Hydrocarbons, C18-C22, n-alkanes, n-alkenes

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 2015 - February 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP compliant

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

yes

Remarks:

There were no deviations that affected the overall integrity of the study or the interpretation of the study results and conclusions. For details see "Any other information on materials and methods incl. tables" below

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Hydrocarbons, C12-C16, n-alkanes, isolkanes, alkenes

EC Number:

810-258-3

Molecular formula:

not applicable; UVCB

IUPAC Name:

Hydrocarbons, C12-C16, n-alkanes, isolkanes, alkenes

Constituent 2

Reference substance name:

Alcohols, C2-33, manuf. of, by-products from overheads

IUPAC Name:

Alcohols, C2-33, manuf. of, by-products from overheads

Test material form:

liquid

Specific details on test material used for the study:

Identification: Alcohols, C2-33, manuf. of, by-products from, overheads (876065-86-0), HF-1000 Solvent
Batch (Lot) No & Date Received: 1408-163, 26 Sept 2015; 1409-103, 10 Jun 2015; 1409-138 11 Dec 2015
- Expiration date of the lot/batch: 18 Sept 2017 (Batch (Lot) No 1408-163); 06 Feb 2018 (Batch /Lot) No 1409-103); 31 Dec 2018 (Batch (Lot) No 1409-138)
Physical Description: Yellow liquid
Purity: 100%
Storage: Ambient laboratory temperature
Stability: The Sponsor has indicated that the test material is considered to be stable for 1 year based on stability work performed on a similar batch. This data was not provided to the Test Facility.

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

The Sprague Dawley rat was chosen as the animal model for this study as it is an accepted rodent species for preclinical toxicity testing by regulatory agencies.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK Limited, Margate, UK
- Age at study initiation: (P) Males: 7-8 wks, Females: 6-7 wks; (F1) 21 days (immediately after weaning)
- Weight at study initiation: (P) Males: 218-290g; Females: 125-174g; (F1) Males: 69-79g; Females: 65-72g
- Housing: 5 animals per cage per sex in appropriately sized suspended polycarbonate cages with stainless steel grid tops and solid bottoms, after stratification 2-3 animals per cage per sex (unless reduced by mortality). A few days prior to pairing for mating, males were transferred to individual cages with solid bottoms. The females were transferred to these cages for mating. On a convenient day after mating, the males were re-caged with their original cage-mates. Females were transferred to individual solid bottomed cages following a positive mating sign or on completion of the mating period, with the exception of F1 females showing positive mating signs between Days 75 to 80 of the F1 Phase were initially housed in pairs and returned to single housing on Day 7 of gestation. Females with litters were retained in this type of cage until termination. Animals were socially housed (when possible) for psychological/environmental enrichment and were provided with items such as a device for hiding in and an object for chewing, except when interrupted by study procedures/activities.
- Diet: SDS VRF-1 breeder diet ad libitum throughout the study, except during designated procedures.
- Water: ad libitum from the public supply
- Acclimation period: 13 days before the commencement of dosing
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-22
- Humidity (%): 44-69
- Air changes (per hr): ten or greater
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: From: 25 May 2015 To: 04 Feb 2016

Administration / exposure

Route of administration:

oral: feed

Details on exposure:

DIET PREPARATION
- Preparation of control item: The control item, SDS VRF1 Diet Ground, was administered as received and stored at ambient laboratory temperature in the dark. Upon receipt, the control item was dispensed weekly for administration to control animals.
- Preparation of test item: A minimum of 200g of stock pre-mix of the test item was prepared ensuring that the ratio of test item:diet in the stock did not exceed a ratio of 1:2. The premix contained the total amount of test item required to make the required volume of the formulatied diet. The test item was weighed into a suitable glass duran and 50 ml of acetone was then added and manually mixed with the test item until a solution was formed. The acetone solution was then slowly added to the blank VRF-1 Diet, in a paddle mixer, and mixed until visibly dry and no lumps of test item were observed. The highest dose group was prepared by adding the appropriate amount of blank VRF-1 diet to a container. The 200 g of prepared stock was transferred to a container with blank VRF-1 diet. The container was then sealed tightly and the formulation was then mixed for ca 20 minutes using a Winkworth Change drum mixer. Diets at lower concentrations were prepared accordingly as a serial dilution from a higher concentration dose group.

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: 14 nights maximum
- Proof of pregnancy: copulary plug in situ and/or sperm in vaginal lavage referred to as day 0 of gestation
- Where evidence of mating was not observed by the end of the pairing period, the female was separated from the male during the morning following the last night of pairing and treated as if it had mated during that night. Procedures for that female continued as if it had mated on the last night of pairing.
- After successful mating each pregnant female was caged individually in solid bottomed cages, with the exception of F1 females showing positive mating signs between Days 75 to 80 of the F1 Phase were initially housed in pairs and returned to single housing on Day 7 of gestation. Females with litters were retained in this type of cage until termination.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were performed by GC with Flame Ionisation Detection using a validated analytical procedure. The method of analysis used is documented in Charles River Laboratories Edinburgh Ltd analytical Procedure AP.433360.02.

CONCENTRATION AND HOMOGENEITY ANALYSIS:
Duplicate sets of top, middle and bottom samples were taken after preparation from each formulation (including control) prepared for dosing during weeks 1, 5, 10, 15 and 20, and sent to the analytical laboratory for analysis (triplicate top, middle and bottom samples from each sampling timepoint as back-up). All results from analytical samples were within the acceptance criteria of -15% to +5% (individual values within or equal to ±20%) of their theoretical concentrations.
For details see table #2 below in section "Any other information on results incl. tables"

STABILITY ANALYSIS:
Stability analyses performed previously in conjunction with Charles River Study 433360 demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study for 15 days. Stability data have been retained in the study records for 433360. Due to a difference in the type of diet used on parts of this study (VRF-1 ground diet with particles size of 4 microns) when compared to study 433360 (standard VRF-1 particle size ≤ 3 microns), a trial formulation was prepared and analysed under the present study prior to dosing to ensure that formulations were comparable. Analytical results for the trial were considered to be within acceptance criteria and the formulations stable for 8 days.

Duration of treatment / exposure:

F0 animals: from 10 weeks prior to mating, and then throughout mating, gestation and lactation until termination after the (F1) litter had reached Day 21 of lactation
F1 animals: for 10 weeks after weaning, and then throughout mating, gestation and lactation until termination after the (F2) litters reached Day 21 of lactation. Diet was available to F1 animals immediately after litter selections/weaning (designated as Day 1 of dosing of F1 phase).

Frequency of treatment:

formulated diets were administered ad libitum

Details on study schedule:

- F1 parental animals not mated until Day 71 after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 21 days of age.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 500 ppm (nominal)

Remarks:

100 mg/kg bw/day nominal, measured values see table #3 below in section "Any other information on results incl. tables"

Doses / concentrations 2

Dose / conc.:

6 500 ppm (nominal)

Remarks:

500 mg/kg bw/day nominal, measured values see table #3 below in section "Any other information on results incl. tables"

Doses / concentrations 3

Dose / conc.:

13 000 ppm (nominal)

Remarks:

1000 mg/kg bw/day nominal, measured values see table #3 below in section "Any other information on results incl. tables"

No. of animals per sex per dose:

F0: 28, F1: 24

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: dose levels were chosen based on data of a dose range finding reproduction study (Charles River Study No. 497123)

Examinations

Parental animals: Observations and examinations:

MORTALITY/ MORIBUNDITY CHECKS
- Time schedule: twice daily, once at the start and once at the end of each working day
Animals were observed for general health/mortality and moribundity, animals were not removed from their cages, unless necessary for identification or confirmation of possible findings.

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once on each dosing day
Animals were not removed from the cages, unless necessary for identification or confirmation of possible findings.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly, beginning week -1
Animals were removed from the cage for examination.

BODY WEIGHT: Yes
- Time schedule for examinations:
F0 males: weighed once during pretreatment and approx. weekly during dosing period (body weights recorded over an 8 day period during Weeks 5-6 and Weeks 15-16)
F0 females: weighed once during pretreatment, and approximately weekly (body weight recorded over an 8 day period during Weeks 5-6) until pairing for mating during the dosing period. Weights were also recorded on Days 0, 7, 14 and 20 of gestation (where Day 0 was the day of detection of positive mating signs) and on Days 1, 7, 14 and 21 of lactation (where Day 0 was the day of birth of the litter). A weekly regimen was applied to females that did not show a positive mating sign, until parturition or termination. Females that showed a positive mating sign but failed to litter reverted to the weekly weighing regimen following the theoretical Day 24 of gestation.
F1 animals: weighed weekly, starting on a suitable day within one week of weaning of the majority of litters until termination for males or until pairing for mating for females. During gestation and lactation, weighing of F1 females followed the same regimen as described for F0 females.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION: Yes, monitored by visual inspection of the water bottles on a regular basis, not measured by weight. No further details given.

OBSERVATION OF FEMALES WITH LITTERS DURING LACTATION.
- The females were allowed to litter normally. Any observed difficulty or prolongation of parturition was recorded. The day of birth of the litter (day on which first pups were born) was designated Day 0 of lactation. The duration of gestation in days was calculated.
- Deficiencies in maternal care were recorded: inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups, or apparently inadequate lactation or feeding.

Oestrous cyclicity (parental animals):

Over a 2 week period prior to the initiation of mating and throughout the mating period, vaginal lavages were taken early each morning and the stages of oestrus observed were recorded.

Sperm parameters (parental animals):

Parameters examined in F0/F1 male parental generations:
Testis weight, epididymis weight from all males, from the first 10 males per group: sperm count in testes, sperm count in epididymides, enumeration of cauda epididymal sperm reserve, sperm morphology (a detailed qualitative histopathological examination of the testes was made, taking into account the tubular stages of the spermatogenic cycle to identify treatment-related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells into the lumen, any cell- or stage-specificity of testicular findings were noted).

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in F1 / F2 offspring:
The numbers of live and dead pups born in each litter were recorded as soon as possible after completion of parturition on Day 0 of lactation. The live pups were counted and examined for the presence of milk in the stomach and for any externally visible abnormalities daily up to at least Day 21 of lactation. These pups were weighed en masse, sexes separate, on Days 1, 4, 7 and 14 of lactation. On Day 21 of lactation they were weighed individually.

GROSS EXAMINATION OF DEAD PUPS:
Offspring Found Dead or Killed (Prematurely) Before Day 14 of Lactation: these animals were sexed and checked for the presence of milk in the stomach and for the presence of any externally visible abnormalities. All pups were fixed in 10% neutral buffered formalin and necropsied. Pups that were found dead on Day 0 of lactation or before examination of litter on Day 1 of lactation were evaluated for vital status at birth.
Offspring (Pre-Weaning) Found Dead or Killed (Prematurely) on or after Day 14 of Lactation: these animals were necropsied (external examination followed by macroscopic examination of the tissues and organs of the cranial, thoracic and abdominal cavities in situ), samples of any grossly abnormal tissues were preserved in 10% neutral buffered formalin.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: no

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: no

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals once the majority of females reached the end of lactation.
- Maternal animals: All surviving animals at or shortly after weaning (ca. Day 21 of lactation)

GROSS NECROPSY
- Gross necropsy consisted of evaluation of the carcass and musculoskeletal system; all external surfaces and orifices; cranial cavity and external surfaces of the brain; and thoracic, abdominal, and pelvic cavities with their associated organs and tissues. The reproductive tract of all adult females was examined for signs of implantation, the number of implantation sites being recorded.
- Animals with unscheduled deaths: Necropsies were conducted with a view to diagnosis of the cause of the animals’ condition or cause of death. An external examination was followed by inspection of the cranial, thoracic and abdominal contents. Representative samples of abnormal tissues and tissues listed in table #1 (see below in section "Any other information on materials and methods incl. tables") were fixed in neutral buffered 10% formalin or other appropriate fixative.

HISTOPATHOLOGY / ORGAN WEIGHTS
- Organs identified as follows from F0 / F1 adults were weighed: brain, epididymis, adrenal glands, pituitary glands, prostate glands, thyroid glands, kindeys, liver, ovaries, spleen, testis uterus (from all paired organs both were weighed).
- Tissue collection and preservation in 10% neutral buffered formalin (unless otherwise indicated) from F0 / F1 animals (for adult males undergoing sperm evaluation the right cauda epididymis and right testes were used for sperm evaluation): see table #1 below in section "Any other information on materials and methods incl. tables".
Histopathological evaluation was performed on all tissues in control group and high dose group (both generations), and on the liver and kidney in low and mid dose group (both generations).
The examination of the ovaries included quantification of the primordial and growing oocytes, and the confirmation of the presence or absence of the corpora lutea.
A detailed qualitative examination of the testes was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment-related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell- or stage-specificity of testicular findings were noted.


HISTOLOGY
Tissues were embedded in paraffin, sectioned, mounted on glas slides, stained with haematoxylin and eosin were subjected to histological examinations as follows:
- all tissues (see table #1 below in section "Any other information on materials and methods incl. tables") for control group and high dose group animals, all animals with unscheduled deaths
- target tissues (liver and kidneys) for low dose and mid dose group animals
Additionally, a Periodic Acid Schiff and Haematoxylin (PAS-H) stained section was prepared from the left testis.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed ca. at Day 21 of lactation.
- These animals were checked for externally visible abnormalities at the time of killing. Any found to have such an abnormality were necropsied (external examination followed by macroscopic examination of the tissues and organs of the cranial, thoracic and abdominal cavities in situ, samples of any grossly abnormal tissues were preserved in 10% neutral buffered formalin or other appropriate fixative).

GROSS NECROPSY / ORGAN WEIGHTS
- Gross necropsy F1 and F2 Weanlings at Scheduled Termination: from each litter, 2 male and 2 female pups were necropsied (external examination followed by macroscopic examination of the tissues and organs of the cranial, thoracic and abdominal cavities in situ), samples of any grossly abnormal tissues were preserved in 10% neutral buffered formalin or other appropriate fixative. From one of the 2 pups of each sex (where they are available), the weights of the brain, spleen and thymus were recorded (as well as a terminal body weight), and these organs were preserved. Representative samples of any abnormal tissues from any of the pups were also preserved. Where 2 pups of either sex were not available for necropsy, or one pup of each sex were not available for organ weight collection, additional pups of the opposite sex were necropsied/weighed such that 4 animals were necropsied and 2 animals were weighed as far as possible.

HISTOPATHOLOGY
The tissues indicated in Table #1 below in section "Any other information on materials and methods incl. tables" were prepared for microscopic examination and weighed, respectively.

Statistics:

The following tests were applied to determine the statistical significance of observed differences between the control group and groups receiving test item (unless otherwise stated, all statistical tests were two-sided and performed at the 5% significance level using in house software, pairwise comparisons were only performed against the control group):
Body weight and food consumption (for F0 and F1 males throughout the study and F0 and F1 females prior to mating) were analysed for homogeneity of variance using the ‘F-Max’ test. If the group variance appeared homogeneous, a parametric ANOVA was used and pairwise comparisons were made using Fisher’s F-protected LSD method via Student’s t-test i.e. pairwise comparisons were made only if the overall F-test was significant. 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 a Kruskal-Wallis non-parametric ANOVA was used and pairwise comparisons were made using chi squared protection (via z tests, the non-parametric equivalent of Student’s t test). Organ weight data were analysed as above, and by analysis of covariance (ANCOVA) using terminal body weight as the covariate.

Reproductive indices:

Fertility index (male) = number siring a litter / number paired
Fertility index (female) = number pregnant / number paired
Gestation index = number bearing live pups / number pregnant

Offspring viability indices:

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
Viability index = number of pups live on Day 4 of lactation / number live of Day 0
Lactation index = number of pups live on Day 21 of lactation / number live on Day 4
Overall survival index = number of pups live on Day 21 of lacatation / total number born (live and dead)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Mortality:

mortality observed, non-treatment-related

Description (incidence):

F0 generation: 3 deaths
- 1 female animal from control group, killed prematurely on Day 1 of lactation due to clinical signs (piloerection, subdued behaviour, pale eyes and skin, on Day 0 of lactation, some pups in the animals litter were found dead or had poor condition), cause of deteriotating condition was considered to be severe necrosis of the kidney.
- 1 male animal from mid dose group, killed prematurely on Day 99 due to clinical signs (shallow respiration, swelling of ventral abdomen, partially closed eyes), the demise of the animal was malignant lymophoma in mutliple tissues, considered to be an incidental finding, since no similar findings were noted in any other animals.
- 1 male animal in high dose group, killed prematurley due to a damaged right eye. Phthisis bulbi was considered to be the cause of the animal's condition, considered as an incidental finding, unrelated to test substance treatment.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In F0 females in the high dose group mean body weights were lower than control from Day 8, continuing until pairing for mating and throughout gestation and lactation (ca. 5-7%). Reduced weight gain during pre-mating period (Days 1-71, ca. 11%, p<0.01), however no obvious reduction in gain noted over the gestation of lactation periods.
In F0 males slightly lower mean weight in the high dose group (1-4% compared to control) throughout the study, effect was too small to be positively attributed to test substance treatment and therefore considered not to be toxicologically relevant.
Group mean body weights and weight gains of mid and low dose group animals were comparable to controls.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Females: at highest dose mean food consumption 10-12% lower than control from Day 8, continuing until pairing for mating and throughout gestation and lactation. Group mean food consumption comparable to controls at mid and low dose groups.
Males: group mean food consumption generally similar between all groups throughout the study. At highest dose statistically lower consumption values were noted sporadically, however, the differences from control (around 2-3g) reflected pretrial differences between the groups, therefore the changes were not positively attributed to treatment.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

In the kidneys, test item-related findings included: higher incidences and severity of basophilic tubules in males at mid and high dose groups and females at high dose group; higher incidences and severity of hyaline droplets and hyaline casts in males at all dose levels and higher incidences mononuclear cell infiltration in males at high dose group. In females, the incidences of hyaline casts and mononuclear cell infiltration did not follow dose related patterns, and were therefore not positively attributed to treatment.
In the liver, a higher incidence of centrilobular hepatocellular hypertrophy was noted in both sexes at mid and high dose groups.
For details see table #6 below in section "Any other information on results incl. tables"

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

effects observed, non-treatment-related

Description (incidence and severity):

Motility was lower than controls in all treated groups, however, there was no dose-related pattern to the changes, therefore, they were judgded to be not positively attributed to treatment.

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

No effects observed in mating performance, fertility and duration of gestation.
Litter size and pup mortality: at high dose group, mean numbers of implantation sites, total live pups born and number of live pups on Days 1-14 of lactation up to approx. 11% lower than controls; litter size comparable to controls at low and mid dose groups; pup survival was comparable to controls at all dose levels (slight inter-group differences considered to be too small to be attributed to treatment, additionally no obvious patterns or trends in the number of pups born alive and the number of pups born dead on Day 0 of lactation observed. For details see table #7 in section “Any other information on results incl. tables”.

Details on results (P0)

In adult females, reduced body weight gain during the pre-mating period correlated with lower mean food consumption throughout the majority of the study at the highest dose level. In addition, a lower number of implantation sites and lower litter size, with correlating lower litter weight, were also noted in the high dose group animals. It was considered that the litter findings were likely secondary to the maternal toxicity, and since there was no change in male or female pup weight or pup survival, these changes were considered not to be adverse.
Higher kidney weights in males correlated with histopathology findings of increased incidences and severity of hyaline droplets (all dose groups) , basophilic tubules (mid and high dose groups), hyaline casts and mononuclear cell infiltration (high dose group), and there was a higher incidence of basophilic tubules in female kidneys in the high dose group animals. These findings were considered not to be adverse as they are considered adaptive responses to the administration of test materials and/or are species specific and are, therefore, not relevant to man.
In both sexes, higher liver weights at mid and high dose groups correlated with an increased incidence of centrilobular hepatocellular hypertrophy. A slightly increased incidence of prominent lobular architecture was noted macroscopically in males at the highest dose level (7 animals compared with 3 control animals), however, this did not always correlate with the histopathology findings, therefore, the macroscopic finding was not positively attributed to treatment with the test item. In studies where dosing periods are greater than 3 months, higher liver weights with correlating hypertrophy are considered not to be adverse if there was an absence of hepatotoxicity findings such as gross liver enlargement, evidence of hepatocyte necrosis, fibrosis or inflammation, evidence of degradation or necrosis of other liver cell types, and/or carcinogenicity. Since those types of changes were not present in this study, the higher weight and hypertrophy noted on this study were, therefore, considered to be non-adverse.
Higher thyroid weight in males at all dose groups were considered not to be adverse since the findings were mild in nature and there were no histopathology correlates or any effects on reproduction which may indicate hormonal effects.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

13 000 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: adaptive liver and kidney weight changes not considered to be relevant to humans

Remarks on result:

other: mean measured dosages: males: 767 mg/kg/day females: 988 mg/kg/day (prior to mating), 881 mg/kg/day (during gestation) or 2293 mg/kg/day (during lactation)

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:

no effects observed

Mortality:

mortality observed, non-treatment-related

Description (incidence):

2 male animals found dead in control group: 1 on Day 89, at necropsy epididymis was enlarged, there was no histopathological correlate, no other histopathological findings, evaluation of some tissues was not possible due to autolysis, cause of death undetermined. The second animal found dead on Day 54, no histopathological findings, evaluation of some tissues not possible due to autolysis, cause of death undetermined.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Group mean body weights were similar in all treated male groups throughout the study and all treated female groups prior to mating.
In high dose group females, mean body weights were lower than controls throughout gestation (up to approximately 8% on Day 20 of gestation) and there was a lower mean weight gain over Days 0-20 of gestation when compared with controls (approximately 17%).
Group mean body weights during gestation were comparable to controls at mid and low dose group animals. During lactation, female group mean body weights were similar in all groups.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Females: at high dose group, mean food consumption was approx. 5-10% lower than controls from Day 29 until pairing for mating (Day 72) and throughout gestation and lactation.
Males: group mean food consumption comparable to controls throughout the study in all dose levels.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Higher kidney weights in males at mid and high dose groups (up to approximately 15% at high dose group), thyroid weights in males at high dose group (approximately 14-17%) and higher liver weights in both sexes at mid and high dose groups (up to approximately 14% in males and 32% in females) were noted, see table #8 below in section "Any other information on results incl. tables"

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

In the kidneys of males, higher incidences and severity of hyaline droplets at all dose groups, basophilic tubules and mononuclear cell infiltration at mid and high dose groups and hyaline casts at high dose group were noted when compared with controls. In females, incidences of findings noted were generally similar in controls and treated groups; therefore the findings were not positively attributed to treatment.
In the liver, centrilobular hepatocellular hypertrophy was noted in both sexes at mid and high dose groups.
For details see table #9 below.

Details on results:

In adult F1 females, reduced body weight gain during gestation for F1 animals correlated with lower mean food consumption throughout the majority of the study at the high dose group. In addition at the high dose group, a lower number of implantation sites and lower litter size, with correlating lower litter weight, were also noted in F1 females. It was considered that the litter findings were likely secondary to the maternal toxicity, and since there was no change in male or female pup weight or pup survival, these changes were considered not to be adverse. Oestrous cycles, mating performance, fertility, duration of gestation, pup weight, pup survival and maternal care were similar between all groups. There were also no obvious differences in these parameters between generations.
In F1 adults, higher kidney weights in males (mid and high dose group) correlated with histopathology findings of increased incidences and severity of hyaline droplets at all dose groups, basophilic tubules at mid and high dose groups, hyaline casts (at high dose group) and mononuclear cell infiltration (mid and high dose group), and there was a higher incidences of basophilic tubules in female kidneys at the high dose group. These findings were considered not to be adverse as they are considered adaptive responses to the administration of test materials and/or are species specific and are, therefore, not relevant to man.
In F1 adults, higher liver weights in both sexes at mid and high dose group correlated with an increased incidence of centrilobular hepatocellular hypertrophy. In studies where dosing periods are greater than 3 months, higher liver weights with correlating hypertrophy are considered not to be adverse if there was an absence of hepatotoxicity findings such as gross liver enlargement, evidence of hepatocyte necrosis, fibrosis or inflammation, evidence of degradation or necrosis of other liver cell types, and/or carcinogenicity. Since those types of changes were not present in this study, the higher weight and hypertrophy noted on this study were, therefore, considered to be non-adverse.
Higher thyroid weight in F1 males at the high dose group were considered not to be adverse since the findings were mild in nature and there were no histopathology correlates or any effects on reproduction which may indicate hormonal effects.

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

Mating performance, fertility and duration of gestation: no effects observed.
Ovary scoring: in the high dose group, total number of ovarian follicles attributable to the number of single layer follicles approximately 30% lower than control. For details see table #10 below in section "Any other information on results incl. tables"
Mean numbers of implantation sites, total live pups born and number of live pups throughout lactation: at the highest dose level lower than controls (up to approximately 15%).
Litter size was comparable to controls at low and mid dose groups, pup survival comparable to controls at all dose levels. Slight intergroup differences were considered to be too small to be attributed to treatment with the test item, additionally there were no obvious patterns of trends in the number of pups presumed born alive and the number of pups presumed born dead on Day 0 of lactation.
For details see table #11 below in section "Any other information on results incl. tables"

Details on results (P1)

A lower number of ovarian follicles in the high dose females were considered not to be adverse since there were no obvious effects on mating, fertility, pregnancy duration or rearing of litters at any dose levels and no histopathology findings in ovaries of the high dose females. Additionally, the magnitude of these changes were generally minor and within background control ranges on similar study types at the Test Facility.

Effect levels (P1)

Key result

Dose descriptor:

NOAEL

Effect level:

13 000 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: decrease number of ovarian follicles with no histopathology and effect on reproductive function

Remarks on result:

other: mean measured dosages: males: 1124 mg/kg/day females: 1200 mg/kg/day (prior to mating), 912 mg/jkg/day (during gestation) or 2256 mg/kg/day (during lactation)

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

The type and distribution of observations amongst pups did not indicate any association with test substance treatment.

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

At the high dose group, mean numbers of implantation sites, total live pups born and number of live pups on Days 1-14 of lactation were lower than controls (up to approximately 11%). Litter size was comparable to controls at low and mid dose groups and pup survival was comparable to controls at all dose levels. Slight inter-group differences were considered to be too small to be attributed to treatment. In addition, there were no obvious patterns or trends in the number of pups presumed born alive and the number of pups presumed born dead on Day 0 of lactation.
For details see table #12 below in section "Any other information on results incl. tables"

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At the high dose group, lower mean litter weight was noted throughout lactation (up to approximately 12%), when compared with controls. Mean litter weight at low and mid dose groups and mean male and female pup weights at all dose levels, were similar to controls throughout lactation.
For details see table #13 below in section "Any other information on results incl. tables"

Sexual maturation:

effects observed, treatment-related

Description (incidence and severity):

Age and body weight of animals at vaginal opening or preputial separation were slightly higher than controls at all dose levels, indicating a slight delay in sexual maturation.
For details see table #14 below in section "Any other information on results incl. tables"

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No organ weight findings in F1 weanlings which were considered to be related to treatment.

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

ca. 13 000 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: mortality ahd body weight changes

Results: F2 generation

General toxicity (F2)

Clinical signs:

no effects observed

Description (incidence and severity):

The type and distribution of observations amongst pups did not indicate any association with test substance treatment.

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

At the high dose group, mean numbers of implantation sites, total live pups born and number of live pups throughout lactation were lower than controls (up to approximately 15%).
Litter size was comparable to controls at low and mid dose groups and pup survival was comparable to controls at all dose levels. Slight intergroup differences were considered to be too small to be attributed to treatment. In addition, there were no obvious patterns of trends in the number of pups presumed born alive and the number of pups presumed born dead on Day 0 of lactation.
For details see table #15 below in section "Any other information on results incl. tables"

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At the high dose group, lower mean litter weight was noted throughout lactation (up to approximately 16%), when compared with control.
Mean litter weight at low and mid dose groups and mean male and female pup weights at all dose levels, were similar to controls throughout lactation.
For details see table #16 below in section "Any other information on results incl. tables"

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

In males at mid and high dose groups, statistically significantly higher mean absolute and covariate brain weights (up to approximately 6% at the high dose group) were observed when compared to controls.
For details see table #17 below in section "Any other information on results incl. tables"

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Details on results (F2)

In F2 weanlings, higher brain weight in males were considered not to be toxicologically significant since the changes were minor (approximately 6% higher than controls) and there were no histopathology correlates.

Effect levels (F2)

Key result

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

ca. 13 000 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: slight brain weight changes in absence of histopathology

Remarks on result:

other:

Remarks:

not considered toxicologically significant

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

Table #2: Summary of formulation analysis results
Timepoint	Dose group	Theoretical concentration (ppm)	mean found concentration (ppm)	RSD	% Difference from theoretical concentration
Week 1	control	0	0	-	-
	low dose	1500	1323	1.0	-11.8
	mid dose	6500	5575	5.7	-14.2
	high dose	13000	11145	5.8	-14.3
Week 5	control	0	0	-	-
	low dose	1500	1342	1.9	-10.5
	mid dose	6500	6321	3.5	-2.8
	high dose	13000	12002	2.7	-7.7
Week 10	control	0	0	-	-
	low dose	1500	1374	5.0	-8.4
	mid dose	6500	5670	2.9	-12.4
	high dose	13000	12241	9.3	-5.8
Week 15	control	0	0	-	-
	low dose	1500	1315	3.9	-12.3
	mid dose	6500	5899	1.2	-9.4
	high dose	13000	11696	3.3	-10.0
Week 20	control	0	0	-	-
	low dose	1500	1440	3.7	-4.0
	mid dose	6500	5586	0.7	-14.1
	high dose	13000	1191	2.2	-7.8
Acceptance criteria				≤10%	-15 to +5%

Table #3: Summary of achieved dose levels
Group / sex		low dose / M	mid dose / M	high dose / M		low dose / F	mid dose / F	high dose / F
Treatment level (ppm)		1500	6500	13000		1500	6500	13000
Target dose level (mg/kg/d)		100	500	1000		100	500	1000
Actual group mean dose level (mg/kg/d)	F0	90	385	767	Prior to mating	122	510	988
					Gestation	102	443	881
					Lactation*	279	1173	2293
	F1	128	551	1124	Prior to mating	141	640	1200
					Gestation	106	440	912
					Lactation*	276	1113	2256
*Results during lactation should be treated with caution as consumption is known to be variable during this time and there is a large amount of scatter and/or diet eaten by pups.

Table #4: Summary of test item-related organ weight findings - F0 adults
	Males				Females
Group	control	low dose	mid dose	high dose	control	low dose	mid dose	high dose
Dose (ppm)	0	1500	6500	13000	0	1500	6500	13000
No. animals per group	28	28	27	27	27	28	28	28
Kidney
Absolute value (g)	3.69	4.00*	4.11***	4.29***	2.36	2.39	2.43	2.40
Absolute (covariance analysis)	3.70	3.89*	4.09***	4.41***	2.35	2.37	2.43	2.42
% of body weight	0.655	0.687*	0.722***	0.784***	0.740	0.745	0.768	0.766
Liver
Absolute value (g)	19.50	20.65	20.86*	22.23***	15.65	16.42	18.01***	20.36***
Absolute (covariance analysis)	19.59	20.00	20.74**	22.93***	15.62	16.24	18.02***	20.57***
% of body weight	3458	3.578	3.665**	4.054***	4.903	5.110	5.686***	6.489***
Thyroid gland
Absolute value (g)	0.0233	0.0266*	0.265*	0.0271**	0.0188	0.0212	0.0190	0.0201
Absolute (covariance analysis)	0.0246	0.266*	0.265*	0.0271**	0.0188	0.0211	0.190	0.0201
% of body weight	0.00415	0.00463*	0.00471*	0.00492**	0.00595	0.00666	0.00600	0.00644
Terminal body weight used as covariance
Significantly different from control group value: * = p<0.05, ** = p<0.01, *** = p<0.001

Table #5: Summary test item-related necropsy findings - F0 adults
	Males				Females
Group	Control	Low dose	Mid dose	High dose	Control	Low dose	Mid dose	High dose
Dose (ppm)	0	1500	6500	13000	0	1500	6500	13000
No. Animals examined	28	28	27	27	27	28	28	28
Liver prominent lobular architecture	3	4	4	7	0	1	0	0

Table #6: Summary of test item related histopathology findings - F0 adults
	Males				Females
Group	control	low dose	mid dose	high dose	control	low dose	mid dose	high dose
Dose (ppm)	0	1500	6500	13000	0	1500	6500	13000
No. of animals examined	28	28	27	27	27	28	28	28
Kidney (No. examined)	(28)	(28)	(27)	(27)	(27)	(28)	(27)	(28)
Hyaline droplets, tubular	5	19	27	27	0	0	0	0
Minimal	5	17	6	3	0	0	0	0
Mild	0	2	21	20	0	0	0	0
Moderate	0	0	0	4	0	0	0	0
Hyaline casts	5	7	6	18	1	4	1	4
Minimal	5	7	6	14	1	4	1	4
Mild	0	0	0	4	0	0	0	0
Basophilic tubules, focal	17	17	20	24	3	4	6	10
Minimal	17	17	18	6	3	4	6	7
Mild	0	0	2	15	0	0	0	3
Moderate	0	0	0	3	0	0	0	0
Mononuclear cell infiltration	14	13	15	20	4	5	6	6
Minimal	14	13	15	17	4	4	6	6
Mild	0	0	0	2	0	1	0	0
Liver (No. examined)	(28)	(28)	(27)	(27)	(27)	(28)	(28)	(28)
Centrilobular hypertrophy, hepatocellular	0	0	3	12	0	0	2	13
Minimal	0	0	3	10	0	0	2	10
Mild	0	0	0	2	0	0	0	3

Table #7: Duration of gestation and overall litter performance: F0 generation, F1 production
Group			Control	Low dose	Mid dose	High dose
Dose (ppm)			0	1500	6500	13000
Number pregnant			26	27	27	28
Duration of gestation (days):
21			8	6	9	9
22			18	21	18	17
Mean duration			21.7	21.8	21.7	21.7**
Number females producing a live litter			26	27	27	28
Gestation index (%)			100	100	100	100
Mean number of implant sites* per pregnancy ± SD			15.4 ± 1.9	15.4 ± 2.8	15.5 ± 2.8	13.8 ± 2.0
Mean total number of pups* born per litter			14.5 ± 2.6	14.3 ± 2.6	14.3 ± 2.6	12.9 ± 1.8
Mean number of live pups* per litter ± SD:
Day 0 of lactation			14.2 ± 2.6	14.2 ± 2.6	14.3 ± 2.6	12.8 ± 1.8
Day 1 of lactation			14.0 ± 2.6	14.1 ± 2.6	14.3 ± 2.6	12.6 ± 1.8
Day 4 of lactation			13.1 ± 3.7	14.0 ± 2.8	14.1 ± 2.4	12.4 ± 2.1
Day 7 of lactation			13.0 ± 3.7	13.9 ± 2.8	13.9 ± 2.3	12.3 ± 2.2
Day 14 of lactation			12.2 ± 3.7	13.0 ± 3.0	12.7 ± 3.2	11.4 ± 2.5
Day 21 of lactation			11.7 ± 3.7	12.5 ± 3.4	12.1 ± 3.5	11.3 ± 2.9
Total number of males* on Day 1 of lactation (%)			188 (54)	185 (49)	173 (45)	174 (49)
Total number of females* on Day 1 of lactation (%)			161 (46)	196 (51)	213 (55)	180 (51)
*= animals rearing young to Day 21 of lactation only
** = excludes two animals where gestation lenght not calculable due to no positive mating sign being noted

Table #8: Summary of test item-related organ weight findings - F1 adults
	Males				Females
Group	control	low dose	mid dose	high dose	control	low dose	mid dose	high dose
Dose (ppm)	0	1500	6500	13000	0	1500	6500	13000
No. animals per group	22	24	24	24	24	24	24	24
Kidney
Absolute value (g)	3.80	3.92	4.31***	4.35***	2.45	2.54	2.57	2.53
Absolute (covariance analysis)	3.79	3.89	4.26***	4.44***	2.45	2.52	2.56	2.56
% of body weight	0.687	0.706	0.770***	0.812***	0.752	0.770	0.787	0.787
Liver
Absolute value (g)	19.44	20.13	21.70**	21.30*	15.46	16.60	18.02***	20.17***
Absolute (covariance analysis)	19.37	19.94	21.39***	21.87***	15.49	16.41	17.91***	20.43***
% of body weight	3.508	3.614	3.865***	3.979***	4.754	5.022	5.497***	6.268***
Thyroid gland
Absolute value (g)	0.0219	0.0234	0.0231	0.0248	0.0202	0.0234	0.0205	0.0206
Absolute (covariance analysis)	0.0219	0.0233	0.229	0.0251**	0.0202	0.0233	0.0205	0.0206
% of body weight	0.00397	0.00422	0.00415	0.00465***	0.00620	0.00712	0.00629	0.0042
Terminal body weight used as covariance
Significantly different from control group value: * = p<0.05, ** = p<0.01, *** = p<0.001

Table #9: Summary of test item related histopathology findings - F1 adults
	Males				Females
Group	control	low dose	mid dose	high dose	control	low dose	mid dose	high dose
Dose (ppm)	0	1500	6500	13000	0	1500	6500	13000
No. of animals examined	22	24	24	24	24	24	24	24
Kidney (No. examined)	(22)	(24)	(24)	(24)	(24)	(24)	(24)	(24)
Hyaline droplets, tubular	2	13	24	23	0	0	0	0
Minimal	2	13	5	23	0	0	0	0
Mild	0	0	19	17	0	0	0	0
Moderate	0	0	0	4	0	0	0	0
Hyaline casts	5	5	5	18	2	3	0	4
Minimal	5	5	5	15	2	3	0	4
Mild	0	0	0	3	0	0	0	0
Basophilic tubules, focal	15	16	21	21	3	4	4	3
Minimal	14	16	16	13	3	4	4	3
Mild	1	0	5	6	0	0	0	0
Moderate	0	0	0	2	0	0	0	0
Mononuclear cell infiltration	9	10	18	19	5	5	5	5
Minimal	9	10	17	15	5	5	5	5
Mild	0	0	1	4	0	0	0	0
Liver (No. examined)	(22)	(24)	(24)	(24)	(24)	(24)	(24)	(24)
Centrilobular hypertrophy, hepatocellular	0	0	4	8	0	0	2	9
Minimal	0	0	4	6	0	0	2	8
Mild	0	0	0	2	0	0	0	1

Table #10: Ovary scoring: mean values ± standard deviation: F1 generation
Follicles	Group/dose levels (ppm)
	control	high dose
	(0)	(13000)
Single layer follicles	22 ± 13	14 ± 9
Multiple layer follicles	3 ± 2	2 ± 2
Antral follicles	2 ± 2	2 ± 2
Total follicles counted	16 ± 15	18 ± 11

Table #11: Duration of gestation and overall litter performance: F1 generation, F2 production
Group			Control	Low dose	Mid dose	High dose
Dose (ppm)			0	1500	6500	13000
Number pregnant			22	23	23	23
Duration of gestation (days):
21			2	3	3	3
22			16	13	19	18
23			4	7	1	2
Mean duration			22.1	22.2	21.9	22.0
Number females producing a live litter			22	23	22	23
Gestation index (%)			100	100	96	100
Mean number of implant sites* per pregnancy ± SD			16.6 ± 2.3	16.3 ± 3.1	16.6 ± 2.4	14.7 ± 2.5
Mean total number of pups* born per litter			15.5 ± 2.3	15.5 ± 3.1	14.7 ± 3.4	13.2 ± 2.5
Mean number of live pups* per litter ± SD:
Day 0 of lactation			15.2 ± 1.9	15.4 ± 3.1	14.6 ± 3.4	13.2 ± 2.5
Day 1 of lactation			15.0 ± 2.0	14.8 ± 3.4	14.5 ± 3.4	12.9 ± 2.6
Day 4 of lactation			14.8 ± 1.9	14.2 ± 3.3	14.3 ± 3.4	12.7 ± 2.6
Day 7 of lactation			14.7 ± 1.8	14.0 ± 3.5	14.3 ± 3.4	12.7 ± 2.5
Day 14 of lactation			14.7 ± 1.8	14.0 ± 3.5	14.2 ± 3.4	12.6 ± 2.5
Day 21 of lactation			14.7 ± 1.8	14.0 ± 3.5	14.2 ± 3.4	12.6 ± 2.5
Total number of males* on Day 1 of lactation (%)			142 (45)	177 (52)	159 (50)	147 (52)
Total number of females* on Day 1 of lactation (%)			174 (55)	164 (48)	160 (50)	136 (48)
* = animals rearing young to Day 21 of lactation only

Table #12: Survival indices: F0 generation, F1 production
Group		control	low dose	mid dose	high dose
Dose (ppm)		0	1500	6500	13000
Birth index	Mean litter index (%)	94	94*	93	93
	Number losing >2 pups	3	2	6	4
	Number of litters	26	25*	27	28
Live birth index	Mean litter index (%)	98	100	100	100
	Number losing >1 pup	1	0	0	0
	Number of litters	26	27	27	28
Viability index	Mean litter index (%)	93	98	99	97
Days 0 - 4	Number losing >3 pups	1	0	1	1
	Number of litters	26	27	27	28
Lactation index	Mean litter index (%)	91	89	86	92
Days 4 - 21	Number losing >1 pup	5	8	10	6
	Number of litters	25	27	27	28
Overall survival index	Mean litter index (%)	84	88	85	88
Birth - 21	Number losing >4 pups	6	3	5	3
	Number of litters	26	27	27	28
* = Litters where birth index not calculable excluded

Table #13: Group mean litter and pup weight: F0 generation, F1 production
Day of lactation	Group/dose level (ppm)
	control	low dose	mid dose	high dose
	0	1500	6500	13000
Litter
Day 1	93 ± 19	95 ± 15	98 ± 13	87 ± 12
Day 4	136 ± 21	138 ± 20	140 ± 18	123 ± 22
Day 7	200 ± 27	205 ± 30	201 ± 27	176 ± 31
Day 14	361 ± 48	376 ± 51	357 ± 64	322 ± 61
Day 21*	587 ± 91	608 ± 77	579 ± 102	530 ± 75
Mean of litter mean pup weight
Males
Day 1	7.0 ± 0.9	7.0 ± 0.7	7.2 ± 0.7	7.1 0.6**
Day 4	10.4 ± 1.5	10.4 ± 1.3	10.4 ± 1.4	10.2 1.3
Day 7	15.3 ± 2.2	15.5 ± 1.8	15.1 ± 2.0	14.8 2.0
Day 14	29.5 ± 4.5	30.4 ± 4.5	29.8 ± 3.9	28.9 3.5
Day 21	50.6 ± 9.5	49.2 ± 8.7	49.0 ± 8.5	47.1 6.4
Females
Day 1	6.4 ± 1.2	6.6 ± 0.7	6.7 ± 0.7	6.7 ± 0.7**
Day 4	9.8 ± 1.4	9.8 ± 1.2	9.8 ± 1.3	9.7 ± 1.3
Day 7	14.7 ± 2.1	14.6 ± 1.9	14.4 ± 1.8	14.1 ± 2.0
Day 14	29.0 ± 4.6	29.5 ± 4.8	28.2 ± 3.8	28.1 3.6
Day 21	47.5 ± 7.7	47.7 ± 8.7	46.6 ± 7.4	45.2 5.9
Means exclude litters where all pups died.
* = excludes litters where not all pups weighed on Day 21 of lactation in error
** = excludes litter where pups mis-sexed on Day 1 of lactation

Table #14: Preputial separations and vaginal openings: F1 generation
	Group/dose level (ppm)
	control	low dose	mid dose	high dose
	0	1500	6500	13000
Females
Age (days) at vaginal openings	33.0 ± 1.7	34.1 ± 1.7	34.8 ± 1.2	35.8 ± 2.4
Weight (g) at vaginal opening	108 ± 12	117 ± 14	116 ± 12	119 ± 16
Males
Age (days) at preputial separation	40.7 ± 1.3	41.0 ± 1.7	41.4 ± 1.5	42.4 ± 2.3
Weight (g) at preputial separation	192 ± 21	194 ± 20	197 ± 19	201 ± 18

Table #15: Survival indices: F1 generation, F2 production
Group		control	low dose	mid dose	high dose
Dose (ppm)		0	1500	6500	13000
Birth index	Mean litter index (%)	94	95	88	90
	Number losing >2 pups	2	2	3	2
	Number of litters	22	23	22	23
Live birth index	Mean litter index (%)	99	99	99	100
	Number losing >1 pup	1	0	1	0
	Number of litters	22	23	23	23
Viability index	Mean litter index (%)	93	92	94	92
Days 0 - 4	Number losing >3 pups	2	2	2	1
	Number of litters	22	23	23	23
Lactation index	Mean litter index (%)	95	98	95	99
Days 4 - 21	Number losing >1 pup	1	1	0	0
	Number of litters	22	23	23	22
Overall survival index	Mean litter index (%)	91	91	93	91
Birth - 21	Number losing >4 pups	3	2	1	1
	Number of litters	22	23	23	23

Table #16: Group mean litter and pup weight: F1 generation, F2 production
Day of lactation	Group/dose level (ppm)
	control	low dose	mid dose	high dose
	0	1500	6500	13000
Litter
Day 1*	103 ± 12	100 ± 25	99 ± 23	90 ± 17
Day 4	150 ± 25	145 ± 37	147 ± 32	133 ± 24
Day 7	221 ± 34	212 ± 51	214 ± 43	194 ± 32
Day 14	415 ± 46	398 ± 78	388 ± 65	347 ± 49
Day 21	661 ± 74	652 ± 127	632 ± 108	575 ± 80
Mean of litter mean pup weight
Males
Day 1*	7.1 ± 0.7	7.0 ± 0.8	7.1 ± 0.7	7.3 ± 0.9
Day 4	10.4 ± 1.6	14.4 ± 1.1	10.8 ± 1.4	11.0 ± 1.5
Day 7	15.5 ± 2.2	15.6 ± 1.6	15.9 ± 2.4	16.0 ± 2.0
Day 14	29.1 ± 2.8	29.7 ± 4.3	29.0 ± 5.3	28.8 ± 4.0
Day 21	46.6 ± 5.3	48.9 ± 7.8	47.4 ± 7.7	48.2 ± 8.3
Females
Day 1	6.7 ± 0.6	6.6 ± 0.7	6.7 ± 0.9*	6.9 ± 0.8
Day 4	10.0 ± 1.3	10.0 ± 1.0	10.2 ± 1.5	10.3 ± 1.5
Day 7	14.8 ± 1.9	14.9 ± 1.4	14.9 ± 2.3	15.1 ± 2.1
Day 14	27.9 ± 2.9	28.8 ± 4.5	27.7 ± 4.7	27.4 ± 4.0
Day 21	44.6 ± 5.3	47.1 ± 6.8	44.7 ± 7.7	45.6 ± 7.6
Means exclude litters where all pups died.
* = excludes litters that were miscounted and/or mis-sexed on Day 1 of lactation

Table #17: Summary of test item-related organ weight findings - F2 Weanlings
	Males				Females
Group	control	low dose	mid dose	high dose	control	low dose	mid dose	high dose
Dose (ppm)	0	1500	6500	13000	0	1500	6500	13000
Brain
Absolute value (g)	1.51	1.52	1.57*	1.61***	1.46	1.48	1.51	1.51
Absolute (covariance analysis)	1.51	1.52	1.56*	1.60***	1.47	1.48	1.51	1.50
% of body weight	2.853	2.854	2.807	2.837	2.884	2.925	2.938	2.936
Terminal body weight used as covariance
Significantly different from control group value: * = p<0.05, ** = p<0.01, *** = p<0.001

Applicant's summary and conclusion

Conclusions:

In conclusion, the dietary administration of Alcohols, C2-33, manuf. Of, by-products from, overheads to rats was well tolerated, and the parental and reproductive No-Observed-Adverse-Effect-Levels (NOAELs) were considered to be 13000 p.p.m (mean achieved dosages of: 767 mg/kg/day in F0 males; 988, 881 or 2293 mg/kg/day in F0 females prior to mating, during gestation and during lactation, respectively; 1124 mg/kg/day in F1 males, and 1200, 912 or 2256 mg/kg/day in F1 females prior to mating, during gestation and during lactation, respectively). A No-Observed-Effect-Level (NOEL) could not be established due to the presence of non-adverse findings in the kidneys of males at all dose levels.

Executive summary:

The study was designed to provide general information concerning the effects of the chemical Alcohols, C2-33, manuf. of, by-products from, overheads (876065-86-0), on reproductive performance when given orally to rats.

F0 animals were randomised into three test groups and one control group. These animals were dosed for 10 weeks prior to mating, and then throughout mating, gestation and lactation until termination after their litters (F1) reached Day 21 of lactation.

From each group, 24 males and 24 females were retained for post weaning assessments. These F1 animals continued dosing for 10 weeks after weaning, and then throughout mating, gestation and lactation until termination after their litters (F2) reached Day 21 of lactation.

The study design was as follows:

Group	Dose level (mg/kg/day)	Dose level (ppm)	Number of F0 animals		Number of F1 animals
			Males	Females	Males	Females
control*	0	0	28	28	24	24
low dose	100	1500	28	28	24	24
mid dose	500	6500	28	28	24	24
high dose	1000	13000	28	28	24	24

* The control diet was untreated SDS VRF-1 ground diet.

Animals from both generations were monitored for clinical signs of toxicity and for effects on body weight, food consumption, oestrous cycle, mating performance, pregnancy performance, difficulty or prolongation of parturition and for deficiencies in maternal care. The offspring were monitored for survival and growth up to weaning. In addition, the following endpoints were evaluated in both generations: gross necropsy findings, organ weights, histopathological evaluation, qualitative examination of the testes and examination of the ovaries and sperm.

There were 5 unscheduled deaths during the course of the study, 3 adult F0 generation animals and 2 adult F1 generation animals. All deaths were considered to be incidental and unrelated to treatment with the test item.

There were no clinical observations in either generation which were considered to be related to treatment with the test item.

At 13000 ppm, reduced mean weight gain was noted in F0 adult females during the premating period (approximate 10%) and in F1 adult females during gestation (approximately 17%), when compared with controls. There were no effects on body weight in males of either generation at any dose level or in females of either generation at 1500 or 6500 ppm.

At 13000 ppm, lower mean food consumption was noted in adult F0 and F1 females throughout the majority of pre-mating, gestation and lactation period (up to approximately 12% or 10% in the F0 and F1 generations, respectively). There were no effects on food consumption in males of either generation at any dose level or in females of either generation at 1500 or 6500 ppm.

Oestrous cycles, mating performance, fertility, duration of gestation and maternal care were similar between all groups in both generations. There were also no obvious differences in these parameters between generations.

At 13000 ppm, lower numbers of implantation sites and lower litter size with correlating lower litter weight, were noted in both generations, when compared with Controls (approximately 11-12% or 15-16% for all parameters in the F1 and F2 litters, respectively). It was considered that these findings were likely secondary to the maternal body weight and food consumption changes. Mean number of implantations sites, litter size and litter weight were similar to controls of both generations at 1500 or 6500 ppm.

Mean pup weights (male and female) and mean pup survival were similar between all groups in both generations. There were also no obvious differences in these parameters between generations.

In F1 adults, a slight delay in sexual maturation, as indicated by slightly higher ages and weights at vaginal opening or preutial separation, was noted at all dose levels.

In F0 generation adults, pathology findings included: higher kidney weights in males at ≥1500 ppm, which correlated with histopathology findings of increased incidences and severity of hyaline droplets (≥1500 ppm), basophilic tubules (≥6500 ppm) and hyaline casts and mononuclear cell infiltration (males at 13000 ppm); higher incidences of basophilic tubules in female kidneys at 13000 ppm; higher liver weights in both sexes at ≥ 6500 ppm, which correlated with a slightly increased incidence of macroscopic prominent lobular architecture at 13000 ppm and centrilobular hepatocellular hypertrophy at ≥ 6500 ppm; and higher thyroid weights in males at ≥ 1500 ppm (no macroscopic or histopathology correlates).

In F1 generation adults, pathology findings included higher kidney weights in males at ≥ 6500 ppm, which correlated with histopathology findings of increased incidences and severity of hyaline droplets (≥1500 ppm) , basophilic tubules and mononuclear cell infiltration (≥6500 ppm) and hyaline casts (13000 ppm); and higher liver weights with correlating centrilobular hepatocellular hypertrophy in both sexes at ≥ 6500 ppm. In F0 females at 13000 ppm, the number and type of ovarian follicles was similar to controls. In F1 females at 13000 ppm, the total number ovarian follicles, attributed to the number of single layer follicles, was approximately 30% lower than controls.

There were no changes in the number of sperm or spermatids, sperm morphology or sperm motility in males of either generation which were considered to be related to treatment with the test item.

There were no toxicologically relevant organ weight, gross necropsy or histopathology findings in the weanlings of either generation (F1 pups or F2 pups) which were considered to be related to treatment with the test item.

None of the aforementioned changes were considered to be adverse, but were considered to be either adaptive or not relevant to man..

In conclusion, the dietary administration of Alcohols, C2-33, manuf. Of, by-products from, overheads (876065-86-0) to rats was well tolerated, and the parental and reproductive No- Observed-Adverse-Effect-Levels (NOAELs) were considered to be 13000 ppm (mean achieved dosages of: 767 mg/kg/day in F0 males and 988, 881 and 2293 mg/kg/day in F0 females prior to mating, during gestation and during lactation, respectively; and 1124 mg/kg/day in F1 males and 1200, 912 and 2256 mg/kg/day in F1 females prior to mating, during gestation and during lactation, respectively). A No-Observed-Effect-Level (NOEL) could not be established due to the presence of non-adverse findings in the kidneys of males at all dose levels.