a specific date on which all pups were at least at PND 21. In addition, the body weight was recorded of each female on the day of acquisition of vaginal patency and of each male on the day of acquisition of balanopreputial separation. For animals of Cohorts 1A and 1B a terminal weight was recorded on the day of scheduled necropsy

FOOD CONSUMPTION (COHORTS 1A, 1B AND 1C)
Food consumption was quantitatively measured weekly, from weaning onwards up to the day prior to scheduled necropsy.

WATER CONSUMPTION (COHORTS 1A, 1B AND 1C)
Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was suspected.

VAGINAL PATENCY (COHORTS 1A, 1B AND 1C)
Vaginal patency (vaginal opening) was monitored daily for all females from PND 25 onwards until vaginal patency was present, by visual inspection of the vaginal area. Body weight was recorded on the day of acquisition of vaginal patency.

BALANOPREPUTIAL SEPARATION (COHORTS 1A, 1B AND 1C)
Balanopreputial separation (prepuce opening) was monitored daily for all males from PND 35 onwards until balanopreputial separation was present, by visual inspection of the genital area. Body weight was recorded on the day of acquisition of balanopreputial separation.

STAGE OF ESTRUS DETERMINATION (COHORTS 1A AND 1B)
Estrous stages were determined by examining the cytology of vaginal lavage sample, taken on the day of scheduled necropsy for Cohorts 1A. Vaginal lavage samples for the determination of estrous stages of Cohort 1B females were not collected on the day of scheduled necropsy.

ESTROUS CYCLE DETERMINATION (COHORT 1A)
Estrous stages were determined by examining the cytology of vaginal lavage samples, taken during two periods. During the first period, daily vaginal lavage was performed for all Cohort 1A females starting on the day of onset of vaginal patency and was minimally continued until the first estrus was determined, in order to determine the time interval between these two events. During the second period, daily vaginal lavage was performed from PND 75 to 88. The estrous cycle data of the first period is not reported.

BODY WEIGHTS (COHORT 1B)
Mated females were weighed individually on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, 14 and 21.

FOOD CONSUMPTION (COHORT 1B)
Food consumption was not determined for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was quantitatively measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, 14 and 21.

ESTROUS CYCLE DETERMINATION (COHORT 1B)
Estrous stages were determined by examining the cytology of vaginal lavage samples. Daily vaginal lavage was performed from start of the mating period until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period.

GENERAL REPRODUCTION DATA (COHORT 1B)
From the mating period onwards, the following parameters were recorded for each female: male number paired with, mating date, confirmation of pregnancy and delivery day. Females were allowed to litter normally. Postnatal day (PND) 1 is defined as the day when a litter is found completed (i.e. membranes and placentas cleaned up, nest built and/or feeding of pups started). The day prior to PND 1 is considered to be the day when the female started to deliver and is defined as PND 0 and used for recording of delivery. Females that were littering were left undisturbed. Cage debris of pregnant females was examined for evidence of premature delivery and pregnant females were examined to detect signs of difficult or prolonged parturition or deficiencies in maternal care.

CLINICAL PATHOLOGY – CULLED PUPS OF THE F1 GENERATION
On PND 4 at culling, blood was collected from two surplus pups per litter (from all litters, if possible) by decapitation, between 7.00 and 10.30 a.m. in the necropsy room, and samples were pooled per litter. If available, blood was collected from one male and one female pup per litter. If only one surplus pup per litter was available at culling, as much as possible blood was collected from this single pup.
Blood samples at a target volume of 0.5 mL were collected into tubes without anticoagulant. Blood samples were processed for serum. Pooled serum of culled PND 4 pups was used for measurement of T4 only.

CLINICAL PATHOLOGY – COHORT 1A
Samples for clinical pathology evaluation are shown in Table 6. Blood of 10 selected animals/sex/group (Table 7) was collected on the day of scheduled necropsy. Samples were collected, between 7.00 and 10.30 a.m., from the retro-orbital sinus under anesthesia using isoflurane in the animal facility.
The selected animals were fasted overnight with a maximum of approximately 24 hours before blood sampling, but water was available.
Urine was collected into a specimen vial from the 10 selected animals/sex/group of animals housed in individual metabolism cages overnight (approximately 15-20 hrs) with absence of food, but water was available.
Haematology - Blood samples at a target volume of 0.5 mL were collected into tubes containing K3-EDTA as anticoagulant. Samples were analyzed for the parameters specified in Table 8.
Coagulation - Blood samples at a target volume of 0.45 mL were collected into tubes containing Citrate as anticoagulant. Samples were processed for plasma, and plasma was analyzed for Prothrombin Time (PT) and Activated Partial Thromboplastin Time (APTT).
Clinical chemistry - Blood samples at a target volume of 0.5 mL were collected into tubes containing Li-Heparin as anticoagulant. Samples at a target volume of 1.0 mL were collected in tubes without anticoagulant (same sample as for thyroid hormone measurement, see below). Blood samples were processed for plasma or serum (bile acids), which was analyzed for the parameters specified in Table 9.
Thyroid hormones - Blood samples at a target volume of 1.0 were collected into tubes without anticoagulant. Blood samples were processed for serum and used for measurement of T3, T4 and TSH.
Urinalysis - Urine samples were analyzed for the parameters specified in Table 10.

CLINICAL PATHOLOGY – COHORT SURPLUS ON PND 22-24
On PND 22, blood was collected from all Cohort Surplus animals (10/sex/group), if possible. Blood was drawn, between 7.00 and 10.30 a.m., by aorta puncture under anesthesia using isoflurane as part of the necropsy procedure.
Blood samples at a target volume of 1.0 mL were collected into tubes without anticoagulant. Blood samples were processed for serum and used for measurement of T3, T4 and TSH.
Postmortem examinations (parental animals):
Terminal procedures are summarised in Table 11.

UNSCHEDULED DEATHS
No F0-animals died during the course of the study.

SCHEDULED EUTHANASIA
Animals surviving until scheduled euthanasia were weighed and deeply anesthetized using isoflurane and subsequently exsanguinated and subjected to a full post mortem examination. See Table 12 for summary of scheduled necropsies. Except for Female No. 142, all animals surviving to scheduled necropsy were fasted overnight with a maximum of 24 hours before necropsy. Water was available.

NECROPSY
All animals were subjected to a full post-mortem examination, with special attention being paid to the reproductive organs.
The numbers of former implantation sites were recorded for all paired females. In case no macroscopically visible implantation sites were present, non-gravid uteri were stained using the Salewski technique in order to detect any former implantation sites and the number of corpora lutea was recorded in addition.
Necropsy procedures were performed by qualified personnel with appropriate training and experience in animal anatomy and gross pathology. A veterinary pathologist, or other suitably qualified person, was available.

ORGAN WEIGHTS AND TISSUE COLLECTION PRESERVATION
The organs identified in Table 14 were weighed at necropsy for all scheduled euthanasia animals. Paired organs were weighed together. In the event of gross abnormalities, in addition to the combined weight, the weight of the aberrant organ was taken and recorded in the raw data. Organ to body weight ratios (using the terminal body weight) were calculated.
Representative samples of the tissues identified in Table 14 were collected from all animals and preserved in 10% neutral buffered formalin (neutral phosphate buffered 4% formaldehyde solution), unless otherwise indicated (for exceptions, see deviation in Appendix 14).
For females which failed to deliver a complete litter, uterine contents (i.e. any fetuses, placenta and implantation sites) was fixed, but was not examined histopathologically.

Postmortem examinations (offspring):
1. TERMINAL PROCEDURES – F1 AND F2 GENERATION UNTIL WEANING

1A) Unscheduled deaths

Pups found dead during the weekend were fixed in identified containers containing 70% ethanol or formaldehyde (see deviation in Appendix 14) as they were not necropsied on the same day.
Stillborn pups and pups found dead between birth and PND 13 were sexed (both externally and internally, if possible) and externally examined with emphasis on developmental morphology. For pups found dead or sacrificed in extremis from PND 14 onwards a limited necropsy was performed including sex determination (both externally and internally).
Descriptions of all external abnormalities were recorded. The stomach of pups not surviving to the scheduled necropsy date were examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated.

1B) Culled pups (PND4)

On PND 4, the pups scheduled for culling (> 8 pups per litter) were euthanized by decapitation. From two extra pups per litter, blood was collected, if possible.
Sex was determined both externally and internally. Pups were externally examined, with particular attention to the external reproductive genitals to examine signs of altered development. Descriptions of all external abnormalities were recorded.

1C) Scheduled euthanasia – F2 generation

Scheduled necropsy of the F2-animals of Cohort 1B was conducted on PND 21-23. The animals were not deprived of food overnight.
From 10 selected litters/group, terminal body weight was determined for one male and one female pup. Subsequently, these pups were deeply anesthetized using isoflurane and subsequently exsanguinated. The animals were subjected to a limited examination, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded. Table 18 list organs to be weight and/or collected. The selected litters were documented in the study files by the study director in advance.
All remaining pups were sacrificed using pentobarbital by intraperitoneal (ip) injection. Also, these pups were subjected to a limited examination, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.

2. TERMINAL PROCEDURES – F1-GENERATION FROM WEANING ONWARDS

Terminal procedures are summarized in Table 14.

A necropsy was conducted for animals that died on study, and specified tissues were saved.

If necessary for humane reasons, animals were euthanized as per Test Facility SOPs. These animals were deeply anesthetized using isoflurane and subsequently exsanguinated. They underwent necropsy, and specified tissues were retained but not weighed.

Spare F1-animals which were not assigned to one of the Cohorts were sacrificed between PND 22-24 by intraperitoneal injection of sodium pentobarbital (Euthasol® 20%). Animals were externally examined, with particular attention to the external reproductive genitals to examine signs of altered development, and sex was determined (both externally and internally). Descriptions of all external abnormalities were recorded.

For all animals, necropsy procedures were performed by qualified personnel with appropriate training and experience in animal anatomy and gross pathology. A veterinary pathologist, or other suitably qualified person, was available. Tissues were preserved in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution) unless otherwise indicated. Organ weights were not recorded for animals euthanized in poor condition or in extremis. Paired organs were weighed together. Organ to body weight ratios (using the terminal body weight) were calculated. The organs identified for weighing and representative samples of the tissues mentioned in Table 15 to Table 18 were collected.

2A) Cohort 1A

Scheduled necropsy of Cohort 1A was conducted on PND 89-95. Cohort 1A animals surviving to scheduled necropsy were deprived of food overnight (with a maximum of approximately 24 hours) before necropsy, but water was available. The animals were weighed and deeply anesthetized using isoflurane and subsequently exsanguinated.
All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs.

Sperm analysis – Cohort 1A:

For all males of Cohort 1A, the following assessments were performed:
Sperm samples were taken from the proximal part of the vas deferens (right) at necropsy. Sperm motility and progressive motility were assessed from all samples. Sperm smears for morphological evaluation were fixed from all samples and stained with hematoxylin and eosin. Abnormal forms of sperm from a differential count of at least 200 spermatozoa (if possible) per animal was recorded. Evaluation was performed for all samples.
One epididymis (right or left (for exception, see deviation in Appendix 14)) was removed, placed in labeled bags, and kept in the freezer at ≤ 15°C. After thawing, the right epididymis was weighed, homogenized and evaluated for sperm numbers. Evaluation was performed for all samples.
As for Animal Nos. 217 (Group 1) and 418 (Group 4), abnormalities were noted in both epididymides, both these organs were fixed in modified Davidson's solution and no evaluation of sperm numbers was performed.

Splenic lymphocyte subpopulation analysis – Cohort 1A:

From 10 selected animals/sex/group (Table 7) of Cohort 1A, splenic lymphocyte subpopulation analysis was performed at termination. One pup (male or female) was selected per litter (20 litters in total).
One half of the spleen was kept on ice until splenic lymphocytes were isolated using 70 µm cell strainers. The other half of the spleen was preserved for histopathological evaluation. Splenocytes were counted with the Coulter Counter Z1. The subpopulations listed in Table 19 were determined in isolated splenic lymphocytes using the BD FACSCanto™ flow cytometer system on the day of necropsy.
The % lymphoid cells of peripheral blood mononuclear cells (PBMC) were determined using the Forward Scatter and Side Scatter.

2B) Cohort 1B

Scheduled necropsy of the F1-Generation of Cohort 1B is listed in Table 20.
The F1-animals of Cohort 1B were not deprived of food overnight before necropsy. The animals were weighed and deeply anesthetized using isoflurane and subsequently exsanguinated.
The numbers of former implantation sites were recorded for all paired females. In case no macroscopically visible implantation sites are present, nongravid uteri were stained using the Salewski technique in order to detect any former implantation sites and the number of corpora lutea were recorded in addition.

2C) Cohort 1C

Scheduled necropsy of Cohort 1C was conducted after positive determination of vaginal patency or balanopreputial separation. Cohort 1C animals were not deprived of food overnight before necropsy and no terminal body weight was recorded.
The animals were deeply anesthetized using isoflurane and subsequently exsanguinated. All animals were subjected to a limited examination, with special attention being paid to the reproductive organs.

2D) Cohort Surplus

Scheduled necropsy of Cohort Surplus was conducted on PND 22-24. Cohort Surplus animals were not deprived of food overnight before necropsy and a terminal body weight was recorded. All animals were subjected to a limited examination, with special attention being paid to the reproductive organs.
The animals were deeply anesthetized using isoflurane and subsequently exsanguinated. Blood samples (1.0 mL) were collected between 8.00 and 11.30 a.m. from all animals by aorta puncture under anesthesia using isoflurane as part of the necropsy procedure. Blood samples were collected into serum tubes for measurement of thyroid-stimulating hormone (TSH) and thyroxine (T4).
Statistics:
STATISTICS FOR DATA COLLECTED IN TOXCAST

Numerical data were analyzed according to sex and occasion. Descriptive statistics: number, mean and standard deviation were reported when possible.
Inferential statistics were performed according to the matrix below, when possible, but excluded semi-quantitative data, and any group with less than 3 observations. All statistical tests were two-sided and conducted at the 5% significance level.
The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1

Continuous datasets with at least 3 groups were compared using Dunnett-test, where permitted by the data. Otherwise, a Steel-test was used. For incidence datasets, an overall Fisher’s exact test was used to compare all groups. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.

STATISTICS FOR DATA COLLECTED/PROCESSED IN PROVANTIS

All statistical tests were two-sided and conducted at the 5% significance level.
The pairwise comparisons of interest are listed below:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1

Parametric, non-parametric, or incidence analyses were used for different datasets, as described in Table 21, but excluded any group with less than 3 observations.
Parametric/non-parametric: Levene’s test was used to assess the homogeneity of group variances. The groups were compared using an overall one-way ANOVA F-test if Levene’s test was not significant or the Kruskal-Wallis test if it was significant. If the overall F test or Kruskal Wallis test was found to be significant, then pairwise comparisons were conducted using Dunnett’s or Dunn’s test, respectively.
Non-parametric: The groups were compared using an overall Kruskal-Wallis test. If the overall Kruskal Wallis test was found to be significant, then the pairwise comparisons were conducted using Dunn’s test.
Incidence: A Fisher’s exact test was used to conduct pairwise group comparisons.
Reproductive indices:
Mating index females (%): Number of females mated x 100/Number of females paired.


Precoital time: Number of days between initiation of cohabitation and confirmation of mating.


Gestation index (%): Number of females with living pups on Day 1 x 100/Number of pregnant females.


Duration of gestation: Number of days between confirmation of mating and the beginning of parturition.


Post-implantation survival index (%): Total number of offspring born x 100/Total number of uterine implantation sites.
Offspring viability indices:
Live birth index (%): Number of live offspring on Day 1 after littering x 100/Total number of offspring born.


Percentage live males at First Litter Check (%): Number of live male pups at First Litter Check x 100/Number of live pups at First Litter Check.


Percentage live females at First Litter Check (%): Number of live female pups at First Litter Check x 100/Number of live pups at First Litter Check.


Viability index (%): Number of live offspring on Day 4 before culling x 100/Number live offspring on Day 1 after littering.



Weaning index (%): Number of live offspring on Day 21 after littering x 100/Number live offspring on Day 4 (after culling)

Percentage live males at weaning (%): Number of live male pups on Day 21 after littering x 100/Number of live pups on Day 21 after littering

Percentage live females at weaning (%): Number of live female pups on Day 21 after littering x 100/Number of live pups on Day 21 after littering
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related clinical signs were noted during daily detailed clinical observations or during weekly arena observations up to 15000 ppm.

Any clinical signs noted during the treatment period occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.
Dermal irritation (if dermal study):
not examined
Description (incidence and severity):
N/A
Mortality:
mortality observed, non-treatment-related
Description (incidence):
No test item-related mortality occurred during the study period.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Please see Table 1 to Table 6 for data.

Body weights and body weight gain were affected by treatment with the test item at 15000 ppm.

In males at 15000 ppm, mean body weight gain was decreased during the first week of treatment. As a result, mean body weight was slightly decreased on Day 8 of the treatment period (5% lower compared to concurrent controls). The apparent lower body weight gain observed during the remainder of the treatment period was considered not a true test item-related effect but secondary to the reduction in body weight gain in the first week of treatment. Statistical significance was achieved on Days 8, 15 and 22 only.

In females at 15000 ppm, mean body weight gain was slightly lower compared to concurrent controls during the 10 weeks pre-mating period (not always statistically significant), resulting in a slightly lower mean body weight at start of the mating period on treatment Day 71 (4% lower compared to concurrent controls). During the post-coitum and lactation period, the same rate for body weight gain was observed for all groups. As such, the apparent lower mean body weight observed at 15000 ppm during post-coitum and lactation (not always statistically significant) was considered not a true test item-related effect but secondary to the reduction in body weight gain during the pre-mating period.

Statistically significant changes observed in body weight and/or body weight gain at 1500 ppm (females) and 5000 ppm (males) were considered unrelated to treatment with the test item as these occurred in absence of a dose-related trend.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Please see Table 7 to Table 9 for relative food consumption data. Please see Table 10 to Table 12 for Test Item intake data.

Food consumption before or after correction for body weight were increased by treatment with the test item from 1500 ppm.

From 1500 ppm, absolute and relative food consumption were increased in both males and females during the pre-mating period (not always statistically significant). A dose-related response was not always present and changes between the different test item-groups were considered slight (mean of means of relative food consumption of the pre-mating period was between 7-12% and 5-9% of control at 1500-15000 ppm for males and females, respectively).

During the post-coitum period, absolute food consumption was considered unaffected by treatment with the test item, while for relative food consumption a dose-dependent increase was observed from 5000 ppm up to post-coitum Day 14-17 (mean of means of de post-coitum period was increased to 6 and 9% of control, at 5000 and 15000 ppm, respectively). The statistically significant increase observed at 1500 ppm during post-coitum Days 17-20 was considered unrelated to treatment with the test item since no trend was apparent regarding dose.

During the lactation period, absolute food consumption was increased (not dose-related and not always statistically significant) from 5000 ppm while for relative food consumption in all treated groups a dose-dependent increase (mean of means up to 11 and 15% of control at 5000 and 15000 ppm, respectively) was observed.

Test Item intake data are summarized in Table 13.
Food efficiency:
not examined
Description (incidence and severity):
N/A
Water consumption and compound intake (if drinking water study):
not specified
Description (incidence and severity):
N/A
Ophthalmological findings:
not examined
Description (incidence and severity):
N/A
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Please see Table 14 & Table 15 for haematology data, and Table 16 & Table 17 for coagulation data.

Test item-related changes in haematology parameters were noted in males at 5000 ppm (males) and 15000 ppm (both sexes):

• Decreased mean reticulocyte count (RETIC; 0.78 and 0.74x of control, at 5000 and 15000 ppm respectively) with concurrent decrease in red blood cell distribution width (RDWG; 0.96 and 0.93x of control, respectively) in males. The magnitude of change in reticulocyte count could at least partly be attributed to the high mean control value which was outside the historical control range*.


• Decreased mean platelet count (PLT) in females at 15000 ppm (0.76x of control).


The slightly increased mean red blood cell distribution width (RDWG) in females at 15000 ppm occurred in absence of corroborative changes in red blood cell count and/or reticulocyte count. Therefore, it was considered to have arisen by chance and not to represent a change of biological significance.

Any other (statistically significant) changes in hematology parameters were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend, were caused by a relatively high control value and/or general overlap and variability in individual values.

Coagulation parameters of treated rats were unaffected by treatment with the test item up to 15000 ppm.

*Historical control data for Wistar Han rats: F0-animals (period 2017-2019):
Reticulocytes (109/L) – males: mean = 198.2; P5 – P95 = 138.60 – 245.20 (n=86).

Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Please see Table 18 & Table 19 for Clinical Chemistry data.

Clinical biochemistry parameters of treated rats were considered affected by treatment with the test item from 15000 ppm.

Test item-related changes in clinical chemistry parameters were noted in males and females at 15000 ppm:

• Increased mean alkaline phosphatase concentration (ALP; 1.42x of control) in males.

• Increased mean cholesterol concentration (CHOL; 1.29x of control) in males with concurrent increase in mean HDL cholesterol (HDL; 1.22x of control) and increase in LDL cholesterol (LDL; 1.29x of control, not statistically significant).

• Increased mean calcium concentration (CA; 1.03x of control) in males.

• Decreased mean bile acid concentration (BILEAC; 0.63x of control, not statistically significant) in males.

• Increased mean triglyceride concentration (TRIG; 1.48x of control, not statistically significant) in females.

The decreased mean inorganic phosphate concentration at 5000 ppm in males, the increased mean bile acid concentration at 1500 ppm in females and the increased mean aspartate aminotransferase concentration at 1500, 5000 and 15000 ppm in females were considered unrelated to treatment with the test item in absence of a dose-related trend.
Endocrine findings:
no effects observed
Description (incidence and severity):
Please see Table 20 & Table 21 for data.

Serum levels of T3, T4 and TSH in F0-males and F0-females were considered not to be affected by treatment with the test item up to 15000 ppm.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Please see Table 22 & Table 23 for data.

Urinalysis parameters were affected by treatment with the test item from 5000 ppm.

In males, urine volume was increased to 2.38x of control at 15000 ppm and specific gravity was decreased with 0.99x and 0.98x of control at 5000 and 15000 ppm, respectively.

Results of the remaining tested urinalysis parameters did not indicate a relation with treatment with the test item.
Behaviour (functional findings):
not examined
Description (incidence and severity):
N/A
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
N/A
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
N/A
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
N/A
Other effects:
not examined
Description (incidence and severity):
N/A
Reproductive function: oestrous cycle:
effects observed, non-treatment-related
Description (incidence and severity):
Length and regularity of the estrous cycle were not affected by treatment with the test item up to 15000 ppm.
All females had regular cycles of 4 days, except for one control female (No. 117) that presented with an irregular cycle. As this was observed for a single female of the control group only, there was no relation with treatment with the test item.
Reproductive function: sperm measures:
effects observed, non-treatment-related
Description (incidence and severity):
Please see Table 29 for sperm measures.

Sperm motility, concentration and morphology parameters were unaffected by treatment with the test item up to 15000 ppm.

A higher epididymal sperm count was recorded for males at 1500 ppm. Since a dose-related trend was absent and an opposite effect would be expected in case of target organ toxicity this was considered not to be related to treatment with the test item.
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
Please see Table 30 to Table 33 for data.

MATING INDEX

Mating index was not affected by treatment with the test item up to 15000 ppm.

The mating indices were 100, 92, 100 and 100% for the control, 1500, 5000 and 15000 ppm groups, respectively.

For two couples (Male/Female Nos. 27/127 and 47/147) in the 1500 ppm group no mating could be confirmed. In the absence of a dose-related incidence, this was considered not related to treatment with the test item

PRE-COITAL TIME

Pre-coital time was not affected by treatment with the test item up to 15000 ppm.

For all mated females mating was confirmed within the first 5 days.

IMPLANTATION SITES

Number of implantation sites was not affected by treatment with the test item up to 15000 ppm.

The mean number of implantation sites was 12.3, 11.6, 11.8 and 11.9 for the control, 1500, 5000 and 15000 ppm groups, respectively.

FERTILITY INDEX

Fertility index was unaffected by treatment with the test item up to 15000 ppm.

The fertility indices were 96, 96, 92 and 92% for the control, 1500, 5000 and 15000 ppm groups, respectively.

One control female (No. 111), one female (No. 146) at 1500 ppm, and two females each (Nos. 157, 168 and 178, 179, respectively) at 5000 and 15000 ppm, were not pregnant. In the absence of a dose-related incidence of non-pregnancy, this was considered not related to treatment with the test item.

GESTATION INDEX AND DURATION

Gestation index and duration of gestation were unaffected by treatment with the test item up to 15000 ppm.

Except for one female (No. 151) in the 5000 ppm group with implantation sites only, all pregnant females had live offspring. The gestation indices were 100, 100, 96 and 100% for the control, 1500, 5000 and 15000 ppm groups, respectively.

All females delivered their pups on post-coitum Day 21 or 22, except for one female at 1500 ppm (No. 142) that delivered its pups (seven dead and one alive) on post-coitum Day 23. As this was observed for a single female of Group 2 only, there was no relation with treatment with the test item.

PARTURITION/MATERNAL CARE

No signs of difficult or prolonged parturition were noted among the pregnant females.

Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.

POST-IMPLANTATION SURVIVAL INDEX

The total number of offspring born compared to the total number of uterine implantations was not affected by treatment with the test item up to 15000 ppm.

Post-implantation survival index was 95, 91, 94 and 96% for the control, 1500, 5000 and 15000 ppm groups, respectively.

For two females at 1500 ppm (Nos. 139 and 143), the number of pups was slightly higher than the number of implantations. This phenomenon is observed from time to time and is caused by normal resorption of these areas during the 21-23 days of lactation. No toxicological relevance was attached to this finding in the current study.

LITTER SIZE

Litter size was not affected by treatment with the test item up to 15000 ppm.

Mean live litter sizes were 11.6, 10.3, 11.6 and 11.3 living pups/litter for the control, 1500, 5000 and 15000 ppm groups, respectively.

One female (No. 142) in the 1500 ppm group had one live pup next to seven dead pups on Lactation Day 1. Given the incidental nature of this finding and in absence of a dose-related trend, it was regarded as unrelated to treatment with the test item.

SEX RATIO

Sex ratio was considered not to be affected by treatment with the test item up to 15000 ppm.

LIVE BIRTH INDEX

The number of live offspring on Day 1 after littering compared to the total number of offspring born was unaffected by treatment with the test item up to 15000 ppm.

The live birth indices were 100, 97, 100, and 100% for the control, 1500, 5000 and 15000 ppm groups, respectively.

One pup of the control group (Litter No. 117, Pup No. 1), seven pups at 1500 ppm (Litter No. 142, Pup Nos. 1-7) and one pup at 5000 ppm (Litter No. 172, Pup No. 1) were found dead at first litter check. No toxicological relevance was attributed to these dead pups since the mortality incidence did not show a dose-related trend and/or remained within the range considered normal for pups of this age.

VIABILITY INDEX

The number of live offspring on Day 4 before culling compared to the number of offspring on Day 1 (viability index) was unaffected by treatment with the test item up to 15000 ppm.

The viability indices were 99, 100, 100 and 99% for the control, 1500, 5000 and 15000 ppm groups, respectively.

Two pups of the control group (Litter No. 113, Pup No. 2, and Litter No. 117, Pup No. 10) were missing on PND 2. Pups missing were most likely cannibalized. One pup of the control group (Litter No. 121, Pup No. 2), and two pups at 15000 ppm (Litter No. 198, Pup Nos. 1 and 3) were found dead on PND 2. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

Note: After Female No. 142 (1500 ppm) was sent to the necropsy room due to suspicion of a total litter loss (seven dead pups), a living pup (No. 8) was found in the cage. Therefore, this female had no total litter loss. As animals are not allowed to return to the pathogenic-free environment of the animal facility after being in the necropsy room, this female and its pup had to be sacrificed.

WEANING INDEX

The number of live offspring at weaning (PND 21) compared to the number of live offspring on Day 4 (after culling) was considered not to be affected by treatment.

The weaning indices were 99, 100, 100 and 100% for the control, 1500, 5000 and 15000 ppm groups, respectively.

One pup of the control group (Litter No. 124, Pup No. 10) was missing on PND 16. Most likely it was cannibalized. No toxicological relevance was attributed to this missing pup as it was a control animal.
N/A
Key result
Dose descriptor:
NOAEL
Remarks:
(on average corresponding to 1197 mg/kg/day in males and 1386 mg/kg/day in females)
Effect level:
>= 15 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other:
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Dose descriptor:
NOAEL
Remarks:
(on average corresponding to 1197 mg/kg/day in males and 1386 mg/kg/day in females)
Effect level:
>= 15 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other:
Remarks on result:
not determinable due to absence of adverse toxic effects
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related clinical signs were noted during daily detailed clinical observations or during weekly arena observations up to 15000 ppm.

Any clinical signs noted during the treatment period occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment with the test item.
Dermal irritation (if dermal study):
not examined
Description (incidence and severity):
N/A
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Female No. 509 (control group, Cohort 1B) was euthanized in Week 7 of treatment due to its poor clinical condition. The most significant clinical findings in this animal included uncoordinated movements, decreased locomotor activity and the macroscopic finding enlarged brain. These findings correlate with the microscopic finding of an invasive cerebellar neoplasia (malignant medulloblastoma) that replaced a large part of the cerebellum and extended and partially replaced the midbrain. Additional microscopic findings included minimal increased hematopoiesis (myeloid) in the bone marrow which was regarded related to the multiple areas of necrosis and neutrophilic inflammation seen within the cerebellar neoplasia. Medulloblastoma is a primitive neuroectodermal tumor (PNET) that forms in the cerebellum and often extends into adjacent regions. It is a rare tumor in rats which is typically reported in younger animals as seen in this case (Weber et al., 2011).

Female No. 579 (1500 ppm group, Cohort 1B) was euthanized in Week 15 (post-coitum Day 22) of treatment due to its poor clinical condition. Microscopic findings of note included severe neutrophilic inflammation of the fetal portion of the placenta with multiple small colonies of bacteria, hemorrhage and lytic necrosis which was suspected to be associated with the reported presence of blood within the cage. Additional microscopic findings included increased hematopoiesis (slight, myeloid) in the bone marrow, extramedullary hematopoiesis in the spleen (marked) and liver (slight) which was consistent with a secondary response to the inflammation present in the placenta and blood loss. Cortical hypertrophy of the zona fasciculata of the adrenal gland (slight) was regarded secondary to stress. Based on the microscopic findings, the alterations in placenta (inflammation, necrosis and hemorrhage) were interpreted to be a significant factor to the moribundity of this animal.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Please see Table 34 to Table 39 for data.


Body weights and body weight gain were affected by treatment with the test item at 15000 ppm.


In males at 15000 ppm, lower mean body weight was observed from start of treatment onwards (not always statistically significant). Mean body weight gain was decreased during Days 8-22 of treatment (up to 22% of control on Day 8 and 6% on Day 22). Since body weight gains recovered to normal levels thereafter, the decreased body weights from Day 29 of the treatment period was considered not a true test item-related effect but secondary to the reduction in body weight gain in the first five weeks of treatment.


In females at 15000 ppm, mean body weight gain was decreased on Day 9 of treatment (10% of control), followed by normal weight gain during the remainder of the pre-mating period. The difference in body weights between high dose and control group during the premating and post-coitum periods was more or less constant from Day 16 onwards (4-8% lower) and was most likely related to the decreased body weights on Day 1 and decreased body weight gain on Day 9 of the pre-mating period.


During the lactation period, a trend towards slightly higher mean body weight gains was observed in females at 15000 ppm, reaching statistical significance on Day 21 of lactation only. This resulted in partial recovery in mean body weight. At the end of the lactation period (Day 21), mean body weight in the high dose females was only 4% lower compared to controls (not statistically significant).


Body weights and body weight gain of animals treated at 1500 or 5000 ppm were considered unaffected by treatment with the test item.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Please see Table 40 to Table 42 for food consumption data. Please see Table 43 to Table 45 for Test Item intake data.


FOOD CONSUMPTION


Food consumption before or after correction for body weight was increased by treatment with the test item from 1500 ppm.


From 1500 ppm onwards, mean values for food consumption (absolute and relative) were increased in both males and females during the pre-mating period (not always statistically significant). A dose-related response was not always present and changes between the different test item-groups were considered slight (mean of means of relative food consumption of the pre-mating period was between 7, 11 and 14% above control in males and 4, 8 and 15% above control in females at 1500, 5000 and 15000 ppm, respectively).


This trend continued in pregnant and lactating females. Mean of means of relative food consumption at 1500, 5000 and 15000 ppm were respectively 5, 14 and 22% above control during post-coitum, and 2, 8 and 17% above control during lactation.


TEST ITEM INTAKE


Mean test article intake over the study period is summarized in Table 46.
Food efficiency:
not examined
Description (incidence and severity):
N/A
Water consumption and compound intake (if drinking water study):
not specified
Description (incidence and severity):
N/A
Ophthalmological findings:
not examined
Description (incidence and severity):
N/A
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Please see Table 47 & Table 48 for haematology data; and Table 49 & Table 50 for coagulation data


HAEMATOLOGY


Test item-related changes in haematology parameters were noted in males from 1500 ppm and in females from 5000 ppm:
• Decreased mean white blood cell (WBC) count in males starting at 1500 ppm, with concurrent decrease in differential WBC count.
• Decreased mean reticulocyte count (RETIC; 0.88x, 0.86x (not statistically significant) and 0.75x of control at 1500, 5000 and 15000 ppm, respectively) with concurrent decrease in red blood cell distribution width (RDWG; 0.96, 0.95 and 0.93x of control, at 1500, 5000 and 15000 ppm respectively) in males.
• Decreased mean haemoglobin concentration (HGB; 0.97 and 0.93x of control, at 5000 and 15000 ppm, respectively) with concurrent decrease in mean concentrations of haematocrit (HCT; 0.93x of control at 15000 ppm) and mean corpuscular haemoglobin (MCH; 0.97x of control at 15000 ppm) in females.
• Increased mean platelet count (PLT) in females at 15000 ppm (1.15x of control).


COAGULATION


Coagulation parameters of treated rats were unaffected by treatment with the test item up to 15000 ppm.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Please see Table 51 & Table 52 for data.


Test item-related changes in clinical chemistry parameters were noted at 15000 ppm:


• Increased mean alkaline phosphatase in males and females (ALP; 1.34x of control).
• Increased mean urea in males (UREA; 1.17x of control).
• Increased mean cholesterol (CHOL; 1.21x of control, not statistically significant) with concurrent increase in LDL cholesterol in males (LDL; 1.16x of control) and HDL cholesterol (HDL; 1.16x of control, not statistically significant).
• Increased mean calcium concentration in males (CA; 1.12x of control).
• Decreased mean bile acids in males and females (BILEAC; 0.44x and 0.68x of control in males and females, respectively).


Any other (statistically significant) changes in hematology parameters were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend and/or were caused by general overlap and variability in individual values.
Endocrine findings:
effects observed, non-treatment-related
Description (incidence and severity):
Please see Table 51 & Table 52 for data.

Serum T3, T4 and TSH levels in males and females were considered unaffected by treatment with the test item up to 15000 ppm. Any changes in thyroid hormone levels occurred in the absence of a dose-related trend and/or were caused by general overlap and variability in individual values.
Urinalysis findings:
no effects observed
Description (incidence and severity):
Please see Table 53 & Table 54 for data.

Urinalysis parameters were unaffected by treatment with the test item up to 15000 ppm.
Behaviour (functional findings):
not examined
Description (incidence and severity):
N/A
Immunological findings:
no effects observed
Description (incidence and severity):
Please see Table 55 & Table 56 for data.

There were no test item-related effects on splenic lymphocyte subpopulations observed by treatment with the test item up to 15000 ppm.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Please see Table 57 to Table 64 for data.

Statistically significant higher liver weights were noted in both sexes and higher thyroid gland weights were noted in males (Cohort 1A) and females (Cohort 1B), as summarized in Table 65.

Cohort 1A (treatment duration 69-74 days):

Statistically significant higher liver weights were noted in males and females at 15000 ppm (absolute and relative to body weight).

Statistically significant higher thyroid gland weights were noted in males at 15000 ppm (absolute and relative to body weight).

Cohort 1B (treatment duration males: 84-91 days, females: 110-134 days)

Statistically significant higher thyroid gland weights were noted in females at 15000 ppm (absolute and relative to body weight). In males higher thyroid gland weights were recorded for all test item-treated dose groups, which did not show a clear dose response and did not reach statistical significance.

There were no other organ weight changes regarded test item-related.

The remaining minor organ weight changes at 15000 ppm which reached statistical significance were regarded to be related to the slightly lower final body weights. These included lower absolute weight of adrenal gland in both sexes (Cohort 1B) and lower absolute pituitary gland of females (Cohort 1B) and lower absolute weight of heart in females (Cohort 1A) and higher relative weight of thymus in females (Cohort 1A).
Gross pathological findings:
no effects observed
Description (incidence and severity):
Cohort 1A (PND 89-95) and Cohort 1B (≥ PND 90):

There were no test item-related gross observations.

Watery fluid in the uterus, found in ten, three, seven and seven females of the control, 1500, 5000 and 15000 ppm groups, respectively, is related to a stage in the estrous cycle and is a normal finding.

All of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.

Cohort 1C (males: ≥ PND 35; females: ≥ PND 25):

There were no test item-related gross observations.

All of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.
Neuropathological findings:
not examined
Description (incidence and severity):
N/A
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related microscopic findings.

All of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Ovarian Follicle Counts

There were no test item-related effects on the ovarian follicle counts and the corpora lutea counts in the F1 15000 ppm group females when compared to control group females. Any variation between group mean counts represented biological variability and were not statistically significant.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
N/A
Other effects:
not examined
Description (incidence and severity):
N/A
Details on results:
N/A
Reproductive function: oestrous cycle:
effects observed, non-treatment-related
Description (incidence and severity):
Length and regularity of the estrous cycle were considered not to have been affected by treatment with the test item up to 15000 ppm.

Most females had regular cycles of 4 to 5 days. An irregular cycle was noted for one female of the control group (No. 485) and one female at 15000 ppm (No. 709). For one female of the control group (No. 481), one female at 1500 ppm (No. 570), four females at 5000 ppm (Nos. 629, 630, 634 and 638) and one female at 15000 ppm (No. 695) the cycle classification could not be determined. In absence of a dose-related incidence and of any apparent correlation to pregnancy status, these findings did not indicate a relation with treatment.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Please see Table 66 for data.

Sperm motility, concentration and morphology parameters were unaffected by treatment with the test item up to 15000 ppm.
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
Please see Table 67 to Table 70 for data.

MATING INDEX

Mating index was not affected by treatment with the test item up to 15000 ppm.

The mating indices were 100% for the control and 95% for the 1500, 5000 and 15000 ppm groups, respectively.

For one couple in each the 1500, 5000 and 15000 ppm group (Male/Female Nos. 308/588, 378/659 and 438/719, respectively) mating could not be confirmed. In the absence of a dose-related incidence, this was considered not related to treatment with the test item.

PRE-COITAL TIME

Precoital time was unaffected by treatment with the test item up to 15000 ppm.

Most females were mated within the first 4 days of the mating period. Three females were mated after 13 days (one female at 1500 ppm and two females at 5000 ppm) and two females after 14 days (one female each at 1500 and 5000 ppm). In the absence of a dose-related incidence, this was considered not related to treatment with the test item.

IMPLANTATION SITES

Number of implantation sites was affected by treatment with the test item at 15000 ppm.

The mean number of implantation sites was 13.1, 12.7, 12.4 and 10.8 for the control, 1500, 5000 and 15000 ppm groups, respectively. Mean number of implantation sites at 15000 ppm was decreased (0.82x of control).

Mean number of implantation sites observed in the 1500 and 5000 ppm groups were comparable with controls and within historical control range*. and at 15000 ppm were only slightly below the lower range of the historical control data based on study means. However, based on the number of implantation sites per animal, the vast majority of values remained within normal historical control data range. Furthermore, it should be noted that the concurrent controls values were significantly higher than the historical control data ranges. Therefore, this change was considered not to be adverse and was within normal biological variation at this laboratory.

FERTILITY INDEX

Fertility index was unaffected by treatment with the test item up to 15000 ppm.

The fertility indices were 95, 95, 100 and 100% for the control, 1500, 5000 and 15000 ppm groups, respectively.

One control female (No. 512) and one female (No. 584) at 1500 ppm, were not pregnant. In the absence of a dose-related incidence of non-pregnancy, this was considered not related to treatment with the test item.

GESTATION INDEX AND DURATION

Gestation index and duration of gestation were unaffected by treatment with the test item up to 15000 ppm.

Except for one female (No. 579) in the 1500 ppm group, that was sacrificed in extremis on post-coitum Day 22, all pregnant females had live offspring. The gestation indices were 100, 94, 100 and 100% for the control, 1500, 5000 and 15000 ppm groups, respectively.

All females delivered their pups on post-coitum Day 20-22, except for one control female (No. 516) that delivered its pups (13 living pups) on post-coitum Day 19.

PARTURITION/MATERNAL CARE

Parturition and maternal care were unaffected by treatment with the test item up to 15000 ppm.

One female (No. 579) at 1500 ppm was sacrificed in extremis on post-coitum Day 22 which may have been due to a difficult parturition. As no signs of difficult or prolonged parturition were observed among the other pregnant females, there was no relation with treatment with the test item.

Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.

POST-IMPLANTATION SURVIVAL INDEX

The total number of offspring born compared to the total number of uterine implantations was not affected by treatment with the test item up to 15000 ppm.

The post-implantation survival indices were 92, 89, 92 and 95% for the control, 1500, 5000 and 15000 ppm groups, respectively.

For one female at 15000 ppm (No. 727), the number of pups was slightly higher than the number of implantations. This phenomenon is observed from time to time and is caused by normal resorption of these areas during the 21 days of lactation. No toxicological relevance was attached to this finding in the current study.

LITTER SIZE

Litter size was affected by treatment with the test item at 15000 ppm.

Mean live litter sizes were 12.0, 11.9, 11.4 and 10.2 living pups/litter for the control, 1500, 5000 and 15000 ppm groups, respectively.

Mean litter size observed in the 1500 and 5000 ppm groups were comparable with controls and within historical control range. At 15000 ppm, mean litter size was slightly below the lower the range of the historical control data, but the litter sizes of the individual litters for the vast majority of litters remained within normal ranges**. Therefore, this change was considered not to be adverse and was within normal biological variation at this laboratory.

SEX RATIO

Sex ratio was considered not to be affected by treatment with the test item up to 15000 ppm.

LIVE BIRTH INDEX

The number of live offspring on Day 1 after littering compared to the total number of offspring born was considered not to be affected by treatment with the test item.

The live birth indices were 100, 99, 100 and 99% for the control, 1500, 5000 and 15000 ppm, respectively.

One pup of the control group (Litter No. 508, Pup No. 4), two pups at 1500 ppm (Litter No. 583, Pup No. 11, and Litter No. 585, Pup No. 7), one pup at 5000 ppm (Litter No. 648, Pup No. 8) and one pup at 15000 ppm (Litter No. 718, Pup No. 3) were found dead at first litter check. For Litter No. 648, Pup No. 8, a malformation of the head and no milk at macroscopic examination was noted. No toxicological relevance was attributed to these dead pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

VIABILITY INDEX

The number of live offspring on Day 4 before culling compared to the number of offspring on Day 1 (viability index) was unaffected by treatment with the test item up to 15000 ppm.

The viability indices were 99, 99, 100 and 99% for the control, 1500, 5000 and 15000 ppm groups, respectively.

Two pups at 1500 ppm (Litter No. 587, Pup No. 1, and Litter No. 590, Pup No. 3) and two pups at 15000 ppm (Litter No. 712, Pup No. 4 and Litter No. 716, Pup No. 4) were found missing on PND 2 or 3. These missing pups were most likely cannibalized. Furthermore, two pups of the control group (Litter No. 503, Pup No. 2, and Litter No. 505, Pup No. 9) and one pup at 1500 ppm (Litter No. 571, Pup No. 8) were found dead on PND 2 or 3. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

WEANING INDEX

The number of live offspring at weaning (PND 21) compared to the number of live offspring on Day 4 (after culling) was considered not to be affected by treatment.

The weaning indices were 100, 99, 100 and 100% for the control, 1500, 5000 and 15000 ppm groups, respectively.

One pup at 1500 ppm (Litter No. 585, Pup No. 3) was found dead on PND 14. No toxicological relevance was attributed to this single incidence in the low dose group only.

Note: During pup observations, one pup at 1500 ppm (Litter No. 586, Pup No. 3) accidently fell on the floor on Lactation Day 20. As a consequence of the injury from this fall, the pup had to be sacrificed on the same day.

* Historical control data for female Wistar Han rats: F1-animals (period 2017-2021):

Number of implantation sites (based on study means): mean = 12.5; Min-Max = 12.2-12.7 (n= 7).
Number of implantation sites (based on individual animals): mean = 12.5; P5-P95 = 10.0-15.0; Min-Max = 4.0-17.0 (n=125).

** Historical control data for female Wistar Han rats: F1-animals (period 2017-2021):

Litter size (based on study means): mean = 11.4; Min-Max = 10.6-12.2 (n=6).
Litter size (based on individual litters): mean = 11.6; P5-P95 = 8.0-15.0; Min-Max = 0 – 17 (n=125).
N/A
Key result
Dose descriptor:
NOAEL
Remarks:
(on average corresponding to 1304 mg/kg/day in males and 1478 mg/kg/day in females)
Effect level:
>= 15 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other:
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Dose descriptor:
NOAEL
Remarks:
(on average corresponding to 1304 mg/kg/day in males and 1478 mg/kg/day in females)
Effect level:
>= 15 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other:
Remarks on result:
not determinable due to absence of adverse toxic effects
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No clinical signs occurred among pups that were considered to be related to treatment with the test item up to 15000 ppm.

The nature and incidence of clinical signs remained within the range considered normal for pups of this age and were therefore considered not to be toxicologically relevant.

Dermal irritation (if dermal study):
not examined
Description (incidence and severity):
N/A
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
The number of live offspring at weaning (PND 21) compared to the number of live offspring on Day 4 (after culling) was considered not to be affected by treatment.

The weaning indices were 99, 100, 100 and 100% for the control, 1500, 5000 and 15000 ppm groups, respectively.

One pup of the control group (Litter No. 124, Pup No. 10) was missing on PND 16. Most likely it was cannibalized. No toxicological relevance was attributed to this missing pup as it was a control animal.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Please see Table 71 for data.

Body weights of pups were affected by treatment with the test item at 15000 ppm.

At 15000 ppm, slightly lower body weights were observed throughout the 3-weeks lactation period (not statistically significant until PND 7 and 13 for females and males, respectively). Weights ranged from 94 to 97% of control for males and from 93 to 95% of control for females from PND 1-21. Male and female body weights combined ranged from 94 to 95% of control from PND 1-21.
Food consumption and compound intake (if feeding study):
not examined
Description (incidence and severity):
Pups were not directly administered test item.
Food efficiency:
not examined
Description (incidence and severity):
N/A
Water consumption and compound intake (if drinking water study):
not examined
Description (incidence and severity):
N/A
Ophthalmological findings:
not examined
Description (incidence and severity):
N/A
Haematological findings:
not examined
Description (incidence and severity):
N/A
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Please see Table 72 & Table 73 for data.

Serum T4 levels in male and female pups, culled at PND 4 were considered not to be affected by treatment with the test item up to 15000 ppm.

Serum T4 in male pups and TSH levels in male and female pups of Cohort Surplus at PND 22 were increased by treatment with the test item at 15000 ppm (21% of control for T4 and 68 and 55% of control for TSH for male and female pups, respectively). All values remained within the historical control data range.

T4 in female pups and T3 levels in male and female pups of Cohort Surplus at PND 22 were unaffected by treatment with the test item.
Urinalysis findings:
not examined
Description (incidence and severity):
N/A
Sexual maturation:
effects observed, treatment-related
Description (incidence and severity):
Please see Table 74 & Table 75 for data.

Sexual maturation was delayed in males at 15000 ppm.

In males at 15000 ppm, the age at attainment of balanopreputial separation was increased (mean of 42.2 days vs. 40.9 days in controls). Body weight at attainment was decreased compared with controls (5% below control mean), indicating that the observed delay in sexual maturation was secondary to the test item-related growth retardation observed at this dose level.

Body weight at attainment vaginal opening was decreased at 15000 ppm. In absence of an increase in age at attainment, the decreased body weight at attainment was considered not related to treatment with the test item but rather the result of the observed test item-related growth retardation at this dose level.

The age at first estrus and time between attainment of vaginal opening and first estrus was considered unaffected by treatment with the test item up to 15000 ppm.
Anogenital distance (AGD):
effects observed, non-treatment-related
Description (incidence and severity):
Please see Table 76 & Table 77 for data.

Anogenital distance (absolute and normalized for body weight) in male and female pups was considered not to be affected by treatment with the test item up to 15000 ppm.

The slightly higher normalized anogenital distance of male pups at 5000 ppm occurred without a dose-related trend and was considered not related to treatment with the test item.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
Please see Table 76 for data.

Treatment with the test item up to 15000 ppm had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Please see Table 78 to Table 81 for data.

COHORT SURPLUS:

No organ weight changes were noted that were regarded test item-related. The lower absolute spleen weight in males at 15000 ppm was regarded to be related to the lower final body weight.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
COHORT SURPLUS:

There were no test item-related gross observations.

All of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.
Histopathological findings:
not examined
Description (incidence and severity):
N/A
Other effects:
not examined
Description (incidence and severity):
N/A
Behaviour (functional findings):
not examined
Description (incidence and severity):
N/A
Developmental immunotoxicity:
not examined
Description (incidence and severity):
N/A
N/A
Key result
Dose descriptor:
NOAEL
Remarks:
(on average corresponding to 1197 mg/kg/day in males and 1386 mg/kg/day in females)
Generation:
F1
Effect level:
>= 15 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Developmental toxicity
Remarks on result:
not determinable due to absence of adverse toxic effects
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No clinical signs occurred among pups that were considered to be related to treatment with the test item up to 15000 ppm.
One pup at 5000 ppm (Litter No. 651, Pup No. 8) was noted to be pale, inactive and had a staggered posture. This may have been related to the relatively large nodule found in the cervix at macroscopic examination. For one pup at 15000 ppm (Litter No. 714, Pup No. 8) the left hindleg was paralyzed from PND 4 to 17.
The nature and incidence of clinical signs remained within the range considered normal for pups of this age, and were therefore considered not to be toxicologically relevant.
Dermal irritation (if dermal study):
not examined
Description (incidence and severity):
N/A
Mortality / viability:
not specified
Description (incidence and severity):
N/A
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Please see Table 82 for data.

Body weights of pups were considered unaffected by treatment with the test item up to 15000 ppm.
Food consumption and compound intake (if feeding study):
not examined
Description (incidence and severity):
Pups terminated at end of lactation.
Food efficiency:
not examined
Description (incidence and severity):
Pups terminated at end of lactation period.
Water consumption and compound intake (if drinking water study):
not examined
Description (incidence and severity):
Pups terminated at end of lactation period.
Ophthalmological findings:
not examined
Description (incidence and severity):
N/A
Haematological findings:
not examined
Description (incidence and severity):
N/A
Clinical biochemistry findings:
not examined
Description (incidence and severity):
N/A
Urinalysis findings:
not examined
Description (incidence and severity):
N/A
Sexual maturation:
not examined
Description (incidence and severity):
N/A
Anogenital distance (AGD):
effects observed, non-treatment-related
Description (incidence and severity):
Please see Table 83 & Table 84 for data.

Anogenital distance (absolute and normalized for body weight) in male and female pups was considered not to be affected by treatment with the test item up to 15000 ppm.

The slightly higher normalized anogenital distance of female pups at 5000 and 15000 ppm occurred without a dose-related trend and therefore was considered not related to treatment with the test item.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
Please see Table 83 for data.

Treatment with the test item up to 15000 ppm had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Please see Table 85 to Table 88 for data.

Brain, thymus and spleen weights (absolute and relative to terminal body weight) were considered not to be affected by treatment with the test item up to 15000 ppm.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No macroscopic findings were noted among pups sacrificed at the end of the lactation period that were considered to be related to treatment with the test item up to 15000 ppm.
For one control pup (Litter No. 504, Pup No. 8), necrosis of the bone of the tail apex was observed and for one pup (Litter No. 651, Pup No. 8) in the 5000 ppm group, a nodule on the cervix was observed on PND 23. At the incidence observed and in absence of a dose-related response, these findings were considered unrelated to treatment with the test item.
Histopathological findings:
not examined
Description (incidence and severity):
N/A
Other effects:
not examined
Description (incidence and severity):
N/A
Behaviour (functional findings):
not examined
Description (incidence and severity):
N/A
Developmental immunotoxicity:
not examined
Description (incidence and severity):
N/A
N/A
Key result
Dose descriptor:
NOAEL
Remarks:
(on average corresponding to 1304 mg/kg/day in males and 1478 mg/kg/day in females)
Generation:
F2
Effect level:
>= 15 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other:
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Reproductive effects observed:
no

Table 1               Body Weights (g). Male – F0



























































































































 



GROUP 1


CONTROL



GROUP 2


1500 PPM



GROUP 3


5000 PPM



GROUP 4


15000 PPM



PRE-MATING



DAY 1


WEEK 1



MEAN


SD


N



148


12.2


25



151


11.9


25



153


13.4


25



151


14.9


25



DAY 8


WEEK 2



MEAN


SD


N



190


12.8


25



191


13.5


25



193


14.9


25



180 *


15.1


25



DAY 15


WEEK 3



MEAN


SD


N



231


14.9


25



231


14.9


25



236


18.3


25



220


19.2


25



DAY 22


WEEK 4



MEAN


SD


N



263


17.2


25



264


17.5


25



273


20.1


25



255


21.3


25



DAY 29


WEEK 5



MEAN


SD


N



288


21.1


25



290


20.1


25



301


22.8


25



281


25.0


25



DAY 36


WEEK 6



MEAN


SD


N



306


23.9


25



310


22.7


25



324


25.0


25



296


36.4


25



DAY 43


WEEK 7



MEAN


SD


N



322


27.0


25



326


25.3


25



342 *


27.3


25



315


31.0


25



DAY 50


WEEK 8



MEAN


SD


N



338


29.5


25



342


26.9


25



359 *


30.4


25



328


36.5


25



DAY 57


WEEK 9



MEAN


SD


N



350


33.1


25



357


29.4


25



373 *


31.7


25



343


36.4


25



DAY 64


WEEK 10



MEAN


SD


N



362


33.8


25



367


30.5


25



385 *


33.9


25



354


37.1


25



MATING



DAY 1


WEEK 1



MEAN


SD


N



371


36.0


25



376


32.4


25



394


34.3


25



361


37.4


25



DAY 9


WEEK 2



MEAN


SD


N



375


35.6


25



379


32.7


25



398


34.5


25



365


36.3


25



DAY 15


WEEK 3



MEAN


SD


N



379


32.3


20



385


36.0


20



408 *


37.6


20



379


39.3


20



*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level


 


'


Table 2                Body Weights (g). Female – F0











































































































































 



GROUP 1


CONTROL



GROUP 2


1500 PPM



GROUP 3


5000 PPM



GROUP 4


15000 PPM



PRE-MATING



DAY 1


WEEK 1



MEAN


SD


N



119


7.9


25



124


6.0


25



120


9.7


25



122


9.8


25



DAY 8


WEEK 2



MEAN


SD


N



139


8.7


25



145


7.4


25



139


12.1


25



136


10.5


25



DAY 15


WEEK 3



MEAN


SD


N



157


8.8


25



165 *


8.0


25



159


12.0


25



155


11.2


25



DAY 22


WEEK 4



MEAN


SD


N



170


12.1


25



180 *


9.6


25



174


12.6


25



169


13.6


25



DAY 29


WEEK 5



MEAN


SD


N



185


10.5


25



191


10.0


25



185


14.2


25



181


13.6


25



DAY 36


WEEK 6



MEAN


SD


N



194


10.4


25



202 *


12.3


25



194


13.6


25



188


13.2


25



DAY 43


WEEK 7



MEAN


SD


N



200


10.4


25



210 *


10.8


25



203


13.5


25



196


13.5


25



DAY 50


WEEK 8



MEAN


SD


N



204


14.1


25



215 *


11.3


25



208


13.1


25



200


15.3


25



DAY 57


WEEK 9



MEAN


SD


N



211


11.7


25



219


11.5


25



212


13.9


25



205


14.5


25



DAY 64


WEEK 10



MEAN


SD


N



215


10.7


25



223


11.9


25



218


13.1


25



210


14.9


25



MATING



DAY 1


WEEK 1



MEAN


SD


N



222


12.7


25



230


11.1


25



222


11.7


25



214


15.7


25



DAY 9


WEEK 2



MEAN


SD


N



---


---


0 x



260


1.4


2



---


---


0 x



 



DAY 15


WEEK 3



MEAN


SD


N



---


---


0 x



265


8.5


2



---


---


0 x



 



DAY 22


WEEK 4



MEAN


SD


N



---


---


0 x



258


2.1


2



---


---


0 x



 



DAY 29


WEEK 5



MEAN


SD


N



 



260


0


2



 



 



*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level


x Explanations for excluded data are listed in the tables of the individual value.


 


Table 3                Body Weights (g). Female – F0, Post-coitum & Lactation



















































































































 



GROUP 1


CONTROL



GROUP 2


1500 PPM



GROUP 3


5000 PPM



GROUP 4


15000 PPM



POST-COITUM



DAY 0



MEAN


SD


N



221


12.3


22



227


12.2


22



222


12.7


22



214


16.3


22



DAY 4



MEAN


SD


N



233


13.7


23



240


11.8


22



235


13.1


22



227


16.9


23



DAY 7



MEAN


SD


N



242


12.7


23



247


12.6


22



240


14.3


22



233


16.9


23



DAY 11



MEAN


SD


N



258


13.0


23



260


13.5


22



253


14.1


22



245 **


16.4


23



DAY 14



MEAN


SD


N



266


15.0


23



268


14.7


22



262


15.4


22



253 *


16.5


23



DAY 17



MEAN


SD


N



289


14.5


23



292


17.1


22



284


22.7


22



276 *


17.9


23



DAY 20



MEAN


SD


N



325


17.1


23



326


21.1


22



317


32.1


22



307 *


18.6


23



LACTATING



DAY 1



MEAN


SD


N



250


16.2


24



255


16.1


21



250


16.4


22



237 *


17.5


23



DAY 4



MEAN


SD


N



266


14.7


24



267


14.7


21



264


16.8


22



251 **


18.2


23



DAY 7



MEAN


SD


N



273


15.9


24



272


15.9


21



271


15.1


22



257 **


18.8


23



DAY 14



MEAN


SD


N



294


15.9


24



291


16.5


21



287


13.4


22



276 **


19.8


23



DAY 21



MEAN


SD


N



282


17.1


24



285


15.5


21



280


15.7


22



275


19.5


23



*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level


Explanations for excluded data are listed in the tables of the individual values


 


Table 4                Body Weight Gain (%). Male – F0



























































































































 



GROUP 1


CONTROL



GROUP 2


1500 PPM



GROUP 3


5000 PPM



GROUP 4


15000 PPM



PRE-MATING



DAY 1


WEEK 1



MEAN


SD


N



0


0.0


25



0


0.0


25



0


0.0


25



0


0.0


25



DAY 8


WEEK 2



MEAN


SD


N



29


3.7


25



26


3.3


25



27


3.3


25



19 **


3.6


25



DAY 15


WEEK 3



MEAN


SD


N



56


7.1


25



53


6.3


25



55


6.3


25



46 **


6.0


25



DAY 22


WEEK 4



MEAN


SD


N



78


12.4


25



75


11.4


25



79


11.3


25



70 *


10.4


25



DAY 29


WEEK 5



MEAN


SD


N



95


16.3


25



92


15.1


25



98


14.7


25



87


14.0


25



DAY 36


WEEK 6



MEAN


SD


N



108


19.5


25



106


17.8


25



113


17.5


25



97


20.0


25



DAY 43


WEEK 7



MEAN


SD


N



118


22.4


25



116


20.1


25



125


20.4


25



110


18.3


25



DAY 50


WEEK 8



MEAN


SD


N



129


24.6


25



127


21.4


25



136


22.2


25



118


20.2


25



DAY 57


WEEK 9



MEAN


SD


N



138


26.8


25



137


23.2


25



145


23.5


25



128


20.1


25



DAY 64


WEEK 10



MEAN


SD


N



145


27.3


25



144


24.5


25



153


25.5


25



135


21.1


25



MATING



DAY 1


WEEK 1



MEAN


SD


N



152


29.0


25



150


26.4


25



159


26.6


25



140


22.3


25



DAY 9


WEEK 2



MEAN


SD


N



154


29.3


25



152


27.1


25



162


26.8


25



143


22.2


25



DAY 15


WEEK 3



MEAN


SD


N



155


28.4


20



158


30.9


20



165


29.1


20



150


21.5


20



*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level


 


Table 5                Body Weight Gain (%). Female – F0











































































































































 



GROUP 1


CONTROL



GROUP 2


1500 PPM



GROUP 3


5000 PPM



GROUP 4


15000 PPM



PRE-MATING



DAY 1


WEEK 1



MEAN


SD


N



0


0.0


25



0


0.0


25



0


0.0


25



0


0.0


25



DAY 8


WEEK 2



MEAN


SD


N



17


3.9


25



16


3.4


25



16


3.8


24



12 **


3.2


25



DAY 15


WEEK 3



MEAN


SD


N



32


4.5


25



32


4.7


25



32


4.9


25



27 **


5.5


25



DAY 22


WEEK 4



MEAN


SD


N



43


5.6


25



45


5.1


25



45


4.8


25



39


6.5


25



DAY 29


WEEK 5



MEAN


SD


N



56


5.4


25



54


6.4


25



54


6.0


25



49 **


7.6


25



DAY 36


WEEK 6



MEAN


SD


N



63


7.2


25



63


7.6


25



62


6.4


25



55 **


9.0


25



DAY 43


WEEK 7



MEAN


SD


N



68


7.3


25



69


6.8


25



69


7.2


25



61 **


9.4


25



DAY 50


WEEK 8



MEAN


SD


N



72


8.3


25



73


6.5


25



73


6.7


25



65 **


9.0


25



DAY 57


WEEK 9



MEAN


SD


N



78


7.9


25



76


7.5


25



77


7.0


25



69 **


9.4


25



DAY 64


WEEK 10



MEAN


SD


N



81


8.0


25



80


8.2


25



81


7.6


25



73 **


9.7


25



MATING



DAY 1


WEEK 1



MEAN


SD


N



87


9.6


25



85


7.4


25



85


7.6


25



76 **


10.5


25



DAY 9


WEEK 2



MEAN


SD


N



--


--


0 x



101


8.8


2



--


--


0 x



 



DAY 15


WEEK 3



MEAN


SD


N



--


--


0 x



105


14.4


2



--


--


0 x



 



DAY 22


WEEK 4



MEAN


SD


N



--


--


0 x



99


9.2


2



--


--


0 x



 



DAY 29


WEEK 5



MEAN


SD


N



 



101


7.7


2



 



 



*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level.


Explanations for excluded data are listed in the tables of the individual values.


 


Table 6                Body Weight Gain (%). Female – F0, Post-coitum & Lactation



















































































































 



GROUP 1


CONTROL



GROUP 2


1500 PPM



GROUP 3


5000 PPM



GROUP 4


15000 PPM



POST-COITUM



DAY 0



MEAN


SD


N



0


0.0


25



0


0.0


25



0


0.0


25



0


0.0


25



DAY 4



MEAN


SD


N



5


1.6


22



6


2.1


22



6


1.7


22



6


2.1


22



DAY 7



MEAN


SD


N



9


1.8


22



9


2.6


22



8


2.2


22



9


2.7


22



DAY 11



MEAN


SD


N



16


2.5


22



15


3.2


22



14


2.7


22



15


3.1


22



DAY 14



MEAN


SD


N



20


3.0


22



18


3.2


22



18


2.6


22



19


3.4


22



DAY 17



MEAN


SD


N



30


4.2


22



29


4.8


22



28


5.5


22



29


4.3


22



DAY 20



MEAN


SD


N



47


5.3


22



44


6.8


22



43


10.2


22



44


5.3


22



LACTATING



DAY 1



MEAN


SD


N



0


0.0


24



0


0.0


21



0


0.0


22



0


0.0


23



DAY 4



MEAN


SD


N



6


3.6


24



5


4.0


21



6


3.8


22



6


3.3


23



DAY 7



MEAN


SD


N



9


3.4


24



7


4.3


21



9


3.5


22



9


4.4


23



DAY 14



MEAN


SD


N



18


3.4


24



14 *


4.4


21



15


4.9


22



17


4.5


23



DAY 21



MEAN


SD


N



13


4.7


24



12


6.9


21



12


4.6


22



16


5.3


23



*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level.


Explanations for excluded data are listed in the tables of the individual values.


 


Table 7                Relative Food Consumption (g/kg/day). Male – F0











































































































































 



GROUP 1


CONTROL



GROUP 2


1500 PPM



GROUP 3


5000 PPM



GROUP 4


15000 PPM



PRE-MATING



DAYS 1-8


WEEKS 1-2



MEAN


SD


N (CAGE)



100


1.5


5



108 *


2.3


5



110 **


1.9


5



111 **


8.5


5



DAYS 8-15


WEEKS 2-3



MEAN


SD


N (CAGE)



93


1.6


5



98 *


2.1


5



99 **


3.3


5



105 **


1.8


5



DAYS 15-22


WEEKS 3-4



MEAN


SD


N (CAGE)



85


1.3


5



89


2.2


5



91 *


2.2


5



92 **


4.4


5



DAYS 22-29


WEEKS 4-5



MEAN


SD


N (CAGE)



79


2.2


5



83 **


1.8


5



87 **


2.1


5



88 **


1.0


5



DAYS 29-36


WEEKS 5-6



MEAN


SD


N (CAGE)



70


1.5


5



74 *


1.7


5



77 **


1.7


5



80 **


4.2


5



DAY 36-43


WEEKS 6-7



MEAN


SD


N (CAGE)



66


1.3


5



71 **


1.2


5



72 **


1.1


5



73 **


2.3


5



DAYS 43-50


WEEKS 7-8



MEAN


SD


N (CAGE)



64


1.4


5



68 **


0.7


5



70 **


1.5


5



73 **


1.6


5



DAYS 50-57


WEEKS 8-9



MEAN


SD


N (CAGE)



59


1.2


5



62 *


1.0


5



66 **


1.4


5



67 **


3.0


5



DAYS 57-64


WEEKS 9-10



MEAN


SD


N (CAGE)



61


0.9


5



66 **


1.1


5



68 **


1.8


5



69 **


2.4


5



DAYS 64-71


WEEKS 10-11



MEAN


SD


N (CAGE)



62


0.8


5



66 **


0.5


5



68 **


1.7


5



69 **


2.6


5



MEAN OF MEANS (PRE-MATING)



MEAN



74



79



81



83



MATING



DAYS 1-9


WEEKS 1-2



MEAN


SD


N (CAGE)



70


8.7


5



75


11.9


5



70


3.7


5



73


3.8


5



DAYS 9-15


WEEKS 2-3



MEAN


SD


N (CAGE)



53


0.7


4



56


3.0


4



58 *


1.2


4



60 **


2.5


4



DAYS 15-22


WEEKS 3-4



MEAN


SD


N (CAGE)



--


--


0



--


--


0



--


--


0



--


--


0



MEAN OF MEANS (MATING)



MEAN



62



65



64



66



*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level.


 


Table 8                Relative Food Consumption (g/kg/day). Female – F0



















































































































 



GROUP 1


CONTROL



GROUP 2


1500 PPM



GROUP 3


5000 PPM



GROUP 4


15000 PPM



PRE-MATING



DAYS 1-8


WEEKS 1-2



MEAN


SD


N (CAGE)



103


1.8


5



111 **


3.8


5



114 **


2.4


5



108 *


1.0


5



DAYS 8-15


WEEKS 2-3



MEAN


SD


N (CAGE)



100


1.8


5



105 *


1.0


5



107 **


1.1


5



110 **


3.4


5



DAYS 15-22


WEEKS 3-4



MEAN


SD


N (CAGE)



94


2.9


5



97


1.4


5



97


1.2


5



98 *


2.1


5



DAYS 22-29


WEEKS 4-5



MEAN


SD


N (CAGE)



89


3.4


5



93 *


1.4


5



98 **


2.1


5



97 **


2.2


5



DAYS 29-36


WEEKS 5-6



MEAN


SD


N (CAGE)



78


1.8


5



85 **


2.0


5



89 **


3.1


5



92 **


2.9


5



DAY 36-43


WEEKS 6-7



MEAN


SD


N (CAGE)



78


1.5


5



84 **


1.4


5



88 **


2.0


5



88 **


3.6


5



DAYS 43-50


WEEKS 7-8



MEAN


SD


N (CAGE)



79


3.0


5



83 *


2.0


5



86 **


1.7


5



88 **


2.9


5



DAYS 50-57


WEEKS 8-9



MEAN


SD


N (CAGE)



72


3.7


5



75


1.5


5



82 **


1.9


5



81 **


2.8


5



DAYS 57-64


WEEKS 9-10



MEAN


SD


N (CAGE)



75


3.7


5



80


2.3


5



84 **


3.4


5



84 **


2.0


5



DAYS 64-71


WEEKS 10-11



MEAN


SD


N (CAGE)



79


2.8


5



83 *


0.9


5



85 **


3.1


5



87 **


2.3


5



MEAN OF MEANS (PRE-MATING)



MEAN



85



89



93



93



MATING



DAYS 1-9


WEEKS 1-2



MEAN


SD


N (CAGE)



--


--


0



 



--


--


0



--


--


0



*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level.


Explanations for excluded data are listed in the tables of the individual values.


 


Table 9                Relative Food Consumption (g/kg/day). Female – F0, Post-coitum & Lactation



















































































































 



GROUP 1


CONTROL



GROUP 2


1500 PPM



GROUP 3


5000 PPM



GROUP 4


15000 PPM



POST-COITUM



DAYS 0-4



MEAN


SD


N



81


9.1


22



81


5.8


22



90 **


5.7


22



91 **


8.6


22



DAYS 4-7



MEAN


SD


N



78


9.9


23



78


6.7


22



84 *


7.8


22



86 **


6.2


23



DAYS 7-11



MEAN


SD


N



77


7.7


23



79


6.2


22



85 **


5.6


22



85 **


6.7


23



DAYS 11-14



MEAN


SD


N



82


7.7


23



83


4.2


22



87 *


7.3


22



91 **


6.8


23



DAYS 14-17



MEAN


SD


N



81


6.7


23



82


6.1


22



87 **


5.9


22



90 **


6.4


23



DAYS 17-20



MEAN


SD


N



83


9.5


23



77 *


7.1


22



80


8.1


22



83


8.5


23



MEAN OF MEANS



MEAN


 



80



80



85



87



LACTATING



DAYS 1-4



MEAN


SD


N



134


19.2


24



138


20.1


21



153 **


18.9


22



153 **


16.6


23



DAYS 4-7



MEAN


SD


N



141


13.0


24



141


15.9


21



156 **


16.4


22



163 **


11.4


23



DAYS 7-14



MEAN


SD


N



187


10.5


24



196


15.6


21



211 **


16.5


22



221 **


14.5


23



DAYS 14-21



MEAN


SD


N



237


15.2


24



240


26.9


21



257 **


22.2


22



264 **


13.4


23



MEAN OF MEANS



MEAN


 



174



179



194



200



*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level.


 


Table 10             Test Item Intake (mg/kg/day). Male – F0











































































































































 



GROUP 1


CONTROL



GROUP 2


1500 PPM



GROUP 3


5000 PPM



GROUP 4


15000 PPM



PRE-MATING



DAYS 1-8


WEEKS 1-2



MEAN


SD


N (CAGE)



0


0.0


5



162


3.5


5



548


9.5


5



1668


126.8


5



DAYS 8-15


WEEKS 2-3



MEAN


SD


N (CAGE)



0


0.0


5



147


3.2


5



497


16.4


5



1572


27.7


5



DAYS 15-22


WEEKS 3-4



MEAN


SD


N (CAGE)



0


0.0


5



134


3.3


5



453


10.9


5



1379


66.6


5



DAYS 22-29


WEEKS 4-5



MEAN


SD


N (CAGE)



0


0.0


5



125


2.7


5



436


10.5


5



1317


15.2


5



DAYS 29-36


WEEKS 5-6



MEAN


SD


N (CAGE)



0


0.0


5



112


2.5


5



387


8.6


5



1195


62.4


5



DAY 36-43


WEEKS 6-7



MEAN


SD


N (CAGE)



0


0.0


5



106


1.7


5



360


5.5


5



1091


34.3


5



DAYS 43-50


WEEKS 7-8



MEAN


SD


N (CAGE)



0


0.0


5



103


1.1


5



349


7.3


5



1088


23.4


5



DAYS 50-57


WEEKS 8-9



MEAN


SD


N (CAGE)



0


0.0


5



93


1.5


5



330


7.0


5



1001


44.9


5



DAYS 57-64


WEEKS 9-10



MEAN


SD


N (CAGE)



0


0.0


5



99


1.7


5



341


8.8


5



1034


35.8


5



DAYS 64-71


WEEKS 10-11



MEAN


SD


N (CAGE)



0


0.0


5



99


0.8


5



339


8.6


5



1036


39.2


5



MEAN OF MEANS (PRE-MATING)



MEAN



0



118



404



1238



MATING



DAYS 1-9


WEEKS 1-2



MEAN


SD


N (CAGE)



0


0.0


5



112


17.9


5



352


18.4


5



1088


56.3


5



DAYS 9-15


WEEKS 2-3



MEAN


SD


N (CAGE)



0


0.0


4



84


4.5


4



290


56.2


4



896


37.8


4



DAYS 15-22


WEEKS 3-4



MEAN


SD


N (CAGE)



--


--


0



--


--


0



--


--


0



--


--


0



MEAN OF MEANS (MATING)



MEAN



0



98



321



992



 


Table 11             Test Item Intake (mg/kg/day). Female – F0



















































































































 



GROUP 1


CONTROL



GROUP 2


1500 PPM



GROUP 3


5000 PPM



GROUP 4


15000 PPM



PRE-MATING



DAYS 1-8


WEEKS 1-2



MEAN


SD


N (CAGE)



0


0.0


5



167


5.7


5



572


11.8


5



1618


15.2


5



DAYS 8-15


WEEKS 2-3



MEAN


SD


N (CAGE)



0


0.0


5



157


1.5


5



533


5.5


5



1643


51.1


5



DAYS 15-22


WEEKS 3-4



MEAN


SD


N (CAGE)



0


0.0


5



146


2.0


5



487


6.0


5



1472


32.2


5



DAYS 22-29


WEEKS 4-5



MEAN


SD


N (CAGE)



0


0.0


5



140


2.0


5



490


10.5


5



1450


32.8


5



DAYS 29-36


WEEKS 5-6



MEAN


SD


N (CAGE)



0


0.0


5



127


3.1


5



446


15.7


5



1375


43.7


5



DAY 36-43


WEEKS 6-7



MEAN


SD


N (CAGE)



0


0.0


5



125


2.0


5



440


10.0


5



1319


54.4


5



DAYS 43-50


WEEKS 7-8



MEAN


SD


N (CAGE)



0


0.0


5



124


3.0


5



429


8.7


5



1327


44.0


5



DAYS 50-57


WEEKS 8-9



MEAN


SD


N (CAGE)



0


0.0


5



113


2.3


5



409


9.6


5



1220


42.2


5



DAYS 57-64


WEEKS 9-10



MEAN


SD


N (CAGE)



0


0.0


5



119


3.5


5



422


16.8


5



1262


30.2


5



DAYS 64-71


WEEKS 10-11



MEAN


SD


N (CAGE)



0


0.0


5



124


1.4


5



427


15.7


5



1303


35.1


5



MEAN OF MEANS (PRE-MATING)



MEAN



0



134



465



1399



MATING



DAYS 1-9


WEEKS 1-2



MEAN


SD


N (CAGE)



--


--


0 x



 



--


--


0 x



--


--


0 x



 


Table 12             Test Item Intake (mg/kg/day). Female – F0, Post-coitum & Lactation



















































































































 



GROUP 1


CONTROL



GROUP 2


1500 PPM



GROUP 3


5000 PPM



GROUP 4


15000 PPM



POST-COITUM



DAYS 0-4



MEAN


SD


N



0


0.0


22



122


8.7


22



448


28.4


22



1358


129.3


22



DAYS 4-7



MEAN


SD


N



0


0.0


23



117


10.0


22



421


39.1


22



1283


92.9


23



DAYS 7-11



MEAN


SD


N



0


0.0


23



118


9.4


22



424


27.9


22



1279


100.5


23



DAYS 11-14



MEAN


SD


N



0


0.0


23



124


6.3


22



434


36.6


22



1361


101.6


23



DAYS 14-17



MEAN


SD


N



0


0.0


23



124


9.1


22



437


29.6


22



1346


95.6


23



DAYS 17-20



MEAN


SD


N



0


0.0


23



116


10.7


22



399


40.5


22



1244


127.6


23



MEAN OF MEANS



MEAN


 



0



120



427



1312



LACTATING



DAYS 1-4



MEAN


SD


N



0


0.0


24



138


20.1


21



510


63.1


22



1530


166.5


23



DAYS 4-7



MEAN


SD


N



0


0.0


24



106


12.0


21



391


41.1


22



1220


85.3


23



DAYS 7-14



MEAN


SD


N



0


0.0


24



118


9.3


21



421


32.9


22



1325


86.9


23



DAYS 14-21



MEAN


SD


N



0


0.0


24



144


16.1


21



515


44.4


22



1585


80.6


23



MEAN OF MEANS



MEAN


 



0



126



459



1415



 


 


Table 13             Summary of Test Item Intake – F0




































































































 



Mean over Means Intake
[mg test item/kg body weight]


(mean range indicated within brackets)



 



Group No.



2



3



4



Nominal Dietary Inclusion Level (ppm)



1500



5000



15000



 



 



Sex



Study Period



 



 



 



Males



Pre-mating



118



(93-162)



404



(330-548)



1238



(1001-1668)



Post-mating



98



(84-112)



321



(290-352)



992



(896-1088)



Mean of Meansa



115



390



1197



 



 



Females



Pre-mating



134



(113-167)



465



(409-572)



1399



(1220-1643)



Post-coitum



120



(116-124)



427



(399-448)



1312



(1244-1361)



Lactation



126



(106-144)



459



(391-515)



1415



(1220-1585)



Mean of Meansa



130



457



1386



a   Mean of means of all periods, weighed for number of treatment days per period:


Males: ((70x mean pre-mating) + (14x mean mating)) / 84


Females: ((70x mean pre-mating) + (20x mean post-coitum) + (20x mean lactation)) / 110



 


Table 14             Haematology. Male – F0


























































































































































Sex: Male



 



 



 



 



 



 



Reporting Hematology



 



 



 



 



WBC



NEUT



LYMPH



MONO



EOS



BASO



LUC



RBC



RETIC



RDWG



HGB



HCT



 



 



 



 



 



 



 



 



 



 



 



 



(10^9/L)



(10^9/L)



(10^9/L)



(10^9/L)



(10^9/L)



(10^9/L)



(10^9/L)



(10^12/L)



(10^9/L)



(%)



(g/L)



(L/L)



 



 



 



 



 



 



 



 



 



 



 



 



[G]



[G1]



[G1]



[G1]



[G1]



[G1]



[G]



[G1]



[G]



[G1]



[G1]



[G1]



 0


 ppm


 Group 1



Mean


SD N



7.396 


2.152 


9        



1.551 


0.557 


9        



5.504 


1.535 


9        



0.152 


0.067 


9        



0.148 


0.105 


9        



0.013 


0.009 


9        



0.030 


0.018 


9        



8.427  0.272 


9        



252.90


36.44 


9      



13.28 


0.64 


9      



145.2 


3.9


9    



0.4310


0.0106


9        



 1500  ppm


 Group 2



Mean


SD


N tCtrl



5.573 


1.022 


10      


0.75   



1.448 


0.476 


10      


0.93   



3.842  **


0.770 


10      


0.70   



0.160 


0.063 


10      


1.05   



0.093 


0.038 


10      


0.63   



0.008 


0.006 


10      


0.60   



0.022 


0.010 


10      


0.73   



8.350  0.470 


10      


0.99   



251.42


84.41 


10      


0.99 



13.09 


0.72 


10      


0.99 



146.6 


3.9 


10    


1.01



0.4332


0.0174


10      


1.01   



 5000  ppm


 Group 3



Mean


SD


N tCtrl



5.695 


0.908 


10      


0.77   



1.333 


0.295 


10      


0.86   



4.107  *


0.744 


10      


0.75   



0.134 


0.049 


10      


0.88   



0.089 


0.034 


10      


0.60   



0.008 


0.004 


10      


0.60   



0.022 


0.009 


10      


0.73   



8.394  0.207 


10      


1.00   



198.37*


29.07 


10      


0.78 



12.76 


0.44 


10      


0.96 



144.8 


3.9 


10    


1.00



0.4303


0.0081


10      


1.00   



 15000  ppm


 Group 4



Mean


SD


N tCtrl



6.066  0.778 


10      


0.82   



1.129  0.232 


10      


0.73   



4.668  0.795 


10      


0.85   



0.114  0.032 


10      


0.75   



0.117  0.025 


10      


0.79   



0.012  0.004 


10      


0.90   



0.027  0.013 


10      


0.90   



8.111  0.557 


10      


0.96   



186.94**


24.30 


10      


0.74 



12.37 **


0.57 


10      


0.93 



141.6 


5.9 


10    


0.98



0.4111 0.0284


10      


0.95   



[G] - Anova & Dunnett: * = p ≤ 0.05; ** = p ≤ 0.01


[G1] - Kruskal-Wallis & Dunn: * = p ≤ 0.05; ** = p ≤ 0.01


 










































































Sex: Male



 



 



Reporting Hematology



MCV



MCH



MCHC



PLT



 



 



 



 



(fL)



(pg)



(g/L)



(10^9/L)



 



 



 



 



[G]



[G]



[G]



[G]



 0


 ppm


 Group 1



Mean


SD


N



51.18 


1.21 


9      



17.22 


0.60 


9      



336.3 


5.8


9    



659.2 


80.6 


9    



 1500  ppm


 Group 2



Mean


SD


N


tCtrl



51.95 


1.75 


10      


1.02 



17.58 


0.70 


10      


1.02 



338.4 


7.0 


10    


1.01



676.3 


85.8 


9    


1.03



 5000  ppm


 Group 3



Mean


SD


N


tCtrl



51.29 


1.71 


10      


1.00 



17.26 


0.60 


10      


1.00 



336.7 


4.3 


10    


1.00



631.1 


79.5 


10    


0.96



 15000  ppm


 Group 4



Mean


SD


N


tCtrl



50.70 


1.24 


10      


0.99 



17.52 


0.87 


10      


1.02 



345.6 


15.2 


10    


1.03



685.2 


73.1 


10    


1.04



[G] - Anova & Dunnett


 


Table 15             Haematology. Female – F0


























































































































































Sex: Female



 



 



 



 



 



 



Reporting Hematology



 



 



 



 



WBC



NEUT



LYMPH



MONO



EOS



BASO



LUC



RBC



RETIC



RDWG



HGB



HCT



 



 



 



 



 



 



 



 



 



 



 



 



(10^9/L)



(10^9/L)



(10^9/L)



(10^9/L)



(10^9/L)



(10^9/L)



(10^9/L)



(10^12/L)



(10^9/L)



(%)



(g/L)



(L/L)



 



 



 



 



 



 



 



 



 



 



 



 



[G]



[G]



[G]



[G]



[G]



[G]



[G1]



[G]



[G]



[G]



[G]



[G]



 0


 ppm


 Group 1



Mean


SD N



4.953  


0.720  


10       



1.332  


0.314  


10       



3.288  


0.595  


10       



0.189  


0.074  


10       



0.117  


0.126  


10       



0.011  


0.007  


10       



0.018  


0.008  


9         



8.763  


0.410  


10       



222.02 40.77  


10       



12.93  


0.76


10       



165.8  


4.3


10     



0.4908


0.0166


10       



 1500  ppm


 Group 2


 



Mean


SD


N tCtrl



5.281  


0.915  


10       


1.07    



1.644  


0.431  


10       


1.23    



3.231  


0.505  


10       


0.98    



0.207  


0.045  


10       


1.10    



0.155  


0.186  


10       


1.32    



0.007  


0.005  


10       


0.64    



0.035  


0.020  


10       


1.97    



8.712  


0.380  


10       


0.99    



209.70 32.26  


10       


0.94  



13.15  


0.93  


10       


1.02  



162.5  


7.0  


10     


0.98



0.4849


0.0208


10       


0.99    



 5000  ppm


 Group 3


 



Mean


SD


N tCtrl



5.024  


0.955  


10       


1.01    



1.810  


0.776  


10       


1.36    



2.914  


0.786  


10       


0.89    



0.175  


0.064  


10       


0.93    



0.096  


0.039  


10       


0.82    



0.006  


0.007  


10       


0.55    



0.024  


0.011  


10       


1.35    



8.435  


0.301  


10       


0.96    



198.20 49.19  


10       


0.89  



13.62  


0.86  


10       


1.05  



157.3  *


6.1  


10     


0.95



0.4749


0.0173


10       


0.97    



 15000  ppm


 Group 4


 



Mean


SD


N tCtrl



5.519   1.617  


10       


1.11    



2.123   1.073  


10       


1.59    



3.036   0.706  


10       


0.92    



0.204   0.083  


10       


1.08    



0.120   0.075  


10       


1.03    



0.008   0.004  


10       


0.73    



0.022  


0.008  


10       


1.24    



8.579  


0.509  


10       


0.98    



188.86 34.10  


10       


0.85  



14.01  *


0.66  


10       


1.08  



157.6  *


6.7  


10     


0.95



0.4739 0.0235


10       


0.97    



[G] - Anova & Dunnett: * = p ≤ 0.05


 










































































Sex: Female



 



 



Reporting Hematology



MCV



MCH



MCHC



PLT



 



 



 



 



(fL)



(pg)



(g/L)



(10^9/L)



 



 



 



 



[G]



[G]



[G]



[G]



 0


 ppm


 Group 1



Mean


SD


N



56.06 


2.11 


10      



18.96 


0.64 


10      



338.2 


6.5


10    



828.6 


42.8


10    



 1500  ppm


 Group 2



Mean


SD


N


tCtrl



55.68 


1.53 


10      


0.99 



18.66 


0.65 


10      


0.98 



335.3 


7.2 


10    


0.99



791.9 


113.1 


10    


0.96



 5000  ppm


 Group 3



Mean


SD


N


tCtrl



56.32 


1.63 


10      


1.00 



18.64 


0.39 


10      


0.98 



331.5 


5.4 


10    


0.98



758.5 


92.4 


10    


0.92



 15000  ppm


 Group 4



Mean


SD


N


tCtrl



55.34 


2.33 


10      


0.99 



18.40 


0.64 


10      


0.97 



332.6 


5.7 


10    


0.98



627.4  **


128.2 


10    


0.76



[G] - Anova & Dunnett: ** = p ≤ 0.01


 


 


Table 16             Coagulation. Male – F0







































Sex: Male



 



Reporting Coagulation



PT


(sec)


[G]



APTT


(sec)


[G]



 0


 ppm


 Group 1



Mean


SD


N



16.89  


1.06


10       



21.39  


3.99  


9       



 1500  ppm


 Group 2


 



Mean


SD


N


tCtrl



17.08  


1.03  


8       


1.01  



22.10  


4.86  


10       


1.03  



 5000  ppm


 Group 3


 



Mean


SD


N


tCtrl



17.24  


0.80  


10       


1.02  



20.44  


1.56  


10       


0.96  



 15000  ppm


 Group 4


 



Mean


SD


N


tCtrl



17.09  


0.45  


9       


1.01  



19.52  


4.64  


9       


0.91  



[G] - Anova & Dunnett


 


Table 17             Coagulation. Female – F0







































Sex: Female



 



Reporting Coagulation



PT


(sec)


[G]



APTT


(sec)


[G]



 0


 ppm


 Group 1



Mean


SD


N



17.75  


2.14


10       



19.97  


2.16


10       



 1500  ppm


 Group 2


 



Mean


SD


N


tCtrl



16.54  


0.76  


10       


0.93  



20.88  


1.02  


10       


1.05  



 5000  ppm


 Group 3


 



Mean


SD


N


tCtrl



16.73  


0.93  


10       


0.94  



19.72  


1.72  


10       


0.99  



 15000  ppm


 Group 4


 



Mean


SD


N


tCtrl



17.07  


0.85  


10       


0.96  



20.72  


1.46  


10       


1.04  



[G] - Anova & Dunnett


 


Table 18             Clinical Chemistry. Male – F0


























































































































































Sex: Male



 



 



 



 



 



 



Reporting Biochemistry



 



 



 



 



ALT



AST



ALP



TPROT



ALB



BILEAC



TBIL



UREA



CREAT



GLUC



CHOL



HDL



 



 



 



 



 



 



 



 



 



 



 



 



(U/L)



(U/L)



(U/L)



(g/L)



(g/L)



(umol/L)



(umol/L)



(mmol/L)



(umol/L)



(mmol/L)



(mmol/L)



(mmol/L)



 



 



 



 



 



 



 



 



 



 



 



 



[G]



[G1]



[G]



[G]



[G]



[G]



[G1]



[G]



[G]



[G]



[G1]



[G1]



 0


 ppm


 Group 1



Mean


SD N



40.2  


4.9


10     



79.1  


5.8


10     



83.1  


17.3


10     



62.59  


1.74


10       



36.95  


1.01


10       



13.67  


8.73


10       



1.81  


0.39


10       



5.43  


0.60


10       



29.9  


3.8


10     



8.396  


1.434  


10       



1.510  


0.193  


10       



0.808  


0.083  


10       



 1500  ppm


 Group 2


 



Mean


SD


N tCtrl



38.2  


5.6  


10     


0.95



85.3  


12.8  


10     


1.08



101.2   18.0  


10     


1.22



63.48  


3.26  


10       


1.01  



37.57  


1.46  


10       


1.02  



15.12  


14.64  


10       


1.11  



1.77  


0.16  


10       


0.98  



5.28  


0.28  


10       


0.97  



29.3  


4.1  


10     


0.98



7.760  


0.706  


10       


0.92    



1.605  


0.344  


10       


1.06    



0.841  


0.148  


10       


1.04    



 5000  ppm


 Group 3


 



Mean


SD


N tCtrl



37.9  


5.4  


10     


0.94



76.5  


7.3  


10     


0.97



94.0  


17.8  


10     


1.13



63.67  


2.59  


10       


1.02  



36.98  


1.03  


10       


1.00  



18.76  


9.37  


10       


1.37  



1.71  


0.28  


10       


0.94  



5.56  


0.58  


10       


1.03  



30.1  


3.8  


10     


1.01



7.841  


0.776  


10       


0.93    



1.492  


0.212  


10       


0.99    



0.781  


0.071  


10       


0.97    



 15000  ppm


 Group 4


 



Mean


SD


N tCtrl



39.8  


7.2  


10     


0.99



95.9  


38.9  


10     


1.21



118.4 **


21.2  


10     


1.42



62.06  


2.49  


10       


0.99  



36.05  


1.34  


10       


0.98  



8.62   3.51  


10       


0.63  



1.93   0.30  


10       


1.07  



5.72   0.49  


10       


1.05  



31.5  


2.2  


10     


1.05



8.331   1.292  


10       


0.99    



1.941  * 0.401  


10       


1.29    



0.985  * 0.167  


10       


1.22    



[G] - Kruskal-Wallis & Dunn: ** = p ≤ 0.01


[G1] - Anova & Dunnett: * = p ≤ 0.05







































































































Sex: Male



 



 



Reporting Biochemistry



 



 



LDL



TRIG



NA



K



CL



CA



PHOS



 



 



 



 



 



 



 



(mmol/L)



(mmol/L)



(mmol/L)



(mmol/L)



(mmol/L)



(mmol/L)



(mmol/L)



 



 



 



 



 



 



 



[G]



[G1]



[G1]



[G1]



[G1]



[G1]



[G1]



 0


 ppm


 Group 1



Mean


SD N



0.329  


0.035  


10       



0.431  


0.127  


10       



144.2  


1.0


10     



3.86  


0.21


10       



107.0  


1.1


10     



2.523  


0.065  


10       



1.766  


0.205  


10       



1500  ppm


Group 2


 



Mean


SD


N tCtrl



0.336  


0.081  


10       


1.02    



0.515  


0.139  


10       


1.19    



143.4  


1.4  


10     


0.99



3.75  


0.19  


10       


0.97  



107.0  


1.6  


10     


1.00



2.563  


0.032  


10       


1.02    



1.649  


0.109  


10       


0.93    



 5000  ppm


 Group 3


 



Mean


SD


N


tCtrl



0.335  


0.087  


10       


1.02    



0.561  


0.195  


10       


1.30    



143.6  


1.3  


10     


1.00



3.70  


0.12  


10       


0.96  



107.1  


1.6  


10     


1.00



2.536  


0.056  


10       


1.01    



1.589  *


0.150  


10       


0.90    



 15000  ppm


 Group 4


 



Mean


SD


N


tCtrl



0.425   0.103  


10       


1.29    



0.502   0.160  


10       


1.16    



143.7  


1.5  


10     


1.00



3.79  


0.14  


10       


0.98  



106.7  


1.9  


10     


1.00



2.598  * 0.071  


10       


1.03    



1.909   0.140  


10       


1.08    



[G] - Kruskal-Wallis & Dunn


[G1] - Anova & Dunnett: * = p ≤ 0.05


 


Table 19             Clinical Chemistry. Female – F0


























































































































































Sex: Female



 



 



 



 



 



 



Reporting Biochemistry



 



 



 



 



ALT



AST



ALP



TPROT



ALB



BILEAC



TBIL



UREA



CREAT



GLUC



CHOL



HDL



 



 



 



 



 



 



 



 



 



 



 



 



(U/L)



(U/L)



(U/L)



(g/L)



(g/L)



(umol/L)



(umol/L)



(mmol/L)



(umol/L)



(mmol/L)



(mmol/L)



(mmol/L)



 



 



 



 



 



 



 



 



 



 



 



 



[G]



[G]



[G1]



[G1]



[G1]



[G1]



[G1]



[G1]



[G1]



[G1]



[G1]



[G1]



 0


 ppm


 Group 1



Mean


SD N



45.7  


6.1


10     



94.3  


8.5


10     



96.6  


81.2


10     



63.62  


2.87


10       



39.79  


1.80


10       



18.85  


8.28


10       



2.14  


0.51


10       



8.96  


1.33


10       



24.9  


4.1


10     



9.104  


0.930  


10       



1.990  


0.280  


10       



1.179  


0.141  


10       



 1500  ppm


 Group 2


 



Mean


SD


N tCtrl



50.0  


4.1  


10     


1.10



120.3  *


22.7  


10     


1.28



75.3  


19.7  


10     


0.78



62.15  


2.64  


10       


0.98  



39.30  


1.86  


10       


0.99  



35.89  *


15.46  


10       


1.90  



2.09  


0.35  


10       


0.98  



8.78  


1.51  


10       


0.98  



24.7  


2.3  


10     


0.99



8.396  


0.792  


10       


0.92    



1.951  


0.288  


10       


0.98    



1.209  


0.196  


10       


1.03    



 5000  ppm


 Group 3


 



Mean


SD


N tCtrl



49.0  


5.8  


10     


1.07



117.2 **


11.6  


10     


1.24



76.8  


18.5  


10     


0.80



60.95  


2.13  


10       


0.96  



38.49  


1.43  


10       


0.97  



22.48  


13.28  


10       


1.19  



1.88  


0.42  


10       


0.88  



9.28  


1.24  


10       


1.04  



23.6  


3.3  


10     


0.95



8.226  


0.616  


10       


0.90    



2.053  


0.170  


10       


1.03    



1.252  


0.094  


10       


1.06    



 15000  ppm


 Group 4


 



Mean


SD


N tCtrl



56.5   12.4  


10     


1.24



117.7 **


24.1  


10     


1.25



111.8   43.3  


10     


1.16



60.21  


3.99  


10       


0.95  



37.43  


2.59  


10       


0.94  



23.64   16.43  


10       


1.25  



2.04   0.45  


10       


0.95  



9.54   1.52  


10       


1.06  



26.5  


4.3  


10     


1.06



8.817   0.874  


10       


0.97    



2.284   0.318  


10       


1.15    



1.353   0.234  


10       


1.15    



[G] - Kruskal-Wallis & Dunn: * = p ≤ 0.05; ** = p ≤ 0.01


[G1] - Anova & Dunnett: * = p ≤ 0.05


 







































































































Sex: Female



 



 



Reporting Biochemistry



 



 



LDL



TRIG



NA



K



CL



CA



PHOS



 



 



 



 



 



 



 



(mmol/L)



(mmol/L)



(mmol/L)



(mmol/L)



(mmol/L)



(mmol/L)



(mmol/L)



 



 



 



 



 



 



 



[G]



[G1]



[G1]



[G1]



[G1]



[G1]



[G1]



 0


 ppm


 Group 1



Mean


SD N



0.356  


0.130  


10       



1.229  


0.437  


10       



140.9  


0.9


10     



3.33  


0.39


10       



102.1  


3.0


10     



2.588  


0.081  


10       



2.236  


0.306  


10       



1500  ppm


Group 2


 



Mean


SD


N tCtrl



0.330  


0.051  


10       


0.93    



1.552  


0.352  


10       


1.26    



140.3  


1.7  


10     


1.00



3.38  


0.28  


10       


1.02  



99.7  


1.8  


10     


0.98



2.575  


0.114  


10       


0.99    



2.568  


0.290  


10       


1.15    



 5000  ppm


 Group 3


 



Mean


SD


N


tCtrl



0.341  


0.046  


10       


0.96    



1.456  


0.386  


10       


1.18    



140.4  


1.1  


10     


1.00



3.47  


0.31  


10       


1.04  



101.4  


2.0  


10     


0.99



2.612  


0.095  


10       


1.01    



2.544  


0.398  


10       


1.14    



 15000  ppm


 Group 4


 



Mean


SD


N


tCtrl



0.355   0.060  


10       


1.00    



1.813   1.212  


10       


1.48    



140.3  


1.3  


10     


1.00



3.70


0.27  


10       


1.11  



101.6  


2.4  


10     


1.00



2.567   0.117  


10       


0.99    



2.510   0.429  


10       


1.12    



[G] - Anova & Dunnett


[G1] - Kruskal-Wallis & Dunn


 


Table 20             Thyroid Hormones. Male – F0













































Sex: Male



 



Rep



orting Special Chemistry



T3


(ng/mL)


[G]



T4


(ng/mL)


[G]



TSH


(mU/L)


[G1]



 0


 ppm


 Group 1



Mean


SD


N



0.402  


0.085  


10         



52.46  


8.54


10       



0.1162  


0.0515  


10           



 1500  ppm


 Group 2


 



Mean


SD


N


tCtrl



0.422  


0.051  


10         


1.05    



49.70  


7.87  


10       


0.95  



0.2174  


0.1875  


10           


1.87      



 5000  ppm


 Group 3


 



Mean


SD


N


tCtrl



0.399  


0.052  


10         


0.99    



56.16  


8.05  


10       


1.07  



0.2475  


0.1190  


10           


2.13      



 15000  ppm


 Group 4


 



Mean


SD


N


tCtrl



0.430  


0.089  


10         


1.07    



52.31  


6.01  


10       


1.00  



0.2587


0.2604  


10           


2.23      



[G] - Anova & Dunnett


[G1] - Kruskal-Wallis & Dunn


 


Table 21             Thyroid Hormones. Female – F0













































Sex: Female



 



Rep



orting Special Chemistry



T3


(ng/mL)


[G]



T4


(ng/mL)


[G]



TSH


(mU/L)


[G1]



 0


 ppm


 Group 1



Mean


SD


N



0.494  


0.122  


10         



47.29  


13.60  


10       



0.2577  


0.3387  


10           



 1500  ppm


 Group 2


 



Mean


SD


N


tCtrl



0.509  


0.136  


10         


1.03    



43.90  


10.01  


10       


0.93  



0.2031  


0.1018  


10           


0.79      



 5000  ppm


 Group 3


 



Mean


SD


N


tCtrl



0.554  


0.157  


10         


1.12    



46.36  


9.27  


10       


0.98  



0.3151  


0.1696  


10           


1.22      



 15000  ppm


 Group 4


 



Mean


SD


N


tCtrl



0.510  


0.148  


10         


1.03    



43.57  


8.57  


10       


0.92  



0.5715


0.4586  


10           


2.22      



[G] - Anova & Dunnett


[G1] - Kruskal-Wallis & Dunn


 


Table 22             Urinalysis. Male – F0














































Sex: Male



 



 



Reporting Urinalysi



s



VOLUME


(mL)


[G]



SPECIFIC


GRAVITY


[G]



URINE pH


[G]



 0


 ppm


 Group 1



Mean


SD


N



4.00 


1.15 


10      



1.0403 


0.0045 


10          



6.70 


0.67 


10      



 1500  ppm


 Group 2



Mean


SD


N


tCtrl



5.70 


3.86 


10      


1.43 



1.0331 


0.0090 


10          


0.99     



6.90 


0.46 


10      


1.03 



 5000  ppm


 Group 3



Mean


SD


N


tCtrl



7.30 


4.11 


10      


1.83 



1.0305  *


0.0075 


10          


0.99     



6.95 


0.28 


10      


1.04 



 15000  ppm


 Group 4



Mean


SD


N


tCtrl



9.50  **


4.60 


10      


2.38 



1.0242  **


0.0067 


10          


0.98     



7.25 


0.26 


10      


1.08 



[G] - Anova & Dunnett: * = p ≤ 0.05; ** = p ≤ 0.01


 


Table 23             Urinalysis. Female – F0














































Sex: Female



 



 



Reporting Urinalysi



s



VOLUME


(mL)


[G]



SPECIFIC


GRAVITY


[G]



URINE pH


[G]



 0


 ppm


 Group 1



Mean


SD


N



11.40 


3.24 


10      



1.0282 


0.0064 


10          



6.30 


0.42 


10      



 1500  ppm


 Group 2



Mean


SD


N


tCtrl



13.85 


4.00 


10      


1.21 



1.0251 


0.0036 


10          


1.00     



6.25 


0.26 


10      


0.99 



 5000  ppm


 Group 3



Mean


SD


N


tCtrl



13.60 


3.66 


10      


1.19 



1.0251 


0.0049 


10          


1.00     



6.30 


0.42 


10      


1.00 



 15000  ppm


 Group 4



Mean


SD


N


tCtrl



13.20 


4.52 


10      


1.16 



1.0257


0.0039 


10          


1.00     



6.45


0.44 


10      


1.02 



[G] - Anova & Dunnett


 


Table 24             Organ Weights (g). Male – F0





























































































































 



GROUP 1


CONTROL



GROUP 2


1500 PPM



GROUP 3


5000 PPM



GROUP 4


15000 PPM



BODY WEIGHT



MEAN


SD


N



363


32


25



365


34


25



384


34


25



354


36


25



BRAIN



MEAN


SD


N



2.02


0.09


25



2.02


0.09


25



2.07


0.08


25



2.02


0.07


25



PITUITARY



MEAN


SD


N



0.0102


0.0013


25



0.0099


0.0011


25



0.0105


0.0012


24



0.0098


0.0012


24



HEART



MEAN


SD


N



0.962


0.101


25



0.990


0.088


25



1.049 **


0.104


25



0.950


0.103


25



LIVER



MEAN


SD


N



7.98


0.93


25



8.16


0.71


25



8.87 **


0.91


25



9.00 **


1.03


25



THYROIDS



MEAN


SD


N



0.0158


0.0028


25



0.0151


0.0029


25



0.0176


0.0031


25



0.0177


0.0030


25



THYMUS



MEAN


SD


N



0.290


0.073


25



0.311


0.088


25



0.304


0.080


25



0.292


0.072


25



KIDNEYS



MEAN


SD


N



2.21


0.21


25



2.69


2.42


25



2.43


0.26


25



2.37


0.25


25



ADRENALS



MEAN


SD


N



0.055


0.009


25



0.056


0.011


25



0.057


0.012


25



0.050


0.008


25



SPLEEN



MEAN


SD


N



0.578


0.095


25



0.633


0.095


25



0.622


0.098


25



0.618


0.117


25



TESTES



MEAN


SD


N



3.36


0.22


25



3.49


0.37


25



3.60 *


0.32


25



3.41


0.30


25



PROSTATE GLAND



MEAN


SD


N



0.874


0.118


25



0.859


0.116


25



0.950


0.181


25



0.838


0.136


25



EPIDIDYMIDES



MEAN


SD


N



1.118


0.088


25



1.143


0.104


25



1.184


0.106


25



1.127


0.106


25



SEMINAL VESICLES



MEAN


SD


N



1.543


0.232


25



1.462


0.234


24



1.489


0.240


25



1.506


0.260


25



*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level


 


 


Table 25             Organ Weights (g). Female – F0













































































































 



GROUP 1


CONTROL



GROUP 2


1500 PPM



GROUP 3


5000 PPM



GROUP 4


15000 PPM



BODY WEIGHT



MEAN


SD


N



235


15


25



234


11


24



230


13


25



227


17


25



BRAIN



MEAN


SD


N



1.89


0.09


25



1.90


0.06


24



1.91


0.07


25



1.88


0.08


25



PITUITARY



MEAN


SD


N



0.0126


0.0018


25



0.0129


0.0024


24



0.0129


0.0022


25



0.0121


0.0019


25



HEART



MEAN


SD


N



0.865


0.072


25



0.856


0.070


24



0.823


0.075


25



0.825


0.087


25



LIVER



MEAN


SD


N



8.76


1.17


25



8.28


1.26


24



8.36


1.34


25



9.27


1.25


25



THYROIDS



MEAN


SD


N



0.0157


0.0030


25



0.0158


0.0021


24



0.0165


0.0033


25



0.0183 *


0.0036


25



THYMUS



MEAN


SD


N



0.234


0.042


25



0.213


0.043


24



0.224


0.046


25



0.203 *


0.045


25



KIDNEYS



MEAN


SD


N



1.87


0.18


25



1.85


0.19


24



1.84


0.19


25



1.87


0.16


25



ADRENALS



MEAN


SD


N



0.069


0.010


25



0.067


0.009


24



0.063


0.007


25



0.060 **


0.008


25



SPLEEN



MEAN


SD


N



0.528


0.075


25



0.507


0.069


24



0.524


0.068


25



0.506


0.067


25



OVARIES



MEAN


SD


N



0.148


0.022


25



0.138


0.020


24



0.136


0.019


25



0.134


0.023


25



UTERUS



MEAN


SD


N



0.685


0.170


25



0.674


0.211


24



0.650


0.234


25



0.670


0.271


25



*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level


 


Table 26             Organ/Body Weight Ratio (%). Male – F0





















































































































 



GROUP 1


CONTROL



GROUP 2


1500 PPM



GROUP 3


5000 PPM



GROUP 4


15000 PPM



BRAIN



MEAN


SD


N



0.56


0.04


25



0.56


0.04


25



0.54


0.04


25



0.58


0.05


25



PITUITARY



MEAN


SD


N



0.0028


0.0002


25



0.0027


0.0002


25



0.0027


0.0002


24



0.0028


0.0002


24



HEART



MEAN


SD


N



0.265


0.019


25



0.272


0.017


25



0.274


0.018


25



0.269


0.018


25



LIVER



MEAN


SD


N



2.20


0.14


25



2.24


0.11


25



2.31 **


0.11


25



2.54 **


0.11


25



THYROIDS



MEAN


SD


N



0.0044


0.0008


25



0.0042


0.0007


25



0.0046


0.0007


25



0.0050 **


0.0008


25



THYMUS



MEAN


SD


N



0.080


0.018


25



0.085


0.022


25



0.079


0.019


25



0.082


0.017


25



KIDNEYS



MEAN


SD


N



0.61


0.04


25



0.75


0.70


25



0.63


0.04


25



0.67


0.05


25



ADRENALS



MEAN


SD


N



0.015


0.002


25



0.015


0.003


25



0.015


0.002


25



0.014


0.002


25



SPLEEN



MEAN


SD


N



0.159


0.020


25



0.174 *


0.027


25



0.162


0.018


25



0.174 *


0.022


25



TESTES



MEAN


SD


N



0.93


0.06


25



0.96


0.09


25



0.94


0.07


25



0.97


0.08


25



PROSTATE GLAND



MEAN


SD


N



0.242


0.034


25



0.236


0.026


25



0.247


0.037


25



0.237


0.029


25



EPIDIDYMIDES



MEAN


SD


N



0.309


0.019


25



0.314


0.027


25



0.309


0.020


25



0.320


0.029


25



SEMINAL VESICLES



MEAN


SD


N



0.427


0.068


25



0.399


0.049


24



0.389


0.054


25



0.429


0.080


25



*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level


 


Table 27             Organ/Body Weight Ratio (%). Female – F0





































































































 



GROUP 1


CONTROL



GROUP 2


1500 PPM



GROUP 3


5000 PPM



GROUP 4


15000 PPM



BRAIN



MEAN


SD


N



0.81


0.04


25



0.81


0.03


24



0.83


0.05


25



0.83


0.06


25



PITUITARY



MEAN


SD


N



0.0054


0.0007


25



0.0055


0.0009


24



0.0056


0.0009


25



0.0053


0.0008


25



HEART



MEAN


SD


N



0.369


0.027


25



0.365


0.027


24



0.358


0.024


25



0.363


0.031


25



LIVER



MEAN


SD


N



3.73


0.39


25



3.53


0.51


24



3.63


0.50


25



4.08 *


0.45


25



THYROIDS



MEAN


SD


N



0.0067


0.0013


25



0.0067


0.0008


24



0.0072


0.0013


25



0.0081 **


0.0016


25



THYMUS



MEAN


SD


N



0.100


0.019


25



0.091


0.019


24



0.097


0.019


25



0.090


0.022


25



KIDNEYS



MEAN


SD


N



0.80


0.07


25



0.79


0.07


24



0.80


0.07


25



0.82


0.04


25



ADRENALS



MEAN


SD


N



0.029


0.004


25



0.028


0.004


24



0.028


0.004


25



0.027 *


0.003


25



SPLEEN



MEAN


SD


N



0.226


0.034


25



0.217


0.030


24



0.228


0.030


25



0.223


0.024


25



OVARIES



MEAN


SD


N



0.063


0.009


25



0.059


0.009


24



0.059


0.009


25



0.059


0.010


25



UTERUS



MEAN


SD


N



0.294


0.080


25



0.288


0.089


24



0.286


0.117


25



0.295


0.120


25



*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level


 


Table 28             Mean Percent Liver, Thyroid Gland and Female Adrenal Gland Weight Differences from Control Group. F0
































































































































 



Males



Females



Dose level (ppm):



1500



5000



15000



1500



5000



15000



 



 



 



 



 



 



 



LIVER



 



 



 



 



 



 



               Absolute



2



11**



13**



-5



-5



6



               Relative to body weight



2



5**



15**



-5



-3



9*



 



 



 



 



 



 



 



THYROID GLANDS



 



 



 



 



 



 



               Absolute



-4



11



12



1



5



17*



               Relative to body weight



-5



5



14**



0



7



21**



 



 



 



 



 



 



 



ADRENAL GLANDS



 



 



 



 



 



 



               Absolute



-



-



-



-3



-9



-13**



               Relative to body weight



-



-



-



-3



-3



-7*



*: P≤0.05, **: P≤0.01


-                 No statistically significant organ weight changes in male adrenal gland.


 


Table 29             Sperm Motility, Concentration, and Morphology – F0





























































































 



GROUP 1


CONTROL



GROUP 2


1500 PPM



GROUP 3


5000 PPM



GROUP 4


15000 PPM



MOTILE SPERM (%)



MEAN


SD


N



64


17


25



66


13


25



60


16


25



63


15


25



PROGRESSIVE SPERM (%)



MEAN


SD


N



27


12


25



25


9


25



23


8


25



25


10


25



SPERM COUNT EPI


(10E6/gram)



MEAN


SD


N



315.5


104.1


25



379.9 *


115.9


25



297.2


99.1


25



338.9


112.4


25



NORMAL MORPHOLOGY (number of cells)



MEAN


SD


N



187


10


25



182


18


25



185


7


25



180


16


25



DETACHED HEAD (number of cells)



MEAN


SD


N



3


3


25



2


2


25



3


3


25



3


4


25



ABNORMAL HEAD (number of cells)



MEAN


SD


N



0


1


25



0


1


25



1


1


25



0


1


25



COILED TAIL (number of cells)



MEAN


SD


N



10


10


25



15


17


25



9


7


25



15


14


25



OTHER TAIL (number of cells)



MEAN


SD


N



0


0


25



0


1


25



1


1


25



0


0


25



ABNORMAL NECK (number of cells)



MEAN


SD


N



1


1


25



0


1


25



1


1


25



1


1


25



COMBINED (number of cells)



MEAN


SD


N



0


0


25



0


0


25



0


0


25



0


0


25



*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level


 


Table 30             Reproductive Data Summary – F0



















































































 



GROUP 1


CONTROL



GROUP 2


1500 PPM



GROUP 3


5000 PPM



GROUP 4


15000 PPM



Males paired



25



25



25



25



Males mated



25



23



25



25



Females paired



25



25



25



25



Females mated



25



23



25



25



Pregnant females



24



22



23



23



Females with implantations only



0



0



1



0



Females with living pups on Day 1



24



22



22



23



Mating Index, females (%)



100



92



100



100



Fertility Index, females (%)



96



96



92



92



Gestation Index (%)



100



100



96



100



 


Table 31             Precoital Time – F0












































































DAY OF THE PAIRING PERIOD



GROUP 1


CONTROL



GROUP 2


1500 PPM



GROUP 3


5000 PPM



GROUP 4


15000 PPM



NUMBER OF FEMALES MATED



 



 



 



 



1



5



8



6



8



2



4



5



8



3



3



13



7



5



7



4



1



3



6



7



5



2



-



-



-



MEDIAN PRECOITAL TIME



3



2



2



3



MEAN PRECOITAL TIME



2.6



2.2



2.4



2.5



N



25



23



25



25



+/++ Steel-test significant at 5% (+) or 1% (++) level


 


Table 32             Implantation Sites Summary – F0





























 



GROUP 1


CONTROL



GROUP 2


1500 PPM



GROUP 3


5000 PPM



GROUP 4


15000 PPM



IMPLANTATIONS



MEAN


SD


N



12.3


2.4


24



11.6


2.4


22



11.8


3.0


23



11.9


1.5


23


      

+/++ Steel-test significant at 5% (+) or 1% (++) level


 


Table 33             Developmental Data – F0



















































































 



GROUP 1


CONTROL



GROUP 2


1500 PPM



GROUP 3


5000 PPM



GROUP 4


15000 PPM



Total number of offspring born



279



234



256



261



Total number of uterine implantation sites



294



256



271



273



Number of live offspring on Day 1 after littering



278



227



255



261



Number of live offspring on Day 4 (before culling)1



275



226



255



259



Number of live offspring on Day 4 (after culling)



190



161



171



183



Number of live offspring on Day 21 after littering



188



161



171



183



Post-implantation survival index (%)



95



91



94



96



Live birth index (%)



100



97



100



100



Viability Index (%)



99



100



100



99



Weaning index (%)



99



100



100



100




  • By mistake, Female No. 142 (Group 2) was diagnosed with Total litter Loss; at necropsy a living pup was found


 


Table 34             Body Weights (g). Male – F1 (Cohorts 1A, 1B, 1C)





























































































































 



GROUP 1


CONTROL



GROUP 2


1500 PPM



GROUP 3


5000 PPM



GROUP 4


15000 PPM



DAY 1


WEEK 1



MEAN


SD


N



53


4.2


60



54


4.1


60



54


4.5


60



51 **


4.6


60



DAY 8


WEEK 2



MEAN


SD


N



77


7.1


60



78


7.6


60



77


8.6


60



68 **


9.2


60



DAY 15


WEEK 3



MEAN


SD


N



123


8.9


60



123


10.3


60



122


10.7


60



111 **


12.9


60



DAY 22


WEEK 4



MEAN


SD


N



170


10.1


60



170


12.0


60



169


12.5


60



154 **


16.2


60



DAY 29


WEEK 5



MEAN


SD


N



213


12.2


60



213


14.1


60



213


14.3


60



198 **


18.9


60



DAY 36


WEEK 6



MEAN


SD


N



255


13.9


40



254


17.8


40



257


16.2


40



242 **


22.3


40



DAY 43


WEEK 7



MEAN


SD


N



291


15.9


40



288


21.3


40



293


17.7


40



277 **


25.0


40



DAY 50


WEEK 8



MEAN


SD


N



318


19.1


40



314


24.2


40



323


19.5


40



305 *


28.1


40



DAY 57


WEEK 9



MEAN


SD


N



340


21.7


40



336


29.3


40



342


25.6


40



322 *


30.4


40



DAY 64


WEEK 10



MEAN


SD


N



357


23.6


40



352


31.6


40



362


26.9


40



345


39.5


40



DAY 71


WEEK 11



MEAN


SD


N



370


24.5


35



367


33.1


35



376


26.9


35



352 *


31.8


35



DAY 77


WEEK 11



MEAN


SD


N



378


30.5


20



371


33.4


22



381


30.5


22



353 *


34.6


22



DAY 85


WEEK 13



MEAN


SD


N



391


30.1


20



382


33.6


20



394


31.8


20



367


35.7


20



DAY 92


WEEK 14



MEAN


SD


N



391


27.9


10



392


36.9


10



399


27.9


10



375


29.6


10



*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level


 


Table 35             Body Weights (g). Female – F1 (Cohorts 1A, 1B, 1C)













































































































































 



GROUP 1


CONTROL



GROUP 2


1500 PPM



GROUP 3


5000 PPM



GROUP 4


15000 PPM



DAY 1


WEEK 1



MEAN


SD


N



52


3.8


60



53


4.4


60



52


4.2


60



48 **


4.3


60



DAY 9


WEEK 2



MEAN


SD


N



76


7.1


60



78


7.6


60



76


7.9


60



69 **


7.0


60



DAY 16


WEEK 3



MEAN


SD


N



112


7.8


60



113


9.1


60



112


9.8


60



104 **


8.5


60



DAY 23


WEEK 4



MEAN


SD


N



140


9.7


60



141


10.2


60



139


10.7


60



130 **


9.8


60



DAY 30


WEEK 5



MEAN


SD


N



160


11.2


40



164


12.3


40



159


13.0


40



148 **


9.5


40



DAY 37


WEEK 6



MEAN


SD


N



178


12.9


40



182


13.9


40



177


14.8


40



164 **


10.6


40



DAY 44


WEEK 7



MEAN


SD


N



190


14.0


40



197


15.6


40



191


15.9


40



181 *


11.9


40



DAY 51


WEEK 8



MEAN


SD


N



200


14.0


39



208 *


17.5


40



201


15.8


40



190 **


12.1


40



DAY 58


WEEK 9



MEAN


SD


N



209


15.6


39



218 *


17.6


40



209


17.5


40



197 **


11.8


40



DAY 65


WEEK 10



MEAN


SD


N



217


15.9


39



226


17.7


40



218


18.3


40



203 **


18.0


40



DAY 71


WEEK 11



MEAN


SD


N



223


16.5


34



234 *


17.5


35



225


18.7


35



213 *


13.9


35



DAY 77


WEEK 11



MEAN


SD


N



225


17.3


19



231


18.4


22



223


17.5


22



211 *


13.1


22



DAY 85


WEEK 13



MEAN


SD


N



 



247


7.2


3



234


18.4


4



251


---


1



DAY 92


WEEK 14



MEAN


SD


N



 



260


---


1



254


---


1 x



255


---


1



DAY 105


WEEK 15



MEAN


SD


N



 



261


---


1



254


--


1 x



264


---


1



DAY 112


WEEK 16



MEAN


SD


N



 



261


--


1



254


--


1



263


--


1



*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level


x Explanations for excluded data are listed in the tables of the individual values


 


Table 36             Body Weight Gain (%). Male – F1 (Cohorts 1A, 1B, 1C)





























































































































 



GROUP 1


CONTROL



GROUP 2


1500 PPM



GROUP 3


5000 PPM



GROUP 4


15000 PPM



DAY 1


WEEK 1



MEAN


SD


N



0


0.0


60



0


0.0


60



0


0.0


60



0


0.0


60



DAY 8


WEEK 2



MEAN


SD


N



45


8.4


60



45


12.6


60



42


13.5


60



35 **


11.8


60



DAY 15


WEEK 3



MEAN


SD


N



130


13.4


60



128


18.2


60



126


18.0


60



119 **


16.6


60



DAY 22


WEEK 4



MEAN


SD


N



219


19.6


60



214


22.5


60



212


22.2


60



206 **


20.3


60



DAY 29


WEEK 5



MEAN


SD


N



299


26.2


60



294


28.8


60



293


27.2


60



292


24.3


60



DAY 36


WEEK 6



MEAN


SD


N



378


33.5


40



371


37.9


40



374


32.4


40



378


30.7


40



DAY 43


WEEK 7



MEAN


SD


N



445


41.3


40



434


45.4


40



441


36.8


40



449


36.8


40



DAY 50


WEEK 8



MEAN


SD


N



496


49.6


40



484


51.2


40



495


40.8


40



504


43.2


40



DAY 57


WEEK 9



MEAN


SD


N



536


55.6


40



523


62.0


40



529


48.7


40



539


48.6


40



DAY 64


WEEK 10



MEAN


SD


N



570


60.7


40



553


66.4


40



567


50.4


40



583


60.2


40



DAY 71


WEEK 11



MEAN


SD


N



597


64.2


35



580


69.8


35



592


49.3


35



607


61.7


35



DAY 77


WEEK 11



MEAN


SD


N



607


65.3


20



592


76.3


22



598


48.5


22



604


59.9


22



DAY 85


WEEK 13



MEAN


SD


N



631


67.2


20



611


76.7


20



625


43.3


20



626


63.2


20



DAY 92


WEEK 14



MEAN


SD


N



651


78.0


10



603


65.6


10



645


40.0


10



647


53.1


10



*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level


 


Table 37             Body Weight Gain (%). Female – F1 (Cohorts 1A, 1B, 1C)













































































































































 



GROUP 1


CONTROL



GROUP 2


1500 PPM



GROUP 3


5000 PPM



GROUP 4


15000 PPM



DAY 1


WEEK 1



MEAN


SD


N



0


0.0


60



0


0.0


60



0


0.0


60



0


0.0


60



DAY 9


WEEK 2



MEAN


SD


N



48


9.2


60



48


12.5


60



46


13.7


60



43 *


10.5


60



DAY 16


WEEK 3



MEAN


SD


N



118


13.9


60



116


17.1


60



115


18.5


60



116


16.8


60



DAY 23


WEEK 4



MEAN


SD


N



173


19.3


60



169


20.3


60



166


21.0


60



170


22.1


60



DAY 30


WEEK 5



MEAN


SD


N



210


24.6


40



210


23.5


40



205


27.3


40



206


23.9


40



DAY 37


WEEK 6



MEAN


SD


N



246


29.5


40



244


27.9


40



239


30.2


40



241


29.1


40



DAY 44


WEEK 7



MEAN


SD


N



268


32.5


40



273


30.0


40



267


32.7


40



275


32.2


40



DAY 51


WEEK 8



MEAN


SD


N



288


35.3


39



295


34.0


40



286


33.1


40



294


32.5


40



DAY 58


WEEK 9



MEAN


SD


N



305


39.2


39



313


34.1


40



301


37.8


40



309


33.0


40



DAY 65


WEEK 10



MEAN


SD


N



321


40.3


39



328


35.8


40



318


37.9


40



322


44.4


40



DAY 71


WEEK 11



MEAN


SD


N



335


40.8


34



343


38.4


35



330


38.9


35



347


39.1


35



DAY 77


WEEK 11



MEAN


SD


N



334


44.3


19



336


41.6


22



327


37.4


22



343


38.0


22



DAY 85


WEEK 13



MEAN


SD


N



 



355


24.3


3



365


34.9


4



434


---


1



DAY 92


WEEK 14



MEAN


SD


N



 



348


---


1



346


---


1 x



443


---


1



DAY 105


WEEK 15



MEAN


SD


N



 



350


---


1



346


--


1 x



462


---


1



DAY 112


WEEK 16



MEAN


SD


N



 



350


--


1



346


--


1



460


--


1



*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level


x Explanations for excluded data are listed in the tables of the individual values


 


Table 38             Body Weight (g). Female – F1 Cohort 1B, Post-coitum & Lactation



















































































































 



GROUP 1


CONTROL



GROUP 2


1500 PPM



GROUP 3


5000 PPM



GROUP 4


15000 PPM



POST-COITUM



DAY 0



MEAN


SD


N



228


16.1


18



240


18.0


18



229


17.3


18



214 *


13.0


19



DAY 4



MEAN


SD


N



241


16.5


18



251


18.3


18



239


17.9


18



225 *


15.8


19



DAY 7



MEAN


SD


N



247


16.0


18



258


19.5


18



246


19.8


18



231 *


15.8


19



DAY 11



MEAN


SD


N



261


18.3


18



272


19.2


18



260


21.9


18



244 *


17.9


19



DAY 14



MEAN


SD


N



271


18.5


18



282


19.8


18



269


24.2


18



253 *


17.9


19



DAY 17



MEAN


SD


N



296


20.7


18



305


19.4


18



290


28.5


18



274 *


18.6


19



DAY 20



MEAN


SD


N



332


23.3


17



341


22.2


18



324


33.6


18



306 **


20.1


19



LACTATING



DAY 1



MEAN


SD


N



256


15.6


18



267


21.9


17



252


22.4


19



235**


20.0


19



DAY 4



MEAN


SD


N



270


18.9


18



281


21.3


17



265


24.1


19



250 *


20.8


19



DAY 7



MEAN


SD


N



279


18.3


18



284


22.0


17



275


21.9


19



260 *


18.9


19



DAY 14



MEAN


SD


N



296


17.7


18



302


22.5


17



291


24.1


19



279 *


20.9


19



DAY 21



MEAN


SD


N



285


19.0


18



290


17.4


17



281


19.4


19



273


17.5


19



*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level


x Explanations for excluded data are listed in the tables of the individual values


 


Table 39             Body Weight Gain (%). Female – F1 Cohort 1B, Post-coitum & Lactation



















































































































 



GROUP 1


CONTROL



GROUP 2


1500 PPM



GROUP 3


5000 PPM



GROUP 4


15000 PPM



POST-COITUM



DAY 0



MEAN


SD


N



0


0.0


18



0


0.0


18



0


0.0


18



0


0.0


19



DAY 4



MEAN


SD


N



6


1.5


18



5


1.8


18



5


1.4


18



5


1.6


19



DAY 7



MEAN


SD


N



8


1.9


18



8


2.3


18



8


1.9


18



8


1.8


19



DAY 11



MEAN


SD


N



15


2.9


18



13


2.7


18



14


3.0


18



14


2.8


19



DAY 14



MEAN


SD


N



19


3.4


18



18


3.0


18



17


3.4


18



18


3.1


19



DAY 17



MEAN


SD


N



30


4.8


18



27


3.6


18



27


4.6


18



28


4.3


19



DAY 20



MEAN


SD


N



46


5.6


17



42


5.8


18



42


6.4


18



43


5.6


19



LACTATING



DAY 1



MEAN


SD


N



0


0.0


18



0


0.0


17



0


0.0


19



0


0.0


19



DAY 4



MEAN


SD


N



5


2.6


18



5


3.2


17



5


2.7


19



6


2.8


19



DAY 7



MEAN


SD


N



9


2.7


18



7


3.0


17



9


2.7


19



11


3.1


19



DAY 14



MEAN


SD


N



16


3.6


18



13


3.9


17



15


3.4


19



18


4.3


19



DAY 21



MEAN


SD


N



11


5.3


18



9


4.6


17



12


4.4


19



16 *


5.3


19



*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level


x Explanations for excluded data are listed in the tables of the individual values


 


Table 40             Relative Food Consumption (g/kg/day). Male – F1 (Cohorts 1A, 1B, 1C)





































































































































 



GROUP 1


CONTROL



GROUP 2


1500 PPM



GROUP 3


5000 PPM



GROUP 4


15000 PPM



DAYS 1-8


WEEKS 1-2



MEAN


SD


N (CAGE)



110


6.1


12



123


21.6


12



130 *


18.3


12



122


14.8


12



DAYS 8-15


WEEKS 2-3



MEAN


SD


N (CAGE)



115


3.0


12



116


4.2


12



119 *


3.7


12



124 **


4.0


12



DAYS 15-22


WEEKS 3-4



MEAN


SD


N (CAGE)



108


3.2


12



113 *


4.1


12



120 **


6.5


12



120 **


4.3


12



DAYS 22-29


WEEKS 4-5



MEAN


SD


N (CAGE)



97


2.3


12



102 **


2.5


12



104 **


3.9


12



111 **


3.7


12



DAYS 29-36


WEEKS 5-6



MEAN


SD


N (CAGE)



89


1.0


8



92 *


1.2


8



96 **


3.2


8



99 **


1.7


8



DAY 36-43


WEEKS 6-7



MEAN


SD


N (CAGE)



81


1.5


8



84 **


1.8


8



88 **


2.4


8



93 **


1.4


8



DAYS 43-50


WEEKS 7-8



MEAN


SD


N (CAGE)



75


1.7


8



79 **


1.6


8



84 **


1.8


8



87 **


1.6


8



DAYS 50-57


WEEKS 8-9



MEAN


SD


N (CAGE)



70


1.1


8



74 **


1.7


8



78 **


2.0


8



81 **


1.6


8



DAYS 57-64


WEEKS 9-10



MEAN


SD


N (CAGE)



65


1.4


8



69 **


1.4


8



72 **


1.6


8



75 *


2.3


8



DAYS 64-71


WEEKS 10-11



MEAN


SD


N (CAGE)



61


0.6


7



65 **


1.4


7



67 **


1.3


7



70 **


1.8


7



DAYS 71-77


WEEK 11



MEAN


SD


N (CAGE)



58


1.0


4



62 *


2.7


5



66 **


1.8


5



70 **


1.2


5



DAYS 77-85


WEEKS 11-13



MEAN


SD


N (CAGE)



57


1.4


4



61 *


3.1


4



65 **


2.2


4



67 **


1.2


4



DAYS 85-92


WEEKS 13-14



MEAN


SD


N (CAGE)



54


1.4


2



64


6.1


2



64


7.3


3



63


0.4


2



DAYS 92-99


WEEKS 14-15



MEAN


SD


N (CAGE)



29


9.2


2



30


8.3


2



26


11.0


3



34


8.6


2



MEAN OF MEANS



MEAN



76



81



84



87



*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level


 


Table 41             Relative Food Consumption (g/kg/day). Female – F1 (Cohorts 1A, 1B, 1C)













































































































 



GROUP 1


CONTROL



GROUP 2


1500 PPM



GROUP 3


5000 PPM



GROUP 4


15000 PPM



DAYS 1-9


WEEKS 1-2



MEAN


SD


N (CAGE)



106


5.1


12



113


7.8


12



119 **


6.6


12



121 **


10.8


12



DAYS 9-16


WEEKS 2-3



MEAN


SD


N (CAGE)



113


2.7


12



117 *


2.4


12



120 **


2.7


12



125 **


3.1


12



DAYS 16-23


WEEKS 3-4



MEAN


SD


N (CAGE)



106


2.1


12



108


2.9


12



11 **


1.9


12



117 **


3.1


12



DAYS 23-30


WEEKS 4-5



MEAN


SD


N (CAGE)



97


1.2


8



100 *


2.5


8



104 **


2.8


8



110 **


2.8


8



DAYS 30-37


WEEKS 5-6



MEAN


SD


N (CAGE)



92


1.0


8



97 **


2.0


8



99 **


3.5


8



105 **


4.2


8



DAY 37-44


WEEKS 6-7



MEAN


SD


N (CAGE)



85


2.6


8



91 **


1.6


8



93 **


2.6


8



98 **


3.0


8



DAYS 44-51


WEEKS 7-8



MEAN


SD


N (CAGE)



82


1.9


8



86 **


1.4


8



91 **


2.0


8



96 **


3.4


8



DAYS 51-58


WEEKS 8-9



MEAN


SD


N (CAGE)



79


2.9


8



81


1.6


8



85 **


1.7


8



93 **


3.7


8



DAYS 58-65


WEEKS 9-10



MEAN


SD


N (CAGE)



76


2.2


8



78


1.4


8



82 **


1.5


8



90 **


5.6


8



DAYS 65-71


WEEKS 10-11



MEAN


SD


N (CAGE)



73


2.7


7



76 *


0.9


7



79 **


2.1


7



87 **


3.1


7



DAYS 71-77


WEEK 11



MEAN


SD


N (CAGE)



71


0.9


4



73


2.0


5



77 *


2.1


5



82 **


5.5


5



MEAN OF MEANS



MEAN



89



93



96



102



*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level


 


Table 42             Relative Food Consumption (g/kg/day). Female – F1 Cohort 1B, Post-coitum & Lactation



















































































































 



GROUP 1


CONTROL



GROUP 2


1500 PPM



GROUP 3


5000 PPM



GROUP 4


15000 PPM



POST-COITUM



DAYS 0-4



MEAN


SD


N



18


1.4


18



19


1.5


18



20


4.7


18



19


2.4


19



DAYS 4-7



MEAN


SD


N



19


2.0


18



23 *


5.0


18



22


6.3


18



24 **


2.9


19



DAYS 7-11



MEAN


SD


N



20


2.2


18



22


1.9


18



23


4.5


18



23


2.1


19



DAYS 11-14



MEAN


SD


N



21


2.2


18



23 *


1.9


18



24 **


3.6


18



23 **


2.3


19



DAYS 14-17



MEAN


SD


N



22


3.2


18



25 *


2.0


18



26 **


2.6


18



26 **


2.1


19



DAYS 17-20



MEAN


SD


N



25


2.0


18



26


1.9


18



27


3.0


18



26


2.9


19



MEAN OF MEANS



MEAN


 



21



23



23



24



LACTATING



DAYS 1-4



MEAN


SD


N



31


3.0


18



32


4.3


17



32


5.4


19



31


2.7


19



DAYS 4-7



MEAN


SD


N



40


3.7


18



42


4.2


17



42


3.8


19



44 **


3.6


19



DAYS 7-14



MEAN


SD


N



54


3.8


18



57


4.8


17



58 *


4.6


19



59 **


4.2


19



DAYS 14-21



MEAN


SD


N



63


4.2


18



67 *


3.9


17



70 **


5.8


19



74 **


4.7


19



MEAN OF MEANS



MEAN


 



47



49



50



52



*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level


x Explanations for excluded data are listed in the tables of the individual values


 


Table 43             Test Item Intake (mg/kg/day). Male – F1 (Cohorts 1A, 1B, 1C)


 





































































































































 



GROUP 1


CONTROL



GROUP 2


1500 PPM



GROUP 3


5000 PPM



GROUP 4


15000 PPM



DAYS 1-8


WEEKS 1-2



MEAN


SD


N (CAGE)



0


0.0


12



185


32.4


12



648


91.3


12



1830


222.5


12



DAYS 8-15


WEEKS 2-3



MEAN


SD


N (CAGE)



0


0.0


12



174


6.2


12



597


18.3


12



1866


59.5


12



DAYS 15-22


WEEKS 3-4



MEAN


SD


N (CAGE)



0


0.0


12



170


6.1


12



600


32.5


12



1805


65.2


12



DAYS 22-29


WEEKS 4-5



MEAN


SD


N (CAGE)



0


0.0


12



152


3.8


12



518


19.3


12



1670


54.8


12



DAYS 29-36


WEEKS 5-6



MEAN


SD


N (CAGE)



0


0.0


8



138


1.9


8



479


15.8


8



1489


25.4


8



DAY 36-43


WEEKS 6-7



MEAN


SD


N (CAGE)



0


0.0


8



126


2.7


8



440


11.8


8



1397


20.4


8



DAYS 43-50


WEEKS 7-8



MEAN


SD


N (CAGE)



0


0.0


8



119


2.4


8



419


9.2


8



1305


24.7


8



DAYS 50-57


WEEKS 8-9



MEAN


SD


N (CAGE)



0


0.0


8



110


2.5


8



390


9.9


8



1210


24.0


8



DAYS 57-64


WEEKS 9-10



MEAN


SD


N (CAGE)



0


0.0


8



103


2.1


8



361


8.2


8



1118


33.9


8



DAYS 64-71


WEEKS 10-11



MEAN


SD


N (CAGE)



0


0.0


7



98


2.0


7



337


6.6


7



1054


26.7


7



DAYS 71-77


WEEK 11



MEAN


SD


N (CAGE)



0


0.0


4



92


4.0


5



330


9.2


5



1051


17.6


5



DAYS 77-85


WEEKS 11-13



MEAN


SD


N (CAGE)



0


0.0


4



92


4.7


4



324


10.9


4



999


18.1


4



DAYS 85-92


WEEKS 13-14



MEAN


SD


N (CAGE)



0


0.0


2



96


9.2


2



318


36.6


3



949


6.1


2



DAYS 92-99


WEEKS 14-15



MEAN


SD


N



0


0.0


2



45


12.4


2



131


54.9


3



511


129.3


2



MEAN OF MEANS (MATING)



MEAN



0



122



421



1304



 


Table 44             Test Item Intake (mg/kg/day). Female – F1 (Cohorts 1A, 1B, 1C)













































































































 



GROUP 1


CONTROL



GROUP 2


1500 PPM



GROUP 3


5000 PPM



GROUP 4


15000 PPM



DAYS 1-9


WEEKS 1-2



MEAN


SD


N (CAGE)



0


0.0


12



170


11.8


12



594


32.8


12



1822


162.5


12



DAYS 9-16


WEEKS 2-3



MEAN


SD


N (CAGE)



0


0.0


12



175


3.5


12



602


13.5


12



1872


47.2


12



DAYS 16-23


WEEKS 3-4



MEAN


SD


N (CAGE)



0


0.0


12



162


4.4


12



554


9.4


12



1750


47.1


12



DAYS 23-30


WEEKS 4-5



MEAN


SD


N (CAGE)



0


0.0


8



150


3.8


8



521


14.1


8



1650


41.6


8



DAYS 30-37


WEEKS 5-6



MEAN


SD


N (CAGE)



0


0.0


8



145


3.0


8



493


17.3


8



1568


63.2


8



DAY 37-44


WEEKS 6-7



MEAN


SD


N (CAGE)



0


0.0


8



136


2.4


8



465


13.0


8



1467


44.8


8



DAYS 44-51


WEEKS 7-8



MEAN


SD


N (CAGE)



0


0.0


8



128


2.1


8



453


9.8


8



1441


51.7


8



DAYS 51-58


WEEKS 8-9



MEAN


SD


N (CAGE)



0


0.0


8



122


2.3


8



426


8.5


8



1396


54.9


8



DAYS 58-65


WEEKS 9-10



MEAN


SD


N (CAGE)



0


0.0


8



117


2.1


8



409


7.7


8



1343


83.5


8



DAYS 65-71


WEEKS 10-11



MEAN


SD


N (CAGE)



0


0.0


7



114


1.4


7



396


10.3


7



1302


46.2


7



DAYS 71-77


WEEK 11



MEAN


SD


N (CAGE)



0


0.0


4



109


3.0


5



386


10.7


5



1237


82.4


5



MEAN OF MEANS



MEAN



0



139



482



1532



 


Table 45             Test Item Intake (mg/kg/day). Female – F1 Cohorts 1B, Post-coitum & Lactation



















































































































 



GROUP 1


CONTROL



GROUP 2


1500 PPM



GROUP 3


5000 PPM



GROUP 4


15000 PPM



POST-COITUM



DAYS 0-4



MEAN


SD


N



0


0.0


18



112


6.7


18



411


90.7


18



1287


128.7


19



DAYS 4-7



MEAN


SD


N



0


0.0


18



132


25.0


18



442


118.3


18



1542


167.8


19



DAYS 7-11



MEAN


SD


N



0


0.0


18



120


11.2


18



435


69.1


18



1389


92.4


19



DAYS 11-14



MEAN


SD


N



0


0.0


18



122


9.0


18



450


50.4


18



1385


101.9


19



DAYS 14-17



MEAN


SD


N



0


0.0


18



121


7.7


18



441


26.1


18



1425


77.3


19



DAYS 17-20



MEAN


SD


N



0


0.0


17



113


6.3


18



416


49.3


18



1299


114.8


19



MEAN OF MEANS



MEAN


 



0



120



433



1388



LACTATING



DAYS 1-4



MEAN


SD


N



0


0.0


18



113


14.0


17



396


48.9


19



1254


96.4


19



DAYS 4-7



MEAN


SD


N



0


0.0


18



110


6.8


17



381


20.0


19



1276


83.1


19



DAYS 7-14



MEAN


SD


N



0


0.0


18



113


4.5


17



400


15.9


19



1283


87.6


19



DAYS 14-21



MEAN


SD


N



0


0.0


18



140


6.4


17



496


32.1


19



1635


84.5


19



MEAN OF MEANS



MEAN


 



0



119



418



1362



 


Table 46             Summary of Test Item Intake – F1





















































































 



Mean over Means Intake
[mg test item/kg body weight]


(mean range indicated within brackets)



 



Group No.



2



3



4



Nominal Dietary Inclusion Level (ppm)



1500



5000



15000



 



 



Sex



Study Period



 



 



 



Males



Treatment



122



(45-185)



421



(131-648)



1304



(511-1866)



 



 



Females



Pre-mating



139



(109-175)



482



(386-602)



1532



(1237-1872)



Post-coitum



120



(112-132)



433



(411-450)



1388



(1287-1542)



Lactation



119



(110-140)



418



(381-496)



1362



(1254-1635)



Mean of Meansa



132



463



1478



a      Mean of means of all periods, weighed for number of treatment days per period:


Females: ((76x mean pre-mating) + (20x mean post-coitum) + (20x mean lactation)) / 116



 


Table 47             Haematology. Male – Cohort 1A


























































































































































Sex: Male



 



 



 



 



 



 



Reporting Hematology



 



 



 



 



WBC



NEUT



LYMPH



MONO



EOS



BASO



LUC



RBC



RETIC



RDWG



HGB



HCT



 



 



 



 



 



 



 



 



 



 



 



 



(10^9/L)



(10^9/L)



(10^9/L)



(10^9/L)



(10^9/L)



(10^9/L)



(10^9/L)



(10^12/L)



(10^9/L)



(%)



(g/L)



(L/L)



 



 



 



 



 



 



 



 



 



 



 



 



[G]



[G]



[G]



[G]



[G]



[G]



[G1]



[G1]



[G]



[G]



[G1]



[G]



 0


 ppm


 Group 1



Mean


SD N



8.771  1.219 


10      



1.514 


0.316 


10      



6.828 


1.085 


10      



0.202 


0.078 


10      



0.130 


0.061 


10      



0.020 


0.008 


10      



0.075 


0.058 


10      



8.700 


0.468 


10      



247.43


31.75 


10      



12.50 


0.47 


10      



155.1 


8.0


10    



0.4454 0.0168


10      



 1500  ppm


 Group 2



Mean


SD


N tCtrl



7.351  *


1.094 


10      


0.84   



1.156  *


0.272 


10      


0.76   



5.855 


1.170 


10      


0.86   



0.158 


0.048 


10      


0.78   



0.109 


0.067 


10      


0.84   



0.013 


0.005 


10      


0.65   



0.063 


0.038 


10      


0.84   



8.676 


0.332 


10      


1.00   



218.79


22.38 


10      


0.88 



11.94  *


0.46 


10      


0.96 



154.6 


5.4 


10    


1.00



0.4464 0.0189


10      


1.00   



 5000  ppm


 Group 3



Mean


SD


N tCtrl



6.914  **


0.779 


10      


0.79   



1.187  *


0.308 


10      


0.78   



5.417  *


0.711 


10      


0.79   



0.159 


0.048 


10      


0.79   



0.105 


0.024 


10      


0.81   



0.011  *


0.006 


10      


0.55   



0.036 


0.012 


10      


0.48   



8.546 


0.394 


10      


0.98   



213.22


40.77 


10      


0.86 



11.91  *


0.46 


10      


0.95 



153.1 


4.4 


10    


0.99



0.4422 0.0154


10      


0.99   



 15000  ppm


 Group 4



Mean


SD


N tCtrl



6.016  ** 1.278 


10      


0.69   



0.834  ** 0.233 


10      


0.55   



4.932  ** 1.159 


10      


0.72   



0.120  ** 0.028 


10      


0.59   



0.080  0.029 


10      


0.62   



0.009  ** 0.007 


10      


0.45   



0.045  0.020 


10      


0.60   



8.341  0.151 


10      


0.96   



185.25**


31.75 


10      


0.75 



11.61 **


0.48 


10      


0.93 



150.1 


3.2 


10    


0.97



0.4309 0.0095


10      


0.97   



[G] - Anova & Dunnett: * = p ≤ 0.05; ** = p ≤ 0.01


[G1] - Kruskal-Wallis & Dunn


 










































































Sex: Male



 



 



Reporting Hematology



MCV



MCH



MCHC



PLT



 



 



 



 



(fL)



(pg)



(g/L)



(10^9/L)



 



 



 



 



[G]



[G]



[G]



[G]



 0


 ppm


 Group 1



Mean


SD


N



51.25 


1.59 


10      



17.84 


0.62 


10      



348.2 


7.5


10    



744.7 


88.9


10    



 1500  ppm


 Group 2



Mean


SD


N


tCtrl



51.46 


1.46 


10      


1.00 



17.82 


0.25 


10      


1.00 



346.3 


7.6 


10    


0.99



729.8 


49.7 


10    


0.98



 5000  ppm


 Group 3



Mean


SD


N


tCtrl



51.78 


2.13 


10      


1.01 



17.95 


0.79 


10      


1.01 



346.3 


7.3 


10    


0.99



688.5 


61.8 


10    


0.92



 15000  ppm


 Group 4



Mean


SD


N


tCtrl



51.66 


1.36 


10      


1.01 



18.00 


0.55 


10      


1.01 



348.4 


6.0 


10    


1.00



705.7 


112.4 


10    


0.95



[G] - Anova & Dunnett


 


Table 48             Haematology. Female – Cohort 1A


























































































































































Sex: Female



 



 



 



 



 



 



Reporting Hematology



 



 



 



 



WBC



NEUT



LYMPH



MONO



EOS



BASO



LUC



RBC



RETIC



RDWG



HGB



HCT



 



 



 



 



 



 



 



 



 



 



 



 



(10^9/L)



(10^9/L)



(10^9/L)



(10^9/L)



(10^9/L)



(10^9/L)



(10^9/L)



(10^12/L)



(10^9/L)



(%)



(g/L)



(L/L)



 



 



 



 



 



 



 



 



 



 



 



 



[G]



[G]



[G]



[G]



[G]



[G]



[G1]



[G]



[G]



[G]



[G]



[G]



 0


 ppm


 Group 1



Mean


SD N



3.999  0.859 


10      



0.779 


0.353 


10      



3.037 


0.611 


10      



0.081 


0.030 


10      



0.071 


0.031 


10      



0.006 


0.005 


10      



0.023 


0.007 


10      



8.230 


0.193 


10      



245.25


47.75 


10      



11.47 


0.29 


10      



153.0 


2.6


10    



0.4431 0.0143


10      



 1500  ppm


 Group 2



Mean


SD


N tCtrl



4.223  1.735 


10      


1.06   



0.763 


0.424 


10      


0.98   



3.193 


1.133 


10      


1.05   



0.122 


0.138 


10      


1.51   



0.095 


0.096 


10      


1.34   



0.004 


0.007 


10      


0.67   



0.045 


0.070 


10      


1.96   



8.031 


0.372 


10      


0.98   



218.53


31.96 


10      


0.89 



11.52 


0.37 


10      


1.00 



149.3 


4.3 


10    


0.98



0.4285 0.0166


10      


0.97   



 5000  ppm


 Group 3



Mean


SD


N tCtrl



4.419  1.027 


10      


1.11   



0.800 


0.560 


10      


1.03   



3.428 


0.806 


10      


1.13   



0.085 


0.040 


10      


1.05   



0.075 


0.036 


10      


1.06   



0.004 


0.005 


10      


0.67   



0.028 


0.011 


10      


1.22   



7.977 


0.249 


10      


0.97   



237.05


48.15 


10      


0.97 



11.54 


0.55 


10      


1.01 



148.8  *


3.3 


10    


0.97



0.4277 0.0117


10      


0.97   



 15000  ppm


 Group 4



Mean


SD


N tCtrl



4.029  1.000 


10      


1.01   



0.571  0.211 


10      


0.73   



3.285  0.814 


10      


1.08   



0.066  0.020 


10      


0.81   



0.069  0.028 


10      


0.97   



0.007  0.005 


10      


1.17   



0.027  0.014 


10      


1.17   



7.919  0.331 


10      


0.96   



209.44


44.72 


10      


0.85 



11.40 


0.50 


10      


0.99 



142.4**


4.6 


10    


0.93



0.4130** 0.0136


10      


0.93   



[G] - Anova & Dunnett: * = p ≤ 0.05; ** = p ≤ 0.01


[G1] - Kruskal-Wallis & Dunn


 










































































Sex: Female



 



 



Reporting Hematology



MCV



MCH



MCHC



PLT



 



 



 



 



(fL)



(pg)



(g/L)



(10^9/L)



 



 



 



 



[G]



[G]



[G]



[G]



 0


 ppm


 Group 1



Mean


SD


N



53.86 


1.83 


10      



18.61 


0.45 


10      



345.7 


7.2


10    



648.1 


41.2 


9    



 1500  ppm


 Group 2



Mean


SD


N


tCtrl



53.43 


1.36 


10      


0.99 



18.61 


0.52 


10      


1.00 



348.5 


5.4 


10    


1.01



648.8 


52.5 


10    


1.00



 5000  ppm


 Group 3



Mean


SD


N


tCtrl



53.65 


1.10 


10      


1.00 



18.66 


0.39 


10      


1.00 



347.7 


4.4 


10    


1.01



738.9 


103.9 


10    


1.14



 15000  ppm


 Group 4



Mean


SD


N


tCtrl



52.20 


1.32 


10      


0.97 



18.01  *


0.42 


10      


0.97 



345.0 


4.9 


10    


1.00



742.8  *


108.7 


10    


1.15



[G] - Anova & Dunnett: * = p ≤ 0.05


 


Table 49             Coagulation. Male – Cohort 1A







































Sex: Male



 



Reporting Coagulation



PT


(sec)


[G]



APTT


(sec)


[G]



 0


 ppm


 Group 1



Mean


SD


N



18.27 


0.67 


10      



19.86 


2.43 


10      



 1500  ppm


 Group 2



Mean


SD


N


tCtrl



17.63 


1.03 


9      


0.97 



19.06 


3.73 


9      


0.96 



 5000  ppm


 Group 3



Mean


SD


N


tCtrl



18.25 


0.86 


10      


1.00 



20.69 


2.72 


10      


1.04 



 15000  ppm


 Group 4



Mean


SD


N


tCtrl



17.62 


0.87 


10      


0.96 



18.50 


1.34 


10      


0.93 



[G] - Anova & Dunnett


 


Table 50             Coagulation. Female – Cohort 1A







































Sex: Female



 



Reporting Coagulation



PT


(sec)


[G]



APTT


(sec)


[G]



 0


 ppm


 Group 1



Mean


SD


N



17.77 


1.25 


10      



19.36 


2.30 


9      



 1500  ppm


 Group 2



Mean


SD


N


tCtrl



17.35 


0.71 


10      


0.98 



20.16 


1.16 


10      


1.04 



 5000  ppm


 Group 3



Mean


SD


N


tCtrl



17.00 


0.68 


9      


0.96 



19.16 


1.84 


9      


0.99 



 15000  ppm


 Group 4



Mean


SD


N


tCtrl



16.93 


1.01 


10      


0.95 



19.23 


2.07 


10      


0.99 



[G] - Anova & Dunnett


 


Table 51             Clinical Chemistry (including Thyroid Hormones). Male – Cohort 1A


























































































































































Sex: Male



 



 



 



 



 



 



Reporting Biochemistry



 



 



 



 



ALT



AST



ALP



TPROT



ALB



BILEAC



TBIL



UREA



CREAT



GLUC



CHOL



HDL



 



 



 



 



 



 



 



 



 



 



 



 



(U/L)



(U/L)



(U/L)



(g/L)



(g/L)



(umol/L)



(umol/L)



(mmol/L)



(umol/L)



(mmol/L)



(mmol/L)



(mmol/L)



 



 



 



 



 



 



 



 



 



 



 



 



[G]



[G]



[G]



[G]



[G]



[G1]



[G]



[G]



[G]



[G]



[G]



[G]



 0


 ppm


 Group 1



Mean


SD N



36.6  


3.7


10     



82.4  


9.9


10     



100.1   17.5


10     



63.32  


1.84


10       



38.86  


1.20


10       



18.16  


21.35  


10       



2.00  


0.27


10       



4.99  


0.38


10       



26.4  


2.5


10     



7.976  


1.034  


10       



1.525  


0.288  


10       



0.980  


0.142  


10       



 1500  ppm


 Group 2


 



Mean


SD


N tCtrl



37.2  


5.5  


10     


1.02



80.6  


12.3  


10     


0.98



105.5   24.8  


10     


1.05



63.27  


2.56  


10       


1.00  



38.50  


1.56  


10       


0.99  



21.23  


17.40  


10       


1.17  



1.73  


0.32  


10       


0.87  



5.17  


0.79  


10       


1.03  



28.1  


3.8  


10     


1.07



8.203  


1.009  


10       


1.03    



1.546  


0.296  


10       


1.01    



1.009  


0.181  


10       


1.03    



 5000  ppm


 Group 3


 



Mean


SD


N tCtrl



37.8  


7.7  


10     


1.03



82.8  


13.1  


10     


1.01



102.8   16.2  


10     


1.03



62.60  


1.73  


10       


0.99  



38.06  


1.13  


10       


0.98  



13.09  


9.49  


10       


0.72  



1.76  


0.24  


10       


0.88  



4.61  


0.52  


10       


0.92  



27.9  


3.1  


10     


1.06



8.098  


1.006  


10       


1.02    



1.608  


0.303  


10       


1.05    



0.970  


0.152  


10       


0.99    



 15000  ppm


 Group 4


 



Mean


SD


N tCtrl



37.2  


3.6  


10     


1.02



80.4  


9.1  


10     


0.98



133.8 **


27.9  


10     


1.34



62.09  


1.88  


10       


0.98  



37.95  


1.33  


10       


0.98  



7.92   3.20  


10       


0.44  



1.91   0.23  


10       


0.96  



5.85  **


0.60  


10       


1.17  



28.9  


3.3  


10     


1.10



8.188   1.433  


10       


1.03    



1.841   0.297  


10       


1.21    



1.132   0.176  


10       


1.16    



[G] - Anova & Dunnett: ** = p ≤ 0.01


[G1] - Kruskal-Wallis & Dunn




































































































































Sex: Male



 



 



 



Reporting Biochemistry



 



 



Reporting Special Chemistry



LDL



TRIG



NA



K



CL



CA



PHOS



T3



T4



TSH



 



 



 



 



 



 



 



 



 



 



(mmol/L)



(mmol/L)



(mmol/L)



(mmol/L)



(mmol/L)



(mmol/L)



(mmol/L)



(ng/mL)



(ng/mL)



(mU/L)



 



 



 



 



 



 



 



 



 



 



[G]



[G]



[G]



[G]



[G]



[G1]



[G]



[G]



[G]



[G]



 0


 ppm


 Group 1



Mean


SD N



0.293  


0.039  


10       



0.580  


0.222  


10       



145.5  


0.8


10     



3.59  


0.20


10       



106.7  


1.1


10     



2.633  


0.033  


10       



2.161  


0.174  


10       



0.520  


0.072  


10       



60.30  


10.47  


10       



0.2072


0.1099


10       



 1500  ppm


 Group 2


 



Mean


SD


N tCtrl



0.298  


0.024  


10       


1.02    



0.717  


0.149  


10       


1.24    



145.1  


0.9  


10     


1.00



3.70  


0.22  


10       


1.03  



106.9  


1.7  


10     


1.00



2.595  


0.052  


10       


0.99    



2.044  


0.158  


10       


0.95    



0.563  


0.094  


10       


1.08    



60.27  


8.60  


10       


1.00  



0.2350


0.1393


10       


1.13    



 5000  ppm


 Group 3


 



Mean


SD


N tCtrl



0.298  


0.043  


10       


1.02    



0.800  *


0.156  


10       


1.38    



144.8  


0.8  


10     


1.00



3.81  *


0.20  


10       


1.06  



106.6  


1.7  


10     


1.00



2.631  


0.040  


10       


1.00    



1.998  


0.200  


10       


0.92    



0.544  


0.077  


10       


1.05    



59.21  


5.78  


10       


0.98  



0.2649


0.2401


10       


1.28    



 15000  ppm


 Group 4


 



Mean


SD


N tCtrl



0.341  * 0.052  


10       


1.16    



0.544   0.147  


10       


0.94    



144.5  


0.7  


10     


0.99



3.59   0.17  


10       


1.00  



105.9  


1.2  


10     


0.99



2.948  ** 0.179  


10       


1.12    



2.110   0.124  


10       


0.98    



0.508   0.069  


10       


0.98    



56.74  


5.79  


10       


0.94  



0.1749 0.1183


10       


0.84    



[G] - Anova & Dunnett: * = p ≤ 0.05


[G1] - Kruskal-Wallis & Dunn: ** = p ≤ 0.01


 


Table 52             Clinical Chemistry (including Thyroid Hormones). Female – Cohort 1A


























































































































































Sex: Female



 



 



 



 



 



 



Reporting Biochemistry



 



 



 



 



ALT



AST



ALP



TPROT



ALB



BILEAC



TBIL



UREA



CREAT



GLUC



CHOL



HDL



 



 



 



 



 



 



 



 



 



 



 



 



(U/L)



(U/L)



(U/L)



(g/L)



(g/L)



(umol/L)



(umol/L)



(mmol/L)



(umol/L)



(mmol/L)



(mmol/L)



(mmol/L)



 



 



 



 



 



 



 



 



 



 



 



 



[G]



[G]



[G1]



[G]



[G]



[G]



[G]



[G]



[G]



[G]



[G]



[G]



 0


 ppm


 Group 1



Mean


SD N



33.4  


5.7


10     



93.4  


25.4


10     



57.1  


10.6


10     



63.96  


2.41


10       



39.78  


1.27


10       



15.74  


10.41  


10       



1.78  


0.34


10       



6.24  


0.45


10       



28.6  


2.7


10     



7.657  


0.675  


10       



1.353  


0.208  


10       



0.875  


0.119  


10       



 1500  ppm


 Group 2


 



Mean


SD


N tCtrl



28.2  *


1.8  


10     


0.84



78.1  


7.8  


10     


0.84



59.2  


7.1  


10     


1.04



65.57  


2.59  


10       


1.03  



41.39  


1.74  


10       


1.04  



14.89  


10.31  


10       


0.95  



2.06  


0.28  


10       


1.16  



6.18  


0.99  


10       


0.99  



31.6  *


2.6  


10     


1.10



7.529  


0.900  


10       


0.98    



1.317  


0.283  


10       


0.97    



0.833  


0.170  


10       


0.95    



 5000  ppm


 Group 3


 



Mean


SD


N tCtrl



33.1  


5.1  


10     


0.99



84.1  


9.7  


10     


0.90



60.7  


14.0  


10     


1.06



63.75  


2.13  


10       


1.00  



40.09  


1.83  


10       


1.01  



13.24  


4.51  


10       


0.84  



2.04  


0.33  


10       


1.15  



6.30  


0.75  


10       


1.01  



29.9  


2.8  


10     


1.04



6.892  


0.803  


10       


0.90    



1.502  


0.236  


10       


1.11    



0.964  


0.131  


10       


1.10    



 15000  ppm


 Group 4


 



Mean


SD


N tCtrl



32.3  


4.5  


10     


0.97



87.6   15.1  


10     


0.94



76.7   22.4  


10     


1.34



63.26  


2.32  


10       


0.99  



39.55  


1.34  


10       


0.99  



10.70  


5.22  


10       


0.68  



1.95   0.31  


10       


1.10  



6.26   0.93  


10       


1.00  



31.7  *


2.0  


10     


1.11



7.137   1.179  


10       


0.93    



1.565   0.308  


10       


1.16    



1.006  


0.145  


10       


1.15    



[G] - Anova & Dunnett: * = p ≤ 0.05


[G1] - Kruskal-Wallis & Dunn


 




































































































































Sex: Female



 



 



 



Reporting Biochemistry



 



 



Reporting Special Chemistry



LDL



TRIG



NA



K



CL



CA



PHOS



T3



T4



TSH



 



 



 



 



 



 



 



 



 



 



(mmol/L)



(mmol/L)



(mmol/L)



(mmol/L)



(mmol/L)



(mmol/L)



(mmol/L)



(ng/mL)



(ng/mL)



(mU/L)



 



 



 



 



 



 



 



 



 



 



[G]



[G]



[G]



[G]



[G]



[G1]



[G]



[G]



[G]



[G]



 0


 ppm


 Group 1



Mean


SD N



0.284  


0.051  


10       



0.394  


0.107  


10       



145.0  


1.1


10     



3.31  


0.26


10       



107.2  


1.9


10     



2.926  


0.206  


10       



1.928  


0.287  


10       



0.495  


0.050  


10       



38.19  


6.21


10       



0.1559


0.1432


10       



 1500  ppm


 Group 2


 



Mean


SD


N tCtrl



0.256  


0.062  


10       


0.90    



0.468  


0.117  


10       


1.19    



144.0  


1.8  


10     


0.99



3.39  


0.28  


10       


1.03  



106.2  


2.5  


10     


0.99



2.813  


0.063  


10       


0.96    



1.855  


0.256  


10       


0.96    



0.589  *


0.056  


10       


1.19    



37.02  


9.12  


10       


0.97  



0.0885


0.0550


10       


0.57    



 5000  ppm


 Group 3


 



Mean


SD


N tCtrl



0.298  


0.046  


10       


1.05    



0.489  


0.115  


10       


1.24    



144.6  


1.3  


10     


1.00



3.18  


0.17  


10       


0.96  



107.1  


2.8  


10     


1.00



2.808  


0.126  


10       


0.96    



1.820  


0.153  


10       


0.94    



0.516  


0.093  


10       


1.04    



37.22  


6.90  


10       


0.97  



0.1657


0.1701


10       


1.06    



 15000  ppm


 Group 4


 



Mean


SD


N tCtrl



0.295   0.053  


10       


1.04    



0.522   0.158  


10       


1.32    



144.0  


1.3  


10     


0.99



3.28   0.24  


10       


0.99  



107.3  


2.0  


10     


1.00



2.784   0.115  


10       


0.95    



1.813   0.268  


10       


0.94    



0.552   0.066  


10       


1.12    



44.10  


6.15  


10       


1.15  



0.2111 0.1694


10       


1.35    



[G] - Anova & Dunnett: * = p ≤ 0.05


[G1] - Kruskal-Wallis & Dunn


 


Table 53             Urinalysis. Male – Cohort 1A














































Sex: Male



 



 



Reporting Urinalysi



s



VOLUME


(mL)


[G]



SPECIFIC


GRAVITY


[G1]



URINE pH


[G1]



 0


 ppm


 Group 1



Mean


SD


N



8.20 


5.83 


10      



1.0257 


0.0093 


10          



6.90 


0.52 


10      



 1500  ppm


 Group 2



Mean


SD


N


tCtrl



9.30 


7.15 


10      


1.13 



1.0286 


0.0143 


10          


1.00     



7.00 


0.24 


10      


1.01 



 5000  ppm


 Group 3



Mean


SD


N


tCtrl



10.55 


3.90 


10      


1.29 



1.0254 


0.0090 


10          


1.00     



6.95 


0.37 


10      


1.01 



 15000  ppm


 Group 4



Mean


SD


N


tCtrl



9.30


3.97 


10      


1.13 



1.0272


0.0068 


10          


1.00     



6.80


0.35 


10      


0.99 



[G] - Anova & Dunnett


[G1] - Kruskal-Wallis & Dunn


 


Table 54             Urinalysis. Female – Cohort 1A














































Sex: Female



 



 



Reporting Urinalysi



s



VOLUME


(mL)


[G]



SPECIFIC


GRAVITY


[G]



URINE pH


[G]



 0


 ppm


 Group 1



Mean


SD


N



5.20 


4.13 


10      



1.0305 


0.0126 


10          



6.40 


0.32 


10      



 1500  ppm


 Group 2



Mean


SD


N


tCtrl



6.80 


4.23 


10      


1.31 



1.0262 


0.0064 


10          


1.00     



6.45 


0.37 


10      


1.01 



 5000  ppm


 Group 3



Mean


SD


N


tCtrl



7.30 


4.65 


10      


1.40 



1.0256 


0.0102 


10          


1.00     



6.50 


0.41 


10      


1.02 



 15000  ppm


 Group 4



Mean


SD


N


tCtrl



6.75


3.16 


10      


1.30 



1.0232


0.0079 


10          


0.99     



6.75


0.42 


10      


1.05 



[G] - Anova & Dunnett


 


Table 55             Splenic Lymphocyte Subpopulation Analysis. Male – Cohort 1A





























































 



GROUP 1


CONTROL



GROUP 2


1500 PPM



GROUP 3


5000 PPM



GROUP 4


15000 PPM



T-CELLS


(%Lymphoid cells)



MEAN


SD


N



42.7


8.2


10



44.1


5.9


10



39.5


5.4


10



46.1


7.4


10



T-HELPER CELLS


(%Lymphoid cells)



MEAN


SD


N



28.2


5.5


10



27.9


4.0


10



26.1


3.9


10



29.1


5.8


10



T-CYTOTOXIC CELLS


(%Lymphoid cells)



MEAN


SD


N



14.0


4.0


10



15.1


4.0


10



12.6


1.7


10



16.0


3.5


10



B-CELLS


(%Lymphoid cells)



MEAN


SD


N



41.0


8.2


10



41.6


6.2


10



45.1


5.6


10



40.3


6.0


10



NK-CELLS


(%Lymphoid cells)



MEAN


SD


N



5.8


1.3


10



5.1


0.7


10



5.5


1.0


10



4.7


1.6


10



Th/Tc



MEAN


SD


N



2.1


0.5


10



1.9


0.5


10



2.1


0.3


10



1.9


0.5


10



+/++ Steel-test significant at 5% (+) or 1% (+/+) level


 


Table 56             Splenic Lymphocyte Subpopulation Analysis. Female – Cohort 1A





























































 



GROUP 1


CONTROL



GROUP 2


1500 PPM



GROUP 3


5000 PPM



GROUP 4


15000 PPM



T-CELLS


(%Lymphoid cells)



MEAN


SD


N



44.5


4.5


10



44.5


7.7


10



44.9


5.1


10



43.8


5.4


10



T-HELPER CELLS


(%Lymphoid cells)



MEAN


SD


N



29.5


3.0


10



29.0


4.1


10



29.6


3.3


10



27.7


2.2


10



T-CYTOTOXIC CELLS


(%Lymphoid cells)



MEAN


SD


N



13.7


2.4


10



14.5


4.8


10



13.7


2.2


10



14.5


4.1


10



B-CELLS


(%Lymphoid cells)



MEAN


SD


N



40.1


3.8


10



39.4


9.2


10



41.4


4.5


10



41.7


5.4


10



NK-CELLS


(%Lymphoid cells)



MEAN


SD


N



5.6


1.4


10



5.7


1.4


10



5.0


1.3


10



5.8


1.6


10



Th/Tc



MEAN


SD


N



2.2


0.4


10



2.1


0.6


10



2.2


0.3


10



2.0


0.5


10



+/++ Steel-test significant at 5% (+) or 1% (+/+) level


 


Table 57             Organ Weights (g). Male – Cohorts 1A













































































































































 



GROUP 1


CONTROL



GROUP 2


1500 PPM



GROUP 3


5000 PPM



GROUP 4


15000 PPM



BODY WEIGHT



MEAN


SD


N



347


17


20



343


33


20



356


23


20



339


31


20



BRAIN



MEAN


SD


N



2.03


0.08


20



2.04


0.08


19



2.03


0.09


20



2.01


0.10


20



PITUITARY



MEAN


SD


N



0.0091


0.0012


20



0.0088


0.0011


19



0.0088


0.0008


20



0.0088


0.0011


20



HEART



MEAN


SD


N



0.953


0.068


20



0.950


0.098


20



0.986


0.082


20



0.935


0.086


20



LIVER



MEAN


SD


N



8.99


0.50


20



8.98


1.02


20



9.37


0.81


20



9.85 *


1.22


20



THYROIDS



MEAN


SD


N



0.0164


0.0031


20



0.0169


0.0036


20



0.0177


0.0033


20



0.0205 **


0.0033


20



THYMUS



MEAN


SD


N



0.401


0.076


20



0.385


0.081


20



0.451


0.084


20



0.437


0.078


20



M LYMPH NODE



MEAN


SD


N



0.346


0.099


10



0.357


0.106


10



0.388


0.112


10



0.312


0.113


10



KIDNEYS



MEAN


SD


N



2.28


0.18


20



2.22


0.20


20



2.35


0.17


20



2.33


0.22


20



ADRENALS



MEAN


SD


N



0.061


0.009


20



0.060


0.009


20



0.060


0.009


20



0.057


0.009


20



SPLEEN



MEAN


SD


N



0.594


0.087


20



0.599


0.094


20



0.647


0.105


20



0.606


0.118


20



TESTES



MEAN


SD


N



3.44


0.55


20



3.60


0.29


20



3.71


0.25


20



3.39


0.50


20



PROSTATE GLAND



MEAN


SD


N



0.691


0.159


20



0.617


0.157


20



0.661


0.112


20



0.606


0.127


20



AXI LYMPH NODE L



MEAN


SD


N



0.07


0.03


10



0.07


0.02


10



0.06


0.02


10



0.07


0.02


10



EPIDIDYMIDES



MEAN


SD


N



1.035


0.124


20



1.051


0.070


20



1.087


0.067


20



1.038


0.106


20



SEMINAL VESICLES



MEAN


SD


N



1.027


0.205


20



0.967


0.194


20



1.008


0.186


20



1.055


0.150


20



*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level


 


Table 58             Organ Weights (g). Female – Cohorts 1A





























































































































 



GROUP 1


CONTROL



GROUP 2


1500 PPM



GROUP 3


5000 PPM



GROUP 4


15000 PPM



BODY WEIGHT



MEAN


SD


N



203


13


20



213


18


20



204


17


20



192 *


12


20



BRAIN



MEAN


SD


N



1.86


0.07


20



1.88


0.07


20



1.87


0.07


20



1.84


0.07


20



PITUITARY



MEAN


SD


N



0.0108


0.0016


20



0.0113


0.0017


19



0.0104


0.0013


20



0.0100


0.0016


19



HEART



MEAN


SD


N



0.667


0.085


20



0.684


0.061


20



0.653


0.064


20



0.615 *


0.042


20



LIVER



MEAN


SD


N



5.48


0.66


20



5.85


0.72


20



5.70


0.52


20



5.95 *


0.49


20



THYROIDS



MEAN


SD


N



0.0144


0.0036


20



0.0131


0.0026


20



0.0145


0.0039


20



0.0152


0.0029


20



THYMUS



MEAN


SD


N



0.370


0.054


20



0.360


0.073


20



0.350


0.065


20



0.394


0.048


20



M LYMPH NODE



MEAN


SD


N



0.275


0.075


10



0.282


0.075


10



0.241


0.087


10



0.239


0.040


10



KIDNEYS



MEAN


SD


N



1.54


0.16


20



1.56


0.14


20



1.50


0.13


20



1.47


0.11


20



ADRENALS



MEAN


SD


N



0.071


0.014


20



0.070


0.012


20



0.070


0.025


20



0.061


0.010


20



SPLEEN



MEAN


SD


N



0.417


0.055


20



0.438


0.053


20



0.438


0.058


20



0.428


0.076


20



OVARIES



MEAN


SD


N



0.132


0.024


20



0.139


0.020


20



0.130


0.013


20



0.128


0.024


20



UTERUS



MEAN


SD


N



0.594


0.202


20



0.489


0.128


20



0.595


0.290


20



0.556


0.224


20



AXI LYMPH NODE L



MEAN


SD


N



0.06


0.02


10



0.06


0.02


10



0.05


0.01


10



0.05


0.01


10



*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level


 


Table 59             Organ/Body Weight Ratio (%). Male – Cohorts 1A





































































































































 



GROUP 1


CONTROL



GROUP 2


1500 PPM



GROUP 3


5000 PPM



GROUP 4


15000 PPM



BRAIN



MEAN


SD


N



0.59


0.02


20



0.60


0.05


19



0.57


0.04


20



0.60


0.04


20



PITUITARY



MEAN


SD


N



0.0026


0.0003


20



0.0026


0.0003


19



0.0025


0.0002


20



0.0026


0.0002


20



HEART



MEAN


SD


N



0.275


0.016


20



0.277


0.016


20



0.277


0.018


20



0.276


0.016


20



LIVER



MEAN


SD


N



2.59


0.11


20



2.62


0.26


20



2.63


0.12


20



2.90 **


0.18


20



THYROIDS



MEAN


SD


N



0.0047


0.0008


20



0.0049


0.0009


20



0.0050


0.0009


20



0.0061 **


0.0012


20



THYMUS



MEAN


SD


N



0.116


0.021


20



0.112


0.017


20



0.126


0.020


20



0.129


0.020


20



M LYMPH NODE



MEAN


SD


N



0.101


0.032


10



0.102


0.025


10



0.108


0.027


10



0.091


0.034


10



KIDNEYS



MEAN


SD


N



0.66


0.04


20



0.65


0.05


20



0.66


0.05


20



0.69


0.05


20



ADRENALS



MEAN


SD


N



0.018


0.002


20



0.017


0.002


20



0.017


0.003


20



0.017


0.002


20



SPLEEN



MEAN


SD


N



0.171


0.022


20



0.175


0.023


20



0.182


0.026


20



0.178


0.027


20



TESTES



MEAN


SD


N



0.99


0.16


20



1.05


0.10


20



1.05


0.09


20



1.00


0.13


20



PROSTATE GLAND



MEAN


SD


N



0.198


0.041


20



0.179


0.038


20



0.187


0.034


20



0.180


0.041


20



AXI LYMPH NODE L



MEAN


SD


N



0.022


0.010


10



0.019


0.005


10



0.017


0.006


10



0.021


0.006


10



EPIDIDYMIDES



MEAN


SD


N



0.298


0.033


20



0.308


0.030


20



0.307


0.026


20



0.307


0.030


20



SEMINAL VESCICLES



MEAN


SD


N



0.295


0.049


20



0.283


0.053


20



0.285


0.056


20



0.313


0.043


20



*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level


 


Table 60             Organ/Body Weight Ratio (%). Female – Cohorts 1A





















































































































 



GROUP 1


CONTROL



GROUP 2


1500 PPM



GROUP 3


5000 PPM



GROUP 4


15000 PPM



BRAIN



MEAN


SD


N



0.92


0.05


20



0.89


0.07


20



0.92


0.08


20



0.96


0.07


20



PITUITARY



MEAN


SD


N



0.0053


0.0007


20



0.0053


0.0006


19



0.0051


0.0005


20



0.0052


0.0008


19



HEART



MEAN


SD


N



0.327


0.026


20



0.323


0.029


20



0.321


0.029


20



0.322


0.019


20



LIVER



MEAN


SD


N



2.69


0.25


20



2.75


0.29


20



2.80


0.20


20



3.11 **


0.21


20



THYROIDS



MEAN


SD


N



0.0071


0.0016


20



0.0062


0.0015


20



0.0072


0.0021


20



0.0079


0.0016


20



THYMUS



MEAN


SD


N



0.182


0.026


20



0.169


0.033


20



0.172


0.029


20



0.206 *


0.027


20



M LYMPH NODE



MEAN


SD


N



0.135


0.038


10



0.133


0.031


10



0.115


0.034


10



0.124


0.021


10



KIDNEYS



MEAN


SD


N



0.76


0.06


20



0.74


0.05


20



0.74


0.05


20



0.77


0.04


20



ADRENALS



MEAN


SD


N



0.035


0.006


20



0.033


0.005


20



0.035


0.013


20



0.032


0.005


20



SPLEEN



MEAN


SD


N



0.205


0.023


20



0.206


0.022


20



0.216


0.032


20



0.223


0.034


20



OVARIES



MEAN


SD


N



0.065


0.010


20



0.066


0.011


20



0.064


0.009


20



0.067


0.011


20



UTERUS



MEAN


SD


N



0.292


0.092


20



0.232


0.069


20



0.294


0.148


20



0.291


0.118


20



AXI LYMPH NODE L



MEAN


SD


N



0.029


0.010


10



0.027


0.007


10



0.022


0.006


10



0.024


0.005


10



*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level


 


Table 61             Organ Weights (g). Male – Cohorts 1B













































































 



GROUP 1


CONTROL



GROUP 2


1500 PPM



GROUP 3


5000 PPM



GROUP 4


15000 PPM



BODY WEIGHT



MEAN


SD


N



401


27


20



394


35


20



403


33


20



37836


20



PITUITARY



MEAN


SD


N



0.0098


0.0014


20



0.0098


0.0015


20



0.0097


0.0012


20



0.0093


0.0011


20



THYROIDS



MEAN


SD


N



0.0168


0.0027


20



0.0196


0.0138


20



0.0189


0.0031


20



0.0201


0.0031


20



ADRENALS



MEAN


SD


N



0.056


0.008


20



0.054


0.009


20



0.054


0.009


20



0.050 *


0.007


20



TESTES



MEAN


SD


N



3.76


0.20


20



3.78


0.24


20



3.78


0.37


20



3.52


0.55


20



PROSTATE GLAND



MEAN


SD


N



0.825


0.138


20



0.839


0.147


20



0.833


0.121


20



0.731


0.108


20



EPIDIDYMIDES



MEAN


SD


N



1.185


0.088


20



1.186


0.072


20



1.190


0.077


20



1.133


0.157


20



SEMINAL VESICLES



MEAN


SD


N



1.438


0.213


20



1.371


0.215


20



1.489


0.292


20



1.430


0.202


20



*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level


 


Table 62             Organ Weights (g). Female – Cohorts 1B





























































 



GROUP 1


CONTROL



GROUP 2


1500 PPM



GROUP 3


5000 PPM



GROUP 4


15000 PPM



BODY WEIGHT



MEAN


SD


N



283


18


19



284


19


19



277


19


20



271


17


20



PITUITARY



MEAN


SD


N



0.0121


0.0013


19



0.0119


0.0016


19



0.0113


0.0011


20



0.0108 *


0.0016


20



THYROIDS



MEAN


SD


N



0.0146


0.0037


19



0.0138


0.0028


19



0.0153


0.0030


20



0.0193 **


0.0050


20



ADRENALS



MEAN


SD


N



0.072


0.010


19



0.069


0.009


19



0.069


0.013


20



0.064 *


0.008


20



OVARIES



MEAN


SD


N



0.136


0.017


19



0.138


0.018


19



0.133


0.022


20



0.124


0.033


20



UTERUS



MEAN


SD


N



0.490


0.139


19



0.542


0.200


19



0.490


0.200


20



0.525


0.213


20



*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level


 


Table 63             Organ/Body Weight Ratio (%). Male – Cohorts 1B





































































 



GROUP 1


CONTROL



GROUP 2


1500 PPM



GROUP 3


5000 PPM



GROUP 4


15000 PPM



PITUITARY



MEAN


SD


N



0.0024


0.0003


20



0.0025


0.0004


20



0.0024


0.0002


20



0.0025


0.0002


20



THYROIDS



MEAN


SD


N



0.0042


0.0007


20



0.0048


0.0028


20



0.0047


0.0009


20



0.0054


0.0011


20



ADRENALS



MEAN


SD


N



0.014


0.002


20



0.014


0.002


20



0.013


0.002


20



0.013


0.001


20



TESTES



MEAN


SD


N



0.94


0.07


20



0.97


0.11


20



0.94


0.09


20



0.93


0.14


20



PROSTATE GLAND



MEAN


SD


N



0.206


0.034


20



0.213


0.032


20



0.207


0.033


20



0.194


0.023


20



EPIDIDYMIDES



MEAN


SD


N



0.296


0.019


20



0.303


0.026


20



0.296


0.024


20



0.300


0.040


20



SEMINAL VESICLES



MEAN


SD


N



0.360


0.058


20



0.350


0.061


20



0.371


0.077


20



0.381


0.060


20



*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level


 


Table 64             Organ/Body Weight Ratio (%). Female – Cohorts 1B





















































 



GROUP 1


CONTROL



GROUP 2


1500 PPM



GROUP 3


5000 PPM



GROUP 4


15000 PPM



PITUITARY



MEAN


SD


N



0.0043


0.0004


19



0.0042


0.0006


19



0.0041


0.0004


20



0.0040


0.0005


20



THYROIDS



MEAN


SD


N



0.0052


0.0013


19



0.0049


0.0012


19



0.0056


0.0011


20



0.0071 **


0.0016


20



ADRENALS



MEAN


SD


N



0.026


0.003


19



0.024


0.003


19



0.025


0.004


20



0.024


0.003


20



OVARIES



MEAN


SD


N



0.048


0.005


19



0.049


0.007


19



0.048


0.009


20



0.046


0.012


20



UTERUS



MEAN


SD


N



0.175


0.054


19



0.191


0.069


19



0.178


0.079


20



0.195


0.082


20



*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level


 


Table 65             Summary of organ weight changes. Cohorts 1A, 1B
































































































































 



Males



Females



Dose level (ppm):



1500



5000



15000



1500



5000



15000



 



 



 



 



 



 



 



LIVER (Cohort 1A)



 



 



 



 



 



 



               Absolute



0



4



10*



7



4



9*



               Relative to body weight



1



2



12**



2



4



16**



 



 



 



 



 



 



 



THYROID GLANDS (Cohort 1A)



 



 



 



 



 



 



               Absolute



3



8



25**



-9



1



6



               Relative to body weight



4



6



30**



-13



1



11



 



 



 



 



 



 



 



THYROID GLANDS (Cohort 1B)



 



 



 



 



 



 



               Absolute



17



13



20



-5



5



32**



               Relative to body weight



14



12



29



-6



8



37**



*: P≤0.05, **: P≤0.01


 


Table 66             Sperm Motility, Concentration, and Morphology – F1





























































































 



GROUP 1


CONTROL



GROUP 2


1500 PPM



GROUP 3


5000 PPM



GROUP 4


15000 PPM



MOTILE SPERM (%)



MEAN


SD


N



75


19


20



74


8


20



77


9


20



69


17


20



PROGRESSIVE SPERM (%)



MEAN


SD


N



31


10


20



27


6


20



30


6


20



29


9


20



SPERM COUNT EPI


(10E6/gram)



MEAN


SD


N



492.3


62.0


19



432.3


77.7


20



518.2


101.1


20



503.7


69.7


18



NORMAL MORPHOLOGY (number of cells)



MEAN


SD


N



184


9


19



182


14


20



180


13


20



175


13


19



DETACHED HEAD (number of cells)



MEAN


SD


N



2


2


19



2


2


20



3


3


20



3


2


19



ABNORMAL HEAD (number of cells)



MEAN


SD


N



1


1


19



1


1


20



1


1


20



1


1


19



COILED TAIL (number of cells)



MEAN


SD


N



12


7


19



14


14


20



15


12


20



19


14


19



OTHER TAIL (number of cells)



MEAN


SD


N



1


2


19



1


1


20



1


1


20



0


1


19



ABNORMAL NECK (number of cells)



MEAN


SD


N



0


0


19



1


1


20



0


1


20



1


1


19



COMBINED (number of cells)



MEAN


SD


N



0


0


19



0


0


20



0


0


20



0


0


19



*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level


+/++ Steel-test significant at 5% (+) or 1% (+/+) level


 


Table 67             Reproductive Data Summary – F1





































































 



GROUP 1


CONTROL



GROUP 2


1500 PPM



GROUP 3


5000 PPM



GROUP 4


15000 PPM



Females paired



19



20



20



20



Females mated



19



19



19



19



Pregnant females



18



18



19



19



Females Killed in Extremis Post-    coitum Day 22



0



1



0



0



Females with living pups on Day 1



18



17



19



19



Mating Index, females (%)



100



95



95



95



Fertility Index, females (%)



95



95



100



100



Gestation Index (%)



100



94



100



100



 


Table 68             Precoital Time – F1



















































































DAY OF THE PAIRING PERIOD



GROUP 1


CONTROL



GROUP 2


1500 PPM



GROUP 3


5000 PPM



GROUP 4


15000 PPM



NUMBER OF FEMALES MATED



 



 



 



 



1



6



4



8



4



2



5



4



3



8



3



1



2



1



2



4



7



7



4



5



13



-



1



2



-



14



-



1



1



-



MEDIAN PRECOITAL TIME



2



3



2



2



MEAN PRECOITAL TIME



2.5



3.8



3.8



2.4



N



19



19



19



19



+/++ Steel-test significant at 5% (+) or 1% (+/+) level


 


Table 69             Implantation Sites Summary – F1





























 



GROUP 1


CONTROL



GROUP 2


1500 PPM



GROUP 3


5000 PPM



GROUP 4


15000 PPM



IMPLANTATIONS



MEAN


SD


N



13.1


2.1


18



12.7


2.8


18



12.4


2.1


19



10.8 ++


1.7


19


      

+/++ Steel-test significant at 5% (+) or 1% (+/+) level


 


Table 70             Developmental Data – F1



















































































 



GROUP 1


CONTROL



GROUP 2


1500 PPM



GROUP 3


5000 PPM



GROUP 4


15000 PPM



Total number of offspring born



217



204



217



195



Total number of uterine implantation sites



236



228



235



205



Number of live offspring on Day 1 after littering



216



202



216



194



Number of live offspring on Day 4 (before culling)



214



199



216



192



Number of live offspring on Day 4 (after culling)



144



135



151



150



Number of live offspring on Day 21 after littering



144



133



151



150



Post-implantation survival index (%)



92



89



92



95



Live birth index (%)



100



99



100



99



Viability Index (%)



99



99



100



99



Weaning index (%)



100



99



100



100



 


Table 71             Body Weights of Pups (g). F1 – Lactation












































































































































DAY



SEX



 



GROUP 1


CONTROL



GROUP 2


1500 PPM



GROUP 3


5000 PPM



GROUP 4


15000 PPM



1



M



MEAN


SD


N



6.5


0.7


24



6.7


0.6


21



6.6


0.8


22



6.3


0.5


23



F



MEAN


SD


N



6.2


0.7


24



6.4


0.7


20



6.3


0.7


22



5.9


0.5


23



M+F



MEAN


SD


N



6.4


0.7


24



6.6


0.6


21



6.5


0.7


22



6.1


0.5


23



4



M



MEAN


SD


N



9.8


1.1


24



10.1


1.1


21



10.0


1.4


22



9.4


1.0


23



F



MEAN


SD


N



9.4


1.1


24



9.8


1.2


20



9.6


1.3


22



8.9


1.0


23



M+F



MEAN


SD


N



9.6


1.1


24



10.0


1.2


21



9.8


1.3


22



9.1


0.9


23



7



M



MEAN


SD


N



16.3


1.5


24



16.6


1.4


21



16.8


1.7


22



15.4


1.4


23



F



MEAN


SD


N



15.8


1.5


24



16.2


1.6


20



16.1


1.6


22



14.8 *


1.4


23



M+F



MEAN


SD


N



16.1


1.5


24



16.5


1.5


21



16.5


1.7


22



15.1


1.4


23



13



M



MEAN


SD


N



31.5


1.8


24



31.7


2.1


21



32.1


2.7


22



29.7 *


2.7


23



F



MEAN


SD


N



30.8


1.8


24



31.1


2.3


20



31.1


2.6


22



28.7 **


2.5


23



M+F



MEAN


SD


N



31.1


1.8


24



31.5


2.2


21



31.6


2.6


22



29.2 *


2.6


23



21



M



MEAN


SD


N



53.8


3.6


24



54.3


3.8


21



54.7


4.2


22



50.9


5.0


23



F



MEAN


SD


N



51.7


3.2


24



52.5


4.0


20



52.7


4.0


22



48.8 *


4.0


23



M+F



MEAN


SD


N



52.8


3.4


24



53.6


3.8


21



53.7


4.1


22



49.8 *


4.4


23



*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level


 


Table 72             Thyroid Hormones. F1 – PND4


































Sex: Female



 



Reporting Special C



T4


(ng/mL)


[G]



 0


 ppm


 Group 1



Mean


SD


N



11.00 


3.14 


22      



 1500  ppm


 Group 2



Mean


SD


N


tCtrl



10.81 


3.90 


16      


0.98 



 5000  ppm


 Group 3



Mean


SD


N


tCtrl



13.09 


2.60 


18      


1.19 



 15000  ppm


 Group 4



Mean


SD


N


tCtrl



11.03 


4.28 


20      


1.00 



[G] - Anova & Dunnett


 


Table 73             Thyroid Hormones. F1 – Cohort Surplus













































Sex: Male



 



Rep



orting Special Chemistry



T3


(ng/mL)


[G]



T4


(ng/mL)


[G]



TSH


(mU/L)


[G]



 0


 ppm


 Group 1



Mean


SD


N



0.952 


0.088 


10        



39.70 


4.58 


10      



0.0928 


0.0362 


10          



 1500  ppm


 Group 2



Mean


SD


N


tCtrl



0.971 


0.063 


10        


1.02   



42.37 


7.82 


10      


1.07 



0.0863 


0.0283 


10          


0.93     



 5000  ppm


 Group 3



Mean


SD


N


tCtrl



0.968 


0.163 


10        


1.02   



40.25 


5.22 


10      


1.01 



0.0939 


0.0531 


10          


1.01     



 15000  ppm


 Group 4



Mean


SD


N


tCtrl



0.967


0.122 


10        


1.02   



47.96  *


9.02 


10      


1.21 



0.1559  *


0.0669 


10          


1.68     



[G] - Anova & Dunnett: * = p ≤ 0.05


 


Table 74             Sexual Maturation – Males
































 



GROUP 1


CONTROL



GROUP 2


1500 PPM



GROUP 3


5000 PPM



GROUP 4


15000 PPM



BALANOPREPUTIAL SEPARATION (BPS)



POST-NATAL DAY



MEAN


SD


N



40.9


1.2


60



41.2


1.4


60



40.8


1.2


60



42.2 ++


1.4


60



BODY WEIGHT (g)



MEAN


SD


N



172


9


60



173


12


60



171


12


60



163 **


13


60



*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level


+/++ Steel-test significant at 5% (+) or 1% (++) level


 


Table 75             Sexual Maturation – Females



















































 



GROUP 1


CONTROL



GROUP 2


1500 PPM



GROUP 3


5000 PPM



GROUP 4


15000 PPM



VAGINAL PATENCY



POST-NATAL DAY



MEAN


SD


N



33.1


2.2


60



32.2


2.1


60



33.3


1.8


60



34.0


2.4


60



BODY WEIGHT (g)



MEAN


SD


N



105


11


60



102


11


60



106


9


60



99 *


12


60



FIRST ESTRUS



POST-NATAL DAY



MEAN


SD


N



37.4


2.2


20



36.4


2.6


20



37.2


1.4


20



38.1


3.1


20



TIME TO FIRST ESTROUS



MEAN


SD


N



3.8


2.1


20



3.7


2.2


20



3.4


1.5


20



3.7


2.0


20



*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level


+/++ Steel-test significant at 5% (+) or 1% (++) level


 


 


Table 76             Anogenital Distance and Nipple Retention – F1








































 



GROUP 1


CONTROL



GROUP 2


1500 PPM



GROUP 3


5000 PPM



GROUP 4


15000 PPM



ANOGENITAL DISTANCE, MALE (mm)



MEAN


SD


N



2.53


0.26


24



2.63


0.24


21



2.70


0.20


22



2.60


0.26


23



ANOGENITAL DISTANCE, FEMALE (mm)



MEAN


SD


N



1.26


0.21


24



1.26


0.23


20



1.34


0.21


22



1.20


0.16


23



 



NUMBER OF NIPPLES



MEAN


MEDIAN (+)


N



0.00


0.00


24



0.00


0.00


21



0.00


0.00


22



0.00


0.00


23



# / ## Fisher's Exact test significant at 5% (#) or 1% (##) level


+/++ Steel-test significant at 5% (+) or 1% (++) level


 


Table 77             Corrected Anogenital Distance – F1





























 



GROUP 1


CONTROL



GROUP 2


1500 PPM



GROUP 3


5000 PPM



GROUP 4


15000 PPM



ANOGENITAL DISTANCE, NORMALIZED, MALE (mm)



MEAN


SD


N



1.35


0.11


24



1.40


0.11


21



1.44 +


0.09


22



1.41


0.13


23



ANOGENITAL DISTANCE, NORMALIZED, FEMALE (mm)



MEAN


SD


N



0.69


0.10


24



0.68


0.12


20



0.72


0.10


22



0.66


0.09


23



+/++ Steel-test significant at 5% (+) or 1% (++) level


 


Table 78             Organ Weights (g). Male – Cohort Surplus













































 



GROUP 1


CONTROL



GROUP 2


1500 PPM



GROUP 3


5000 PPM



GROUP 4


15000 PPM



BODY WEIGHT



MEAN


SD


N



60


4


10



58


4


10



58


4


9



55 *


3


10



BRAIN



MEAN


SD


N



1.51


0.11


10



1.49


0.04


10



1.48


0.05


10



1.46


0.07


10



THYMUS



MEAN


SD


N



0.248


0.034


10



0.248


0.042


10



0.249


0.037


10



0.216


0.038


10



SPLEEN



MEAN


SD


N



0.316


0.041


10



0.320


0.040


10



0.298


0.060


10



0.260 **


0.032


10



*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level


 


Table 79             Organ Weights (g). Female – Cohort Surplus













































 



GROUP 1


CONTROL



GROUP 2


1500 PPM



GROUP 3


5000 PPM



GROUP 4


15000 PPM



BODY WEIGHT



MEAN


SD


N



56


3


10



57


4


10



55


3


10



53


3


10



BRAIN



MEAN


SD


N



1.44


0.06


10



1.46


0.04


10



1.42


0.05


10



1.41


0.06


10



THYMUS



MEAN


SD


N



0.225


0.036


10



0.245


0.044


10



0.237


0.026


10



0.229


0.034


10



SPLEEN



MEAN


SD


N



0.293


0.052


10



0.288


0.033


10



0.289


0.044


10



0.266


0.045


10



*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level


 


Table 80             Organ/Body Weight Ratio (%). Male – Cohort Surplus





































 



GROUP 1


CONTROL



GROUP 2


1500 PPM



GROUP 3


5000 PPM



GROUP 4


15000 PPM



BRAIN



MEAN


SD


N



2.54


0.25


10



2.59


0.16


10



2.54


0.12


9



2.69


0.15


10



THYMUS



MEAN


SD


N



0.415


0.040


10



0.427


0.051


10



0.431


0.055


9



0.396


0.063


10



SPLEEN



MEAN


SD


N



0.530


0.064


10



0.557


0.073


10



0.485


0.054


9



0.477


0.043


10



*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level


 


Table 81             Organ/Body Weight Ratio (%). Female – Cohort Surplus





































 



GROUP 1


CONTROL



GROUP 2


1500 PPM



GROUP 3


5000 PPM



GROUP 4


15000 PPM



BRAIN



MEAN


SD


N



2.59


0.19


10



2.58


0.12


10



2.61


0.12


10



2.66


0.19


10



THYMUS



MEAN


SD


N



0.405


0.063


10



0.433


0.062


10



0.434


0.040


10



0.430


0.051


10



SPLEEN



MEAN


SD


N



0.526


0.088


10



0.510


0.050


10



0.529


0.072


10



0.499


0.063


10



*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level


 


Table 82             Body Weights of Pups. F2












































































































































DAY



SEX



 



GROUP 1


CONTROL



GROUP 2


1500 PPM



GROUP 3


5000 PPM



GROUP 4


15000 PPM



1



M



MEAN


SD


N



6.3


0.6


18



6.5


0.6


17



6.4


0.6


19



6.2


0.5


19



F



MEAN


SD


N



6.0


0.4


18



6.1


0.7


17



6.1


0.6


19



5.9


0.4


19



M+F



MEAN


SD


N



6.2


0.5


18



6.3


0.6


17



6.2


0.6


19



6.0


0.5


19



4



M



MEAN


SD


N



9.5


1.0


18



9.7


1.3


17



9.6


0.9


19



9.5


0.7


19



F



MEAN


SD


N



9.2


0.8


18



9.3


1.2


17



9.1


1.0


19



9.1


0.7


19



M+F



MEAN


SD


N



9.3


0.9


18



9.5


1.2


17



9.3


0.9


19



9.3


0.7


19



7



M



MEAN


SD


N



16.3


1.2


18



16.4


1.6


17



15.9


1.2


19



15.8


1.2


19



F



MEAN


SD


N



15.9


0.9


18



15.7


1.3


17



15.3


1.3


19



15.3


1.0


19



M+F



MEAN


SD


N



16.1


1.0


18



16.1


1.4


17



15.6


1.2


19



15.6


1.1


19



13



M



MEAN


SD


N



30.9


1.7


18



31.6


2.9


17



30.3


2.1


19



29.7


2.3


19



F



MEAN


SD


N



30.3


1.6


18



30.5


2.3


17



29.5


2.2


19



29.1


2.0


19



M+F



MEAN


SD


N



30.6


1.6


18



31.1


2.5


17



29.9


2.2


19



29.4


2.1


19



21



M



MEAN


SD


N



52.4


2.7


18



53.2


4.3


17



51.2


3.7


19



50.1


4.2


19



F



MEAN


SD


N



50.4


2.1


18



50.9


3.6


17



49.3


3.5


19



48.4


3.3


19



M+F



MEAN


SD


N



51.4


2.2


18



52.0


3.8


17



50.3


3.6


19



49.4


3.7


19



*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level


 


Table 83             Anogenital Distance and Nipple Retention – F2








































 



GROUP 1


CONTROL



GROUP 2


1500 PPM



GROUP 3


5000 PPM



GROUP 4


15000 PPM



ANOGENITAL DISTANCE, MALE (mm)



MEAN


SD


N



2.58


0.15


18



2.75


0.21


17



2.76


0.25


19



2.70


0.25


19



ANOGENITAL DISTANCE, FEMALE (mm)



MEAN


SD


N



1.08


0.06


18



1.18


0.18


17



1.22


0.12


19



1.19


0.17


19



 



NUMBER OF NIPPLES



MEAN


MEDIAN (+)


N



0.00


0.00


18



0.00


0.00


17



0.00


0.00


19



0.00


0.00


19



# / ## Fisher's Exact test significant at 5% (#) or 1% (##) level


+/++ Steel-test significant at 5% (+) or 1% (++) level


 


Table 84             Corrected Anogenital Distance – F2





























 



GROUP 1


CONTROL



GROUP 2


1500 PPM



GROUP 3


5000 PPM



GROUP 4


15000 PPM



ANOGENITAL DISTANCE, NORMALIZED, MALE (mm)



MEAN


SD


N



1.40


0.09


18



1.48


0.13


17



1.49


0.13


19



1.47


0.14


19



ANOGENITAL DISTANCE, NORMALIZED, FEMALE (mm)



MEAN


SD


N



0.60


0.03


18



0.64


0.10


17



0.67 ++


0.06


19



0.66 ++


0.10


19



+/++ Steel-test significant at 5% (+) or 1% (++) level


 


Table 85             Organ Weights (g). Male – F2













































 



GROUP 1


CONTROL



GROUP 2


1500 PPM



GROUP 3


5000 PPM



GROUP 4


15000 PPM



BODY WEIGHT



MEAN


SD


N



52


3


10



51


4


10



51


5


10



50


4


10



BRAIN



MEAN


SD


N



1.45


0.04


10



1.48


0.06


10



1.46


0.03


10



1.41


0.06


10



THYMUS



MEAN


SD


N



0.210


0.029


10



0.211


0.030


10



0.205


0.042


10



0.212


0.022


10



SPLEEN



MEAN


SD


N



0.265


0.073


10



0.241


0.043


10



0.248


0.042


10



0.243


0.039


10



*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level


 


Table 86             Organ Weights (g). Female – F2













































 



GROUP 1


CONTROL



GROUP 2


1500 PPM



GROUP 3


5000 PPM



GROUP 4


15000 PPM



BODY WEIGHT



MEAN


SD


N



50


2


10



49


4


10



49


4


10



48


3


10



BRAIN



MEAN


SD


N



1.44


0.03


10



1.41


0.07


10



1.43


0.04


10



1.40


0.02


10



THYMUS



MEAN


SD


N



0.201


0.023


10



0.205


0.044


10



0.216


0.035


10



0.196


0.021


10



SPLEEN



MEAN


SD


N



0.247


0.027


10



0.249


0.052


10



0.248


0.042


10



0.253


0.032


10



*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level


 


Table 87             Organ/Body Weight Ratio (%). Male – F2





































 



GROUP 1


CONTROL



GROUP 2


1500 PPM



GROUP 3


5000 PPM



GROUP 4


15000 PPM



BRAIN



MEAN


SD


N



2.83


0.21


10



2.91


0.25


10



2.90


0.27


10



2.86


0.20


10



THYMUS



MEAN


SD


N



0.409


0.060


10



0.415


0.052


10



0.407


0.087


10



0.430


0.047


10



SPLEEN



MEAN


SD


N



0.511


0.123


10



0.471


0.059


10



0.490


0.064


10



0.490


0.069


10



*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level


 


Table 88             Organ/Body Weight Ratio (%). Female – F2





































 



GROUP 1


CONTROL



GROUP 2


1500 PPM



GROUP 3


5000 PPM



GROUP 4


15000 PPM



BRAIN



MEAN


SD


N



2.90


0.14


10



2.87


0.15


10



2.91


0.29


10



2.89


0.12


10



THYMUS



MEAN


SD


N



0.404


0.047


10



0.413


0.067


10



0.438


0.073


10



0.408


0.062


10



SPLEEN



MEAN


SD


N



0.496


0.051


10



0.502


0.087


10



0.502


0.083


10



0.522


0.058


10



*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level


 


 


 


 


 

Conclusions:
In conclusion, based on the results of this Extended One Generation Reproductive Toxicity Study (including Cohorts 1A, 1B (extended to a F2 generation) and 1C), the following No Observed Adverse Effect Levels (NOAELs) of Tall Oil were established:

GENERAL TOXICITY

-P0 generation:
At least 15000 ppm (on average corresponding to 1197 mg/kg/day in males and 1386 mg/kg/day in females).

-P1 generation:
At least 15000 ppm (on average corresponding to 1304 mg/kg/day in males and 1478 mg/kg/day in females).

REPRODUCTION TOXICITY

-P0 generation
At least 15000 ppm (on average corresponding to 1197 mg/kg/day in males and 1386 mg/kg/day in females).

-P1 generation
At least 15000 ppm (on average corresponding to 1304 mg/kg/day in males and 1478 mg/kg/day in females).

DEVELOPMENTAL TOXICITY

-F1 generation
At least 15000 ppm (on average corresponding to 1197 mg/kg/day in males and 1386 mg/kg/day in females).

-F2 generation
At least 15000 ppm (on average corresponding to 1304 mg/kg/day in males and 1478 mg/kg/day in females).
Executive summary:

INTRODUCTION


A key guideline OECD 443 Extended One Generation Reproductive Toxicity Study, including Cohorts 1A, 1B (extended to a F2 generation), Cohort 1C, and Cohort Surplus was conducted with Tall Oil. The objectives were to evaluate the pre- and postnatal effects of Tall Oil on development of Wistar Han rats. Additionally, systemic toxicity in pregnant and lactating females, and young and adult offspring of Wistar Han rats, was evaluated.


EXPERIMENTAL PROCEDURES


Animals (25 per sex per treatment group) were administered Tall Oil by dietary administration (pellet diet) with diet concentrations of 1500, 5000 and 15000 ppm. During the lactation period when relative food consumption increases significantly, diet concentrations were decreased on three occasions to maintain a stable mean test item intake throughout the different study periods. The rats of the control group received similarly prepared pellets without the test item.


The following observations were made for exposed parental animals (P0 and F1 from PND 22 onwards (reported under P1 in RSS)).



  • Mortality/Moribundity checks

  • Clinical observations

  • Arena observations

  • Body weights

  • Food consumption and test item intake

  • Water consumption

  • Sexual maturation (P1 only)

  • Estrous cycle determination prior to mating and during mating up to evidence of copulation (only Cohort 1B from P1)

  • General reproduction data

  • Haematology & Coagulation (only Cohort 1A from P1)

  • Clinical chemistry (only Cohort 1A from P1)

  • Thyroid hormones (only Cohort 1A and Cohort Surplus from P1)

  • Urinalysis (only Cohort 1A from P1)

  • Gross necropsy findings

  • Sperm analysis (only Cohort 1A from P1)

  • Splenic lymphocyte subpopulation analysis (P0 not evaluated; only Cohort 1A from P1)

  • Organ weights (only Cohort 1A, 1B, & Cohort Surplus from P1)

  • Histopathology (only Cohort 1A, 1B, & Cohort Surplus from P1)


 


The following observations were made for pups of the F1 and F2 generations until weaning.



  • Mortality/moribundity checks

  • Clinical observations

  • Body weights

  • Sex determination

  • Anogenital distance

  • Nipple retention in males

  • Thyroid hormones (pups culled on PND4 – F1 only)

  • Gross necropsy of F1 & F2 pups culled on PND4, F1 pups not assigned to cohorts, & F2 pups


 


A number of reproduction and developmental variables and indices were calculated for P0 and P1 Cohort 1B females, including:



  • Mating Index

  • Pre-coital time

  • Gestation Index

  • Duration of gestation

  • Post-implantation survival index

  • Litter size

  • Live birth Index

  • Percentage live males at first litter check

  • Percentage live females at first litter check

  • Viability Index

  • Weaning Index

  • Percentage live males at weaning

  • Percentage live females at weaning


 


RESULTS


-P0 GENERATION


No parental toxicity was observed up to the highest dose level tested (15000 ppm).


A test item-related decrease in body weights and body weight gain was observed in both males and females at 15000 ppm. Effect sizes were small and not always statistically significant. Moreover, effects were more pronounced during the early period of treatment after which body-weight gain tended to recover. Notably, during the post-coitum and lactation periods, the same rate of body weight gain was observed for all treatment groups. Overall, the effects on body weights were considered non-adverse.


Increased food consumption was observed from 1500 ppm onwards in both males and females. In absence of an adverse effect on body weight and as changes were regarded as slight, the increased food consumption was considered non-adverse.


Changes were recorded in clinical pathology parameters. Statistically significant changes in clinical chemistry parameters in both males and females were generally small in magnitude or occurred in the absence of a clear dose-response relationship. However, there were a few notable haematology and urinalysis findings:



  • A dose-dependent, statistically significant, reduction in reticulocyte counts in male animals. A similar trend for dose-dependent reduction in reticulocyte counts was seen in females but did not reach statistical significance. Concomitant dose-dependent and statistically significant decreases (male) or increases (female) in RDWG were observed.

  • In male animals only, a dose-dependent, statistically significant increase in urine volume was observed, and this was accompanied by corresponding reductions in specific gravity.


All changes in clinical pathology parameters occurred in absence of a microscopic correlate at the organ level. These changes were therefore considered to be non-adverse.


A notable organ weight change was a dose-dependent, statistically significant increase in absolute and relative liver weight in male animals. This was accompanied by an accentuated lobular pattern in the liver of males exposed to 15000 ppm of test item. An increase in absolute and relative liver weights were also observed in female animals exposed to the high dose of the test item. Hepatomegaly, such as this, is generally considered an adaptive rather than a toxicological response. It was therefore considered non-adverse. Statistically significant, dose-dependent, increases in relative thyroid weight were observed in both sexes but was more pronounced in females. Such changes are possibly secondary to adaptive liver changes and were considered non-adverse. Other changes in organ weight occurred in the absence of clear dose-dependent responses and/or were small in effect size.


No treatment related changes were observed in any of the other parameters examined during the study, including mortality, clinical observations, coagulation, thyroid hormones, and histopathology.


-F1 GENERATION (up to PND 22)


No reproductive or developmental toxicity was observed.


No treatment-related changes were noted in any of the following reproductive or developmental parameters investigated in this study (estrous cycle, sperm analysis and spermatogenic profiling, mating and fertility indices, precoital time, number of implantations, histopathological examination of reproductive organs, sex ratio, anogenital distance, nipple retention).


Throughout the lactation period, pup body weights (male, female, and combined) were consistently lower in the 15000 ppm treatment group. However, these changes were small in magnitude (maximum of 7% compared to control) and were not always statistically significant. Moreover, body weight changes were not observed in F2 pups (see below). Therefore, these changes were considered non-adverse.


T4 hormone in PND4 pups were unaffected by treatment. In Cohort Surplus, both T4 and TSH levels were statistically significantly elevated in the 15000 ppm treatment group. However, no clear dose-dependent trend was observed, and all values remained within the range of historical control data. Therefore, these changes were considered non-adverse.


 


-P1 GENERATION


No parental toxicity was observed up to the highest dose level tested (15000 ppm).


As observed for P0, a test item-related decrease in body weights and body weight gain was observed in both males and females at 15000 ppm. As body weight gains recovered to normal levels in the following weeks, the lower body weights for the remainder of the study were considered secondary to the lower body weight at start of treatment together with the decreased body weight gains during the first weeks of treatment and were considered non‑adverse.


Similar to P0, increased food consumption was observed from 1500 ppm onwards in both males and females. In absence of an adverse effect on body weight and as changes were regarded as slight, the increased food consumption was considered non-adverse.


At 15000 ppm, males reached balanopreputial separation at a slightly older age than concurrent controls. The delay in sexual maturation was attributed to the lower body weights of these animals. Moreover, there was no effects on mating and fertility of the F1-animals. Therefore, this change was regarded non-adverse.


Changes were recorded in clinical pathology parameters. Similar to the P0 generation, statistically significant changes in clinical chemistry parameters in both males and females were generally small in magnitude or occurred in the absence of a clear dose-response relationship. Unlike the P0 generation, there were no statistically significant changes in urinalysis parameters. There were a few notable haematology findings, and these did not always match those seen in the P0 generation. Notable changes included:



  • Dose-dependent, statistically significant, decrease in WBC, exclusively in male animals, accompanied by reductions in differential counts (neutrophils, lymphocytes, monocytes, eosinophils, and basophils).

  • Similar to P0, a dose-dependent, statistically significant, reduction in reticulocyte counts was observed in male animals. Concomitant dose-dependent and statistically significant decreases in RDWG were observed.

  • Dose-dependent, statistically significant, decreases in haemoglobin, haematocrit, and mean corpuscular haemoglobin was observed exclusively in females.


All changes in clinical pathology parameters occurred in absence of a microscopic correlate at the organ level. These changes were therefore considered to be non-adverse.


Similar to P0, a notable organ weight change was a dose-dependent, statistically significant increase in absolute and relative liver weight in male animals. A dose-dependent increase in relative liver weights were also observed in female animals. Hepatomegaly, such as this, is generally considered an adaptive rather than a toxicological response. It was therefore considered non-adverse. Statistically significant, dose-dependent, increases in absolute and relative thyroid weight were observed in males but, unlike the P0 generation, this was less pronounced in females. Such changes are possibly secondary to adaptive liver changes and were considered non-adverse. Other changes in organ weight occurred in the absence of clear dose-dependent responses and/or were small in effect size.


No treatment related changes were observed in any of the other parameters examined during the study, including mortality, clinical observations, coagulation, thyroid hormones, splenic lymphocyte subpopulation analysis, gross necropsy findings and histopathological examinations (including ovarian follicle and corpora lutea counts).


 


-F2 GENERATION (up to PND 22)


No reproductive or developmental toxicity was observed.


At 15000 ppm, the mean number of implantation sites was decreased in F1-females (Cohort 1B) that were used to generate the F2-Generation.  This was considered not to be adverse as the majority of animals had implantation sites which were within the normal historical control data range and the concurrent controls values were significantly higher than the historical control data ranges.


The decreased litter size of F1-females at 15000 ppm were considered a direct result of the lower number of implantation sites (see above) and was considered not to be adverse at 15000 ppm.  For most litters, the litter sizes were within the normal historical ranges.  


No treatment-related changes were noted in any of the following reproductive or developmental parameters investigated in this study (estrous cycle, sperm analysis and spermatogenic profiling, remaining mating and fertility indices, precoital time, histopathological examination of reproductive organs, sex ratio, anogenital distance, nipple retention, pup body weights).

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
The OECD 443 (Meijer, 2022) is assigned a Klimisch score of 1. It followed the most recently adopted test guideline (Adopted 25th June 2018). Moreover, there were no deviations that impacted upon the integrity of the study.

The OECD 422 study (Clubb, 2003) is assigned a Klimisch score of 1. It was based on the version of the test guideline available at the time (Adopted 22nd March 1996). There are some differences in the information reported compared with stipulations in the current version of OECD 422 (Adopted 29th July 2016). These are:
• Functional Observations not performed
• Thyroid hormones not measured
• Study terminated on PND6, rather than continuing until minimally PND13
• Anogenital distance was not reported

There were no deviations from the study plan that impacted upon the integrity of the study.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

N/A

Effects on developmental toxicity

Description of key information

OECD 414 (rat)


A key Guideline OECD 414 prenatal developmental toxicity study in rats was conducted to evaluate the potential of the test material (Tall Oil) to induce developmental toxicity after maternal exposure during the critical period of organogenesis and to characterize maternal toxicity at the exposure levels tested (Bressers, 2021a). The test was carried out according to the requirements of the guideline and in compliance with GLP.


Time-mated female Wistar Han rats (22 females/dose) were exposed to the test material at 0, 100, 300, or 1000 mg/kg/day via oral gavage once daily, 7 days a week, from Day 6 to Day 20 post-coitum, inclusive.


The only indication of developmental toxicity was a minor, albeit statistically significant, reduction in mean fetal weight in the high dose group compared to controls (to 94% of mean control value). However, as the low fetal body weights were observed in the absence of retarded growth or ossification parameters, and with all values were within the available historical control data, this was considered to be non-adverse.


Based on the lack of adverse treatment-related effects observed at the highest dose tested in this prenatal developmental toxicity study, the developmental No Observed Adverse Effect Level (NOAEL) for Tall Oil in rats was determined to be ≥1000 mg/kg/day.


OECD 414 (rabbit)


A key Guideline OECD 414 prenatal developmental toxicity study in rabbits was conducted to evaluate the potential of the test material (Tall Oil) to induce developmental toxicity after maternal exposure during the critical period of organogenesis and to characterize maternal toxicity at the exposure levels tested (Bressers, 2021b). The test was carried out according to the requirements of the guideline and in compliance with GLP.


Time-mated female New Zealand White rabbits (22 females/dose) were exposed to the test material at 0, 100, 300, or 600 mg/kg/day via oral gavage once daily, 7 days a week, from Day 6 to Day 28 post-coitum, inclusive.


Only two signs of developmental toxicity were seen. Slightly lower fetal body weights and placental weights were observed in male and female fetuses at 600 mg/kg/day, with a more pronounced effect in females. Although no statistical significance was reached and values remained within the available historical control data, lower placental weights were observed for female fetuses that presented with a slightly lower body weight. Based on the small magnitude of change, this effect was considered to be non-adverse.


An increased incidence for unossified tarsals was observed in fetuses at 600 mg/kg/day when compared to concurrent controls. This was caused by four specific fetuses originating from three different litters. These fetuses were observed with body weights below their mean litter weights and the mean group litter weight. Therefore, this effect was considered to be a consequence of low fetal weights rather than being a direct treatment-related effect.


Based on the lack of adverse treatment-related effects observed at the highest dose tested in this prenatal developmental toxicity study, the developmental No Observed Adverse Effect Level (NOAEL) for Tall Oil in rabbits was determined to be ≥600 mg/kg/day.

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2019-10-11 to
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
None of the deviations were considered to have impacted the overall integrity of the study or the interpretation of the study results and conclusions.
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Kraton Chemical BV (The Netherlands); Batch No. AN-400-125
- Expiration date of the lot/batch: 2023-01-01
- Purity test date: 2019-01-17

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In freezer (≤ -15°C) protected from light container, flushed with nitrogen
- Stability under test conditions: Stable, maximum temperature: 40°C, maximum duration: 60 minutes
- Solubility and stability of the test substance in the solvent/vehicle: Stable in vehicle (1% (w/v) Aqueous carboxymethyl cellulose with 0.1% (v/v) Tween 80) for at least 24 hours at room temperature protected from light. Confirmed over the concentration range 1 to 200 mg/mL (emulsions) (Test Facility Reference No. 20180610).

FORM AS APPLIED IN THE TEST (if different from that of starting material) : Amber transparent liquid

OTHER SPECIFICS:
- other information: Sample will crystallize upon prolonged storage at ambient or sub-ambient conditions and will become hazy. Sample should become transparent after 4-8 hrs at ambient temperature. Otherwise, heat sample gently (max 40°C) until sample is transparent again. Shake sample bottle gently before use to assure sample homogeneity.
Species:
rabbit
Strain:
New Zealand White
Remarks:
time-mated females
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River (Chatillon sur Chalaronne, France)
- Age at study initiation: 17-19 weeks old
- Weight at study initiation: between 2942 and 4002 g at the initiation of dosing
- Fasting period before study: Not specified
- Housing: individually in cages with perforated floors (Ebeco, Germany, dimensions 67 x 62 x 55 cm) equipped with water bottles.
- Diet (e.g. ad libitum): Pelleted diet for rabbits (KLIBA NAFAG Rabbit Diet 3409 maintenance and breeding, from Kliba NAFAG Granovit AG, Kaiseraugst, Swizerland) ad libitum throughout the study, except during designated procedures. Additionally, pressed hay (Tecnilab-BMI bv, Someren, The Netherlands) was provided during the study period.
- Water (e.g. ad libitum): Municipal tap water was freely available to each animal via water bottles/containers.
- Acclimation period: at least 2 days before the commencement of dosing

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 20°C
- Humidity (%): 54 to 80%
- Air changes (per hr): Ten or greater air changes per hour with 100% fresh air (no air recirculation)
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 2019-10-11 To: 2019-11-08
Route of administration:
oral: gavage
Vehicle:
other: 1% (w/v) Aqueous carboxymethyl cellulose with 0.1% (v/v) Tween 80
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Test material dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. Test material was pre-weighed in amber vials, flushed with nitrogen gas and stored in the freezer (≤-15°C). The pre-weighed test material was removed from the freezer the night before the intended day of use allowing complete thawing of the test material. The dosing formulations were prepared daily as an emulsion and were dosed within 24 hours after completion of the preparation of the test material.

Test material dosing formulations were kept at room temperature and protected from light until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing. Adjustment was made for specific gravity of the test material.

VEHICLE
- Justification for use and choice of vehicle (if other than water): 1% (w/v) Aqueous carboxymethyl cellulose with 0.1% (v/v) Tween 80
- Concentration in vehicle: 20, 60, 120 mg/mL for the 100, 300, and 600 mg/Kg/day dose groups, respectively
- Amount of vehicle (if gavage): 5 mL/Kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Sample Collection and Analysis
Dose formulation samples were collected for analysis as indicated below:

Occasion Concentration Homogeneity
Week 1 all groups Groups 2, 3, and 4a

a: The homogeneity results obtained from the top, middle and bottom for the Group 2, 3 and 4 preparations were averaged and utilized as the concentration results.

Concentration Analysis
Duplicate sets of samples (approximately 500 mg) were sent to the analytical laboratory. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 15% for emulsions of target concentration.

Homogeneity Analysis
Duplicate sets of samples (approximately 500 mg) were sent to the analytical laboratory. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was ≤10%.

Stability Analysis
Stability analyses performed previously in conjunction with the method development and validation study (Test Facility Reference No. 20180610) demonstrated that the test material is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study. Stability data have been retained in the study records for Test Facility Reference No. 20180610.
Details on mating procedure:
Time-mated female New Zealand White rabbits were received from Charles River (Chatillon sur Chalaronne, France) on Oct 11, 2019. The females arrived on Day 1-4 post-coitum (Day 0 post-coitum is defined as the day of successful mating).
Duration of treatment / exposure:
once daily via oral gavage
Frequency of treatment:
once daily oral gavage for 7 days a week from Day 6 to Day 28 post-coitum, inclusive
Duration of test:
from Day 6 to Day 28 post-coitum, inclusive
Dose / conc.:
0 mg/kg bw/day
Remarks:
Group 1 (Control)
Dose / conc.:
100 mg/kg bw/day
Remarks:
Group 2
Dose / conc.:
300 mg/kg bw/day
Remarks:
Group 3
Dose / conc.:
600 mg/kg bw/day
Remarks:
Group 4
No. of animals per sex per dose:
22 females per dose level
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The oral route of exposure was selected because this is the intended route of human exposure during manufacture, handling or use of the test material. The dose levels were selected based on the results of the dose range finding study (Test Facility Reference No. 20180608), and in an attempt to produce graded responses to the test material.

- Rationale for animal assignment: Animals were randomly assigned to groups.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily, beginning on Day 6 post-coitum and lasting up to the day prior to necropsy. The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity and the maximum grade was predefined at 1, 2, 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored.

BODY WEIGHT: Yes
- Time schedule for examinations: individually weighed on Days 6, 9, 12, 15, 18, 21, 24, 27 and 29 post-coitum. In order to monitor the health status, Animal No. 69 was also weighed on Day 11 post-coitum

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
Food consumption was quantitatively measured for Days 6-9, 9-12, 12-15, 15-18, 18-21, 21-24, 24-27 and 27-29 post-coitum.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Water consumption was monitored on regular basis throughout the study by visual inspection of the water bottles/containers.

POST-MORTEM EXAMINATIONS: Yes (Animals surviving until scheduled euthanasia were euthanized by intravenous injection of pentobarbital (approx. 1 mL/kg Euthasol
® 20%) on Day 29 post-coitum)
- Sacrifice on Day 29 post-coitum
- Organs examined: All animals (including animals found dead or sacrificed before planned necropsy and females with early delivery) were subjected to an external, thoracic and abdominal examination, with special attention being paid to the reproductive organs. All macroscopic abnormalities were recorded, collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution). The liver and uterus were weighed at necropsy for all animals (except for females that were euthanized in poor condition). The adrenal gland, brain, kidneys, ovaries, thymus, spleen, thyroid gland and pituitary gland were weighed 7 days after fixation for all scheduled euthanasia animals. Organs of animals that were sacrificed in extremis were weighed after at least 7 days of fixation. Paired organs were weighed together. Organ weight as a percent of body weight (using the body weight on Day 29 post-coitum) was calculated.

OTHER:
- Clinical Pathology
Blood of all animals (except for animals found dead) was collected on the day of necropsy. Samples were collected from the ear artery.

Haematology: Blood samples at a target volume of 0.5 mL were collected into tubes containing K3-EDTA as anticoagulant. Samples were analyzed for the parameters specified in Table 2 presented in the section ‘Any other information on materials and methods incl. tables’.

Clinical Chemistry: Blood samples at a target volume of 0.5 mL (plasma) were collected into tubes containing Li-Heparin as anticoagulant. Serum samples at a target volume of 0.25 mL were collected in tubes without anticoagulant. Blood samples were processed for plasma or serum (bile acids), which was analyzed for the parameters specified in Table 3 presented in the section ‘Any other information on materials and methods incl. tables’.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes (Each ovary and uterine horn of all animals was dissected and examined as quickly as possible)
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: Placental weights; number and distribution of live and dead fetuses; number and distribution of embryo-fetal deaths
Fetal examinations:
External, visceral, and skeletal findings were recorded as developmental variations (alterations in anatomic structure that are considered to have no significant biological effect on animal health or body conformity and/or represent slight deviations from normal) or malformations (those structural anomalies that alter general body conformity, disrupt or interfere with normal body function, or may be incompatible with life).

- External examinations: Yes: [all per litter]
Each viable fetus was examined in detail to detect macroscopic visible abnormalities and their weight was determined (not for fetuses of animals sacrificed before planned necropsy). For late resorptions and recognizable fetuses of females euthanized in extremis, a gross external examination wasperformed (if possible)

- Soft tissue examinations: Yes: Soft tissue cephalic examination was executed for approximately half of the fetuses of the same litter for all groups. All fetuses were internally sexed and examined for visceral anomalies by dissection in the fresh (non-fixed) state. The thoracic and abdominal cavities were opened and dissected. This examination included the heart and major vessels. Fetal kidneys were examined and graded for renal papillae development.

The heads were removed from approximately one-half of the fetuses in each litter and placed in Bouin's solution for soft-tissue examination of all groups using the Wilson sectioning technique. The heads from the remaining one-half of the fetuses in each litter of all groups were examined by a
mid-coronal slice.

- Skeletal examinations: Yes: [all per litter in Groups 1 and 4]
All eviscerated fetuses, following fixation in 96% aqueous ethanol, were macerated in potassium hydroxide and processed for double staining with Alcian Blue 8GX and Alizarin Red S.

Subsequently, the skeletal examination was done on all fetuses from Groups 1 and 4. Since no possible treatment related effects in the high dose group were seen, skeletal examination was not extended to the fetuses from the low and mid dose group.

A few bones were not available for skeletal examination because they were accidentally damaged or lost during processing. The missing bones were listed in the raw data; evaluation by the fetal pathologist and Study Director determined there was no influence on the outcome of the individual or overall skeletal examinations, or on the integrity of the study as a whole.

- Head examinations: Yes: [all per litter]
The heads were removed from approximately one-half of the fetuses in each litter and placed in Bouin's solution for soft-tissue examination of all groups using the Wilson sectioning technique. The heads from the remaining one-half of the fetuses in each litter of all groups were examined by a mid-coronal slice.
Statistics:
For information on statistics, please refer to the section 'Any other information on materials and methods incl. tables’
Indices:
Maternal Indices
Body Weight Gains: Calculated against the body weight on Day 6 post-coitum.

Corrected Body Weight Gains: Body weight determined on Day 29 post-coitum minus the body weight on Day 6post-coitum and the weight of gravid uterus.

Relative Food Consumption: Calculated against the body weight for scheduled intervals.

Organ Weight Relative to Body Weight: Calculated against the body weight on Day 29 post-coitum.

Reproduction and Developmental Variables

1) Preimplantation loss (%): ((number of corpora lutea - number of implantation sites) / (number of corpora lutea)) x 100

2) Post-implantation loss (%): ((number of implantation sites - number of live fetuses) / (number of implantation sites)) x 100

3) Viable fetuses affected/litter (%): ((number of viable fetuses affected/litter) / (number of viable fetuses/litter)) x 100
Historical control data:
Charles River Den Bosch historical data (Study date range: 2014 – 2018) on the background incidence of fetal malformations and developmental variations in this species from the same strain and source has been presented in Appendix 3 of the study report.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Reduced faeces production was observed for 9/21 females in the control group, for 10/22 females treated at 100 mg/Kg/day, for 15/21 females at 300 mg/Kg/day and for 14/20 females at 600 mg/Kg/day. Piloerection was observed in 2/20 females at 600 mg/Kg/day while red fluid on the manure tray was observed for 2/22 females at 300 mg/Kg/day. Female Nos. 58 and 83 (300 and 600 mg/Kg/day, respectively) were observed with watery discharge from the nose on a single day. For Female No. 83 this was observed one day prior to the observed slow breathing, but for the other female, no breathing difficulties were observed. From the study daybook, it can be retrieved that for Female No. 58 the presence of watery discharge from the nose was due to the dosing procedure (Watery discharge from the nose was directly observed after extubating), most likely this was also the case for the other animal. Therefore, this was regarded to be unrelated to the test material itself.

Incidental observations included quick breathing, alopecia, missing nails, wounds/scabs and overgrown/broken teeth. These findings occurred within the range of background findings to be expected for rabbits of this age and strain which are housed and treated under the conditions in this study. At the incidence observed and since the effects were also observed in concurrent control animals, these were not considered to be of any toxicological relevance.

Of the animals that were killed in extremis, Female No. 3 in the control group was observed with red fluid on the manure tray, absent food consumption and 5% body weight loss between Days 0 and 6 post-coitum. Necropsy revealed intussusception of the colon, together with many dark red foci on the colon. Female No. 61 in the 300 mg/Kg/day dose group exhibited body weight loss of 6% compared to Day 6 post-coitum and nearly absent food consumption (9 grams in total over ten days). Clinical signs included piloerection on the last two days prior to necropsy and the animal was found lean the day prior to her preterm sacrifice. In addition, severely reduced faeces production was observed for seven days. Relevant gross lesions were an accentuated lobular pattern of the liver and a gelatinous, small thymus. Female No. 69 in the 600 mg/Kg/day exhibited severe body weight loss (up to -14% on Day 11 post-coitum compared to Day 0 post-coitum), absent food consumption and clinical signs including reduced faeces production, diarrhoea, piloerection, hunched posture and lean appearance. Gross lesions included a small spleen. Female No. 70 in the 600 mg/Kg/day dose group was observed to have breathing difficulties(quick breathing, labored respiration and rales), piloerection, severely reduced faeces production and pale appearance one day prior or on the day of early sacrifice, severe body weight loss between Days 12 and 15 post coitum (-7%) and nearly absent food consumption from Day 12 post-coitum onwards. Additionally, increased concentrations of alanine aminotransferase, aspartate aminotransferase, urea, cholesterol, HDL cholesterol, LDL cholesterol, triglycerides, bile acids, potassium and inorganic phosphate were observed. Chloride and calcium concentration were also lower compared to both controls and animals treated at 600 mg/Kg/day.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
No treatment-related mortality was observed during the study. However, four animals died during the study period. In the control group, one animal (Female No. 3) was sacrificed in extremis on Day 7 post-coitum. In the 300 mg/Kg/day dose group, one animal (Female No. 61) was sacrificed in extremis on Day 21 post-coitum. Since the effects observed were seen only in a single animal at the mid-dose with no accompanied dose-response, this was considered to be unrelated to treatment. Two females were sacrificed in extremis in the 600 mg/Kg/day dose group. One animal (Female No. 69) was sacrificed in extremis on Day 11 post-coitum and exhibited severe body weight loss. However, since the body weight loss was already present prior to the initiation of dosing, this early sacrifice
was not related to exposure to the test material. The second animal in the high dose group (Female No. 70) was sacrificed in extremis on Day 15 post-coitum. Necropsy confirmed a gavage related trauma as the animal was observed with perforation of the right caudal lobe of the left lung, chest cavity containing watery fluid, black-brown discoloration of the left lung, small left lung, dark-red discoloration of the right lung, adhesion of the pericardium to the heart and thymus, tan discoloration of the thymus, gelatinous thymus and reddish foci on the liver.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related effects were observed at 100 and 300 mg/Kg/day. At 600 mg/Kg/day, body weight gain was slightly lower compared to controls during the complete treatment period, albeit without reaching statistical significance. No relevant changes were observed in body weight or corrected body weight gain.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Normal values for food consumption and relative food consumption were noted for females treated at 100 mg/Kg/day. Mean food consumption before or after allowance for body weight was lower at 300 and 600 mg/Kg/day compared to concurrent controls, reaching statistical significance on Days 15-18 post-coitum and Days 6-24 post-coitum, respectively. All values remained within available historical control data.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
At 100 and 300 mg/Kg/day, a statistically significantly lower red blood cell distribution width (RDW) was observed(0.96x of control for both dose levels). In absence of a dose response-relationship this was not considered to be treatment-related. One animal (Female No. 27) in the 100 mg/Kg/day dose group was observed to have an increased reticulocyte count compared to control means. However, as this only concerns one female at the low dose, this was not considered to be treatment-related. Other parameters were within comparable range of concurrent control animals.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
At 600 mg/Kg/day, triglycerides were statistically significantly increased compared to controls (1.52x of control). Additionally, at 100 and 600 mg/Kg/day, cholesterol levels were statistically significantly higher compared to concurrent controls (1.48x of control for both dose levels). Chloride levels at 100 and 300 mg/Kg/day were statistically significantly increased compared to concurrent controls (1.02x of control). In the absence of a dose response and/or considering the small magnitude of change, this was not considered to be toxicologically relevant.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Liver:body weight ratios were increased with statistical significance for females treated at 300 and 600 mg/Kg/day (1.1x of control for both dose levels). Additionally, ovary weights (absolute only) were increased with statistical significance in females treated at 100 mg/Kg/day(1.1x of control). However, in the absence of a dose-response relationship, this was not considered to be treatment-related.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Macroscopic observations at necropsy did not reveal any alterations that were considered to be treatment-related. Two non-pregnant females (Female No. 58 at 300 mg/Kg/day and Female No. 81 at 600 mg/Kg/day) were observed with ovaries that were reduced in size. At the incidence observed and in the absence of a treatment related effect on ovary weights, this was considered unrelated to treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Number of abortions:
no effects observed
Description (incidence and severity):
All surviving females were pregnant and had litters with viable fetuses.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
The numbers of pre- and post-implantation loss in the control and test groups were comparable and in the range of normal biological variation.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
Females sacrificed in extremis were all pregnant at the time of necropsy. All remaining females were pregnant and had litters with viable fetuses.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Female Nos. 27 (100 mg/Kg/day), 58 (300 mg/Kg/day), and 71, 81 and 88 (600 mg/Kg/day) were nongravid. As dosing was only initiated after the implantation occurred, these were not considered to be treatment-related.
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 600 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: No adverse treatment-related systemic toxicity effects obseved at the highest dose tested.
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
There were no effects on fetal body weights (both sexes) observed subsequent to treatment up to 300 mg/Kg/day. At 600 mg/Kg/day, fetal body weights (both males and females) were lower compared to controls, however without reaching statistical significance and values remaining within the available historical control data.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
The numbers of fetuses (litters) available for fetal morphological examination were 206 (21), 186 (21), 187 (20) and 176 (17) in the control, 100, 300 and 600 mg/Kg/day groups, respectively.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The male:female ratio was unaffected by treatment up to 600 mg/Kg/day.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
There were no test material-related effects on litter size in any dose group.
Changes in postnatal survival:
no effects observed
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
External variations were not observed and there were no test material-related effects on external morphology following treatment up to 600 mg/Kg/day. At 300 mg/Kg/day, a facial cleft occurred in one fetus (Fetus No.A056-05). In addition, the late resorption in litter A045 was observed with exencephaly and gastroschisis. At 100 mg/Kg/day, two malformations occurred that both affected the same fetus (No. A030-01). This fetus missed the head and had two flexed carpals. Due to the single occurrence and occurrence at low and mid dose levels, all these malformations were considered chance findings.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related effects on skeletal morphology were observed at 600 mg/Kg/day. Skeletal malformations occurred in two control and two high-dose fetuses. The high-dose fetuses were observed with sternal anomaly (A087-01) or vertebral centra anomaly (A082-08), in addition to the visceral malformations that were observed in these fetuses. The single occurrence and fact that these skeletal malformations are among historical control data does not indicate a treatment effect and were therefore, considered chance findings.

Two control fetuses were observed with costal cartilage anomaly (A006-07) or bent limb bones (A014-04) and were as such considered to be spontaneous in origin. Of the variations, unossified tarsals occurred more frequently at 600 mg/Kg/day than at the control level. Incidences were 1.2% and 2.3% per litter in Groups 1 and 4, respectively. Although this increase was not statistically significant, the high dose value was above the historical control maximum value of 2.1% per litter. All other skeletal variations that occurred were considered to be unrelated to treatment as they occurred infrequently, in control fetuses only and/or at frequencies that were within the range of available historical control data.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related effects on visceral morphology were observed following treatment up to 600 mg/Kg/day. Visceral malformations occurred in 6 (4), 2 (2), 4 (3) and 3 (3) fetuses (litters) in the control, 100, 300 and 600 mg/Kg/day groups, respectively.

At 600 mg/Kg/day, one fetus (No. A087-01) was observed with the following malformations: malpositioned kidney, retroesophageal aortic arch and left carotid originating from the pulmonary trunk. In addition to this fetus, two other fetuses at this dose level were observed with malformations. One fetus (No.A082-08) had a narrow aortic arch and atrial septum defect, and another fetus (No. A086-07) exhibited internal hydrocephaly. Two of the above malformations were also observed in fetuses at 300 mg/Kg/day, namely malpositioned kidney (Fetus Nos. A047-05 and A047-06) and internal hydrocephaly (Fetus No. A045-10). The other affected fetus in this group had transposition of the great vessels and was also noticed with a facial cleft (Fetus No. A056-05).

At 100 mg/Kg/day, the fetus without head and flexed carpals (A030-01) appeared to have abnormal liver lobation and diaphragmatic hernia. The latter anomaly also occurred in another fetus (No. A042-03) of this group, whereas abnormal liver lobation was observed in four control fetuses (Nos. A004-01, A004-02, A004-07 and A007-13). Two other control fetuses had either tetralogy of Fallot or malpositioned testis (Nos. A012-01 and A013-01, respectively). Although two fetuses were observed with internal hydrocephaly at the mid and high dose groups (A045-10 at 300 mg/Kg/day and A086-07 at 600 mg/Kg/day), both dams originating from different litters and were bred by different males, this malformation was considered to be unrelated to treatment. This malformation is at a low incidence present in the historical control data. Moreover, in the last six prenatal developmental studies in rabbits, this malformation was observed in four studies in which five fetuses were observed with internal hydrocephaly. Based on this recent trend, this malformation was considered to be within the historical context.

The single occurrence and/or group distribution of these malformations does not indicate a test material-relationship and all except two (great vessel transposition and malposition of the left carotid) were observed previously in historical control fetuses and were therefore considered incidental findings. All variations observed were considered unrelated to treatment with the test material as they occurred in the absence of a dose-related trend, infrequently, in control fetuses only and/or at frequencies that were within the range of available historical control data.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
There were no effects on placenta weights (both sexes) observed following treatment up to 300 mg/Kg/day. At 600 mg/Kg/day, placenta weights (both males and females) were lower compared to controls, however without reaching statistical significance.
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 600 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse treatment-related developmental toxicity effects obseved at the highest dose tested.
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

Table 6. Summary of Body Weights (gram): F0 Generation

 

 

Group 1

Control

Group 2

100 mg/Kg

Group 3

300 mg/Kg

Group 4

600 mg/Kg

Post Coitum

Day 0

Mean

3504

3508

3563

3527

St. Dev.

257.6

302.4

319.1

315.8

N

22

21

21

19

 

Day 6

Mean

3483

3530

3522

3418

St. Dev.

242.6

271.6

248.2

243.3

N

22

21

21

19

 

Day 9

Mean

3528

3621

3583

3453

St. Dev.

248.5

275.2

271.1

256.1

N

21

21

21

19

 

Day 12

Mean

3597

3686

3651

3497

St. Dev.

228.0

275.0

261.5

235.3

N

21

21

21

18

 

Day 15

Mean

3696

3781

3726

3566

St. Dev.

223.5

268.4

264.7

258.0

N

21

21

21

18

 

Day 18

Mean

3725

3823

3748

3646

St. Dev.

221.3

283.9

251.0

221.6

N

21

21

21

17

 

Day 21

Mean

3767

3853

3772

3649

St. Dev.

233.7

272.4

252.8

233.7

N

21

21

21

17

 

Day 24

Mean

3817

3878

3824

3695

St. Dev.

231.7

252.2

253.6

256.5

N

21

21

20

17

 

Day 27

Mean

3867

3946

3867

3734

St. Dev.

205.6

245.5

231.2

287.9

N

21

21

20

17

 

Day 29

Mean

3910

3991

3925

3792

St. Dev.

204.4

240.9

237.8

309.0

N

21

21

20

17

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Table 7. Summary of Body Weight Gain (%): F0 Generation

 

 

Group 1

Control

Group 2

100 mg/Kg

Group 3

300 mg/Kg

Group 4

600 mg/Kg

Post Coitum

Day 6

Mean

0

0

0

0

St. Dev.

0.0

0.0

0.0

0.0

N

22

21

21

19

 

Day 9

Mean

2

3

2

1

St. Dev.

1.0

1.5

2.1

2.5

N

21

21

21

19

 

Day 12

Mean

4

4

4

2

St. Dev.

1.4

1.9

2.4

2.6

N

21

21

21

18

 

Day 15

Mean

7

7

6

4

St. Dev.

2.1

2.4

3.6

4.2

N

21

21

21

18

 

Day 18

Mean

8

8

7

6

St. Dev.

2.5

2.8

3.9

4.7

N

21

21

21

17

 

Day 21

Mean

9

9

7

6

St. Dev.

2.5

2.7

4.9

5.3

N

21

21

21

17

 

Day 24

Mean

10

10

9

8

St. Dev.

3.8

3.1

4.2

6.1

N

21

21

20

17

 

Day 27

Mean

12

12

10

9

St. Dev.

4.2

3.5

5.1

8.4

N

21

21

20

17

 

Day 29

Mean

13

13

12

11

St. Dev.

5.4

4.2

5.4

9.2

N

21

21

20

17

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Table 8. Summary of Food Consumption (g/animal/day): F0 Generation

 

 

Group 1

Control

Group 2

100 mg/Kg

Group 3

300 mg/Kg

Group 4

600 mg/Kg

Post Coitum

Day 6-9

Mean

149

158

135

99**

St. Dev.

40.9

39.2

40.3

44.5

N

22

21

21

19

 

Day 9-12

Mean

148

149

136

103**

St. Dev.

23.3

26.1

34.8

40.7

N

21

21

21

19

 

Day 12-15

Mean

121

124

95

77**

St. Dev.

30.1

32.7

48.3

36.9

N

21

21

21

18

 

Day 15-18

Mean

146

143

109**

98**

St. Dev.

22.3

31.9

44.2

52.0

N

21

21

21

18

 

Day 18-21

Mean

150

151

124

113*

St. Dev.

29.0

31.7

43.9

47.5

N

21

21

21

17

 

Day 21-24

Mean

132

115

108

99**

St. Dev.

27.8

29.3

38.4

33.3

N

21

21

21

17

 

Day 24-27

Mean

111

114

98

95

St. Dev.

35.8

36.3

29.7

43.6

N

21

21

20

17

 

Day 27-29

Mean

109

110

109

115

St. Dev.

49.7

32.7

28.5

42.5

N

21

21

20

17

 

Mean of Means

 

133

133

114

100

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Table 9. Summary of Relative Food Consumption (g/Kg Body Weight/day): F0 Generation

 

 

Group 1

Control

Group 2

100 mg/Kg

Group 3

300 mg/Kg

Group 4

600 mg/Kg

Post Coitum

Day 6-9

Mean

44

43

37

29**

St. Dev.

6.0

10.1

10.4

12.8

N

21

21

21

19

 

Day 9-12

Mean

41

40

37

31**

St. Dev.

5.3

6.0

8.8

9.2

N

21

21

21

18

 

Day 12-15

Mean

33

33

25*

21**

St. Dev.

7.6

7.6

12.0

10.5

N

21

21

21

18

 

Day 15-18

Mean

39

37

29**

28**

St. Dev.

5.8

7.4

11.5

12.8

N

21

21

21

17

 

Day 18-21

Mean

40

39

33

31*

St. Dev.

6.6

7.7

11.4

12.7

N

21

21

21

17

 

Day 21-24

Mean

35

30

29

27**

St. Dev.

7.3

6.7

7.0

8.7

N

21

21

20

17

 

Day 24-27

Mean

29

29

25

25

St. Dev.

9.4

9.2

7.7

11.1

N

21

21

20

17

 

Day 27-29

Mean

28

28

28

30

St. Dev.

12.6

8.3

7.0

10.7

N

21

21

20

17

 

Mean of Means

 

36

35

31

28

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Table 10. Summary of Haematology Parameters: F0 Generation

 

 

Group 1

Control

Group 2

100 mg/Kg

Group 3

300 mg/Kg

Group 4

600 mg/Kg

End of Treatment

WBC

10E9/L

Mean

5.9

6.1

6.9

6.7

St. Dev.

1.4

1.5

1.7

2.0

N

21

19

19

15

 

Heterophils

10E9/L

Mean

1.2

1.2

1.3

1.5

St. Dev.

0.2

0.3

0.4

0.6

N

21

19

19

15

 

Lymphocytes

10E9/L

Mean

4.4

4.5

5.2

4.8

St. Dev.

1.2

1.3

1.6

1.5

N

21

19

19

15

 

Monocytes

10E9/L

Mean

0.1

0.1

0.1

0.2

St. Dev.

0.1

0.0

0.1

0.1

N

21

19

19

15

 

Eosinophils

10E9/L

Mean

0.0

0.0

0.0

0.1

St. Dev.

0.0

0.0

0.0

0.0

N

21

19

19

15

 

Basophils

10E9/L

Mean

0.1

0.2

0.1

0.2

St. Dev.

0.0

0.1

0.0

0.1

N

21

19

19

15

 

Large Unstained Cells (LUC)

10E9/L

Mean

0.0

0.0

0.0

0.0

St. Dev.

0.0

0.0

0.0

0.0

N

21

19

19

15

 

Red Blood Cells

10E12/L

Mean

6.02

5.97

6.11

6.12

St. Dev.

0.51

0.33

0.51

0.41

N

21

19

19

15

 

Reticulocytes

10E9/L

Mean

65.7

81.4

55.3

64.0

St. Dev.

39.0

65.0

27.6

31.8

N

21

19

19

15

 

RDW

%

Mean

13.8

13.3*

13.3*

13.7

St. Dev.

0.7

0.7

0.5

0.5

N

21

19

19

15

 

Haemoglobin

mmol/L

Mean

7.8

7.8

7.9

7.8

St. Dev.

0.6

0.4

0.5

0.5

N

21

19

19

15

 

Haematocrit

L/L

Mean

0.385

0.382

0.390

0.380

St. Dev.

0.032

0.021

0.027

0.022

N

21

19

19

15

 

MCV

fL

Mean

63.9

63.9

64.0

62.2

St. Dev.

2.4

1.6

2.7

2.5

N

21

19

19

15

 

MCH

fmol

Mean

1.29

1.30

1.29

1.27

St. Dev.

0.05

0.04

0.05

0.06

N

21

19

19

15

 

MCHC

mmol/L

Mean

20.22

20.37

20.24

20.42

St. Dev.

0.43

0.42

0.38

0.50

N

21

19

19

15

 

Platelets

10E9/L

Mean

408

423

377

411

St. Dev.

109

86

128

114

N

21

19

19

15

+/++ Steel-test significant at 5% (+) or 1% (++) level

* / ** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Table 11. Summary of Clinical Chemistry Parameters: F0 Generation

 

 

Group 1

Control

Group 2

100 mg/Kg

Group 3

300 mg/Kg

Group 4

600 mg/Kg

End of Treatment

Alanine aminotransferase

U/L 

Mean

30.9

32.9

24.2

27.9

St. Dev.

12.4

13.7

6.4

10.8

N

21

22

20

17

 

Aspartate aminotransferase U/L 

Mean

44.1

43.5

33.7

53.4

St. Dev.

29.3

33.9

11.5

54.0

N

21

22

20

17

 

Alkaline Phosphatase

U/L

Mean

43

40

43

48

St. Dev.

20

21

24

34

N

21

22

20

17

 

Total Protein

g/L

Mean

42.3

43.1

41.0

42.2

St. Dev.

3.4

4.9

2.6

3.6

N

21

22

20

17

 

Albumin

g/L

Mean

29.6

29.8

28.2

28.6

St. Dev.

2.6

3.7

2.0

2.6

N

21

22

20

17

 

Total bilirubin

µmol/L

Mean

2.5

2.5

2.5

2.5

St. Dev.

0.4

0.4

0.4

0.3

N

21

22

20

17

 

Urea

mmol/L

Mean

7.3

7.1

7.1

7.2

St. Dev.

1.2

0.8

1.5

1.5

N

21

22

20

17

 

Creatinine

µmol/L

Mean

96.5

96.1

100.6

100.6

St. Dev.

9.1

10.8

10.8

11.3

N

21

22

20

17

 

Glucose

mmol/L

Mean

7.08

7.28

7.34

7.23

St. Dev.

0.60

0.56

0.54

0.62

N

21

22

20

17

 

Cholesterol

mmol/L

Mean

0.25

0.37*

0.30

0.37*

St. Dev.

0.11

0.20

0.09

0.10

N

21

22

20

17

 

HDL cholesterol

mmol/L

Mean

0.09

0.14

0.11

0.14

St. Dev.

0.04

0.13

0.03

0.05

N

21

22

20

17

 

LDL cholesterol

mmol/L

Mean

0.10

0.13

0.10

0.14

St. Dev.

0.05

0.09

0.04

0.05

N

21

22

20

17

 

Triglycerides

mmol/L

Mean

0.64

0.68

0.72

0.97**

St. Dev.

0.18

0.19

0.21

0.52

N

21

22

20

17

 

Bile Acids

µmol/L

Mean

7.2

6.4

5.7

5.9

St. Dev.

3.2

5.2

3.8

2.9

N

21

22

20

17

 

Sodium

mmol/L

Mean

140.9

141.5

142.2

141.3

St. Dev.

2.1

1.7

2.1

2.3

N

21

22

20

17

 

Potassium

mmol/L

Mean

5.10

5.19

5.22

5.27

St. Dev.

0.55

0.57

0.41

0.64

N

21

22

20

17

 

Chloride

mmol/L

Mean

106

108*

108*

107

St. Dev.

3

2

2

3

N

21

22

20

17

 

Calcium

mmol/L

Mean

3.14

3.18

3.17

3.16

St. Dev.

0.8

0.14

0.10

0.22

N

21

22

20

17

 

Inorganic Phosphate

mmol/L

Mean

1.49

1.47

1.49

1.46

St. Dev.

0.20

0.15

0.14

0.22

N

21

22

20

17

* / ** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Table12. Summary of Macroscopic Findings: F0 Generation

 

Group 1

Control

Group 2

100 mg/Kg

Group 3

300 mg/Kg

Group 4

600 mg/Kg

Intercurrent Death

 

Animals Examined

1

 

1

2

Animals Affected</