Registration Dossier
Registration Dossier
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EC number: 232-304-6 | CAS number: 8002-26-4 A complex combination of tall oil rosin and fatty acids derived from acidulation of crude tall oil soap and including that which is further refined. Contains at least 10% rosin.
Endpoint summary
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
- 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. - 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
- 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
- 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. - 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). - 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
- 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
- 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
- By mistake, Female No. 142 (Group 2) was diagnosed with Total litter Loss; at necropsy a living pup was found
- 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).
Referenceopen allclose all
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 |
| 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 |
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 (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 |
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|
| Reporting Biochemistry |
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| ||
ALT | AST | ALP | TPROT | ALB | BILEAC | TBIL | UREA | CREAT | GLUC | CHOL | HDL | ||
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| ||
(U/L) | (U/L) | (U/L) | (g/L) | (g/L) | (umol/L) | (umol/L) | (mmol/L) | (umol/L) | (mmol/L) | (mmol/L) | (mmol/L) | ||
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| ||
[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 | ||
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|
| ||
(mmol/L) | (mmol/L) | (mmol/L) | (mmol/L) | (mmol/L) | (mmol/L) | (mmol/L) | ||
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| ||
[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 |
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|
| Reporting Biochemistry |
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| ||
ALT | AST | ALP | TPROT | ALB | BILEAC | TBIL | UREA | CREAT | GLUC | CHOL | HDL | ||
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| ||
(U/L) | (U/L) | (U/L) | (g/L) | (g/L) | (umol/L) | (umol/L) | (mmol/L) | (umol/L) | (mmol/L) | (mmol/L) | (mmol/L) | ||
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| ||
[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 |
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|
|
LIVER |
|
|
|
|
|
|
Absolute | 2 | 11** | 13** | -5 | -5 | 6 |
Relative to body weight | 2 | 5** | 15** | -5 | -3 | 9* |
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|
|
THYROID GLANDS |
|
|
|
|
|
|
Absolute | -4 | 11 | 12 | 1 | 5 | 17* |
Relative to body weight | -5 | 5 | 14** | 0 | 7 | 21** |
|
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|
|
|
|
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 |
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 |
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 (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 |
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|
| Reporting Hematology |
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| ||
WBC | NEUT | LYMPH | MONO | EOS | BASO | LUC | RBC | RETIC | RDWG | HGB | HCT | ||
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|
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| ||
(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) | ||
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[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 |
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|
|
|
|
| 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) | ||
|
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|
|
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|
| ||
[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 | ||
|
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| ||
(fL) | (pg) | (g/L) | (10^9/L) | ||
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|
| ||
[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 |
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| Reporting Biochemistry |
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| ||
ALT | AST | ALP | TPROT | ALB | BILEAC | TBIL | UREA | CREAT | GLUC | CHOL | HDL | ||
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| ||
(U/L) | (U/L) | (U/L) | (g/L) | (g/L) | (umol/L) | (umol/L) | (mmol/L) | (umol/L) | (mmol/L) | (mmol/L) | (mmol/L) | ||
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| ||
[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 |
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|
| Reporting Biochemistry |
|
| Reporting Special Chemistry | ||||
LDL | TRIG | NA | K | CL | CA | PHOS | T3 | T4 | TSH | ||
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| ||
(mmol/L) | (mmol/L) | (mmol/L) | (mmol/L) | (mmol/L) | (mmol/L) | (mmol/L) | (ng/mL) | (ng/mL) | (mU/L) | ||
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| ||
[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 |
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|
| Reporting Biochemistry |
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| ||
ALT | AST | ALP | TPROT | ALB | BILEAC | TBIL | UREA | CREAT | GLUC | CHOL | HDL | ||
|
|
|
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|
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|
| ||
(U/L) | (U/L) | (U/L) | (g/L) | (g/L) | (umol/L) | (umol/L) | (mmol/L) | (umol/L) | (mmol/L) | (mmol/L) | (mmol/L) | ||
|
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|
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| ||
[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 | ||
|
|
|
|
|
|
|
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|
| ||
(mmol/L) | (mmol/L) | (mmol/L) | (mmol/L) | (mmol/L) | (mmol/L) | (mmol/L) | (ng/mL) | (ng/mL) | (mU/L) | ||
|
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| ||
[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 |
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
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
- 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
Referenceopen allclose all
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 |
1 |
|
1 |
2 |
|
||||
Heart |
|
|
|
|
Grown together with: |
||||