Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

2-Generation Rat Diet: Not a reproductive toxin. OECD 416; Reliability = 1

Link to relevant study records
Reference
Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
yes
Remarks:
Characterization of the test substance was conducted in accordance with the U.S. EPA GLP regulations, which are compatible with current OECD and Japanese MAFF GLP
GLP compliance:
yes
Specific details on test material used for the study:
99.3% purity
Species:
rat
Strain:
other: Crl:CD(SD)
Details on species / strain selection:
The Crl:CD(SD) rat was selected as the test system because of its known ability to respond to toxic effects on reproductive capacity and its history of use as a rodent species in these evaluations.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at arrival: approximately 75 days (males) and 68 days (females)
- Weight at study initiation: 324-390 g (males); 205-238 g (females)
- Housing: P1 generation rats were individually housed in stainless-steel, wire-bottomed cages except during the cohabitation and postpartum periods. During cohabitation, each pair of rats were housed in the male rat's cage. Beginning no later than day 20 of presumed gestation; female rats were individually housed in nesting boxes. Each dam and delivered litter were housed in a common nesting box during the postpartum period. After weaning, F1 generation rats were individually housed in stainless steel, wire-bottomed cages except during the cohabitation and postpartum periods. During cohabitation, each pair of male and female rats was housed in the male rat’s cage. Beginning no later than DG 20, F1 generation female rats were individually housed in nesting boxes until they either naturally delivered litters or were sacrificed (DG 25). Each dam and delivered litter was housed in a common nesting box during the postpartum period. Nesting material (bed-o'cobs®) was provided.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: not reported

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25°C (66-77°F)
- Humidity (%): 30-70%
- Air changes (per hr): at least 10 per hour
- Photoperiod (hrs dark / hrs light): 12 hour light and 12 hour dark
Route of administration:
oral: feed
Vehicle:
other: Certified Rodent Diet ® #5002
Details on exposure:
Male and female rats were offered the test substance via the diet beginning at approximately eleven to twelve weeks of age, at least 70 days before cohabitation, and continuing until sacrifice. A constant concentration of the test substance in the diet was offered to the rats, and the mg/kg/day dosages consumed were calculated and presented for periods corresponding to body weight and feed consumption observations.

F1 generation pups may have been exposed to the test substance during maternal gestation (in utero exposure), via maternal milk, and via maternal feed during the postpartum period. F1 generation rats were exposed to the same dosage level of the test substance as their respective P1 generation sires and dams beginning at weaning (approximately 70 days before cohabitation) and continuing through sacrifice.

F2 generation pups were not directly given the test substance but may have been exposed in utero during gestation or via maternal milk and maternal feed during the postpartum period.
Details on mating procedure:
Estrous cycling was evaluated by examination of vaginal cytology for 21 days before the scheduled cohabitation period and then until spermatozoa were observed in a smear of the vaginal contents and/or a copulatory plug was observed in situ during the cohabitation period.
The estrous cycle was also evaluated by examination of vaginal cytology prior to sacrifice on DL 22.

After at least 70 days of exposure to the test substance/carrier, within each dosage group, consecutive order was used to assign rats to cohabitation, one male rat per female rat. The cohabitation period consisted of a maximum of 21 days. Female rats with spermatozoa observed in a smear of the vaginal contents and/or a copulatory plug in situ were considered to be at DG 0 and assigned to individual housing. Female rats not mated within the first 14 days of cohabitation were assigned alternate male rats that have mated (same dosage group) and remained in cohabitation for a maximum of seven additional days. Female rats not mated after the completion of the 21-day cohabitation period were considered to be at DG 0 and assigned to individual housing.

Female F1 rats were evaluated for the age of vaginal patency, beginning on day 28 postpartum. Male F1 rats were evaluated for the age of preputial separation, beginning on day 39 postpartum. The body weight was recorded for each rat on the day the criterion was achieved/recorded.

Estrous cycling was evaluated by examination of vaginal cytology for 21 days before initiation of cohabitation until spermatozoa were observed in a smear of the vaginal contents and/or a copulatory plug was observed in situ during the cohabitation period. The estrous cycle was also
evaluated by examination of vaginal cytology prior to sacrifice on DL 22.

At 79 to 95 days of age and after at least 70 days of exposure to the test substance/carrier, the F1 generation rats within each dosage group were assigned to cohabitation, one male rat per female rat, based on a random unit table, with the exclusion of sibling matings. The cohabitation
period consisted of a maximum of 21 days. Female rats with spermatozoa observed in a smear of the vaginal contents and/or a copulatory plug observed in situ were considered to be at DG 0 and assigned to individual housing. Female rats not mated within the first 14 days of cohabitation were assigned alternate male rats from the same dosage group that had mated. Female rats not mated within the first 14 days of cohabitation were assigned alternate male rats that have mated (same dosage group) and remained in cohabitation for a maximum of seven additional days. Female rats not mated after the completion of the 21-day cohabitation period were considered to be at DG 0 and assigned to individual housing.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Diet samples were extracted with acetonitrile and test substance stability samples were dissolved in acetonitrile. Concentrations of the test substance in diet sample extracts and test substance solutions were measured by high performance liquid chromatography (HPLC) with ultraviolet (UV) detection.
Duration of treatment / exposure:
2 Generations
Frequency of treatment:
Daily
Details on study schedule:
- F1 parental animals not mated until at least 10weeks after selected from the F1 litters.
Dose / conc.:
75 ppm
Dose / conc.:
300 ppm
Dose / conc.:
1 000 ppm
Dose / conc.:
2 500 ppm
No. of animals per sex per dose:
30
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: Dietary concentrations up to and including 2500 ppm were provided by the Sponsor based on data present from previous studies.
Positive control:
No
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
P1 Generation: Rats were observed for viability at least twice each day of the study and for clinical observations and general appearance at least weekly during the acclimation and dosage period.

F1 Generation: Pups were observed for viability at least twice each day and for clinical observations and general appearance at least weekly during the postweaning period.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
P1 Generation: Rats were evaluated for adverse clinical signs observed during parturition, duration of gestation (DG 0 to the day the first pup was observed), litter sizes (all pups delivered) and pup viability at birth. Maternal behavior was evaluated on DLs 1, 5, 8, 15 and 22.

F1 Generation: Each litter was evaluated for viability at least once daily. The pups in each litter were counted once daily. Clinical observations were recorded once daily during the preweaning period. Pup body weights were recorded on DLs 1 (liveborn pups), 5, 8, 15 and 22 postpartum (body weights only). Rats were evaluated for adverse clinical signs observed during parturition, duration of gestation (DG 0 to the day the first pup was observed), litter sizes (all pups delivered) and pup viability at birth. Maternal behavior was evaluated on DLs 1, 5, 8, 15 and 22.

BODY WEIGHT: Yes
- Time schedule for examinations:
P1 Generation: Body weights were recorded at least weekly for male rats during the acclimation period, weekly during the dosage period and a terminal weight on the day sacrifice occurred. Feed consumption values for male rats were recorded weekly during the dosage period. Body weights were recorded for female rats at least weekly during the acclimation period, weekly during the dosage period, on days 0, 7, 14, 21 and 25 of the gestation period and on days 1, 5, 8, 15 and 22 (terminal body weight recorded) of the postpartum period.

F1 Generation: After weaning, body weights for male rats were recorded weekly during the dosage period and on the day sacrifice occurred. After weaning, body weights were recorded for female rats weekly during the dosage period, on days 0, 7, 14, 18, 21 and 25 of the gestation period and on days 1, 5, 8, 15 and 22 (terminal body weight) of the postpartum period.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
P1 Generation: Feed consumption values were recorded for female rats at least weekly during the acclimation period, weekly during the dosage period, on days 0, 7, 10, 14, 18, 21 and 25 of the gestation period and on days 1, 5, 8, 11 and 15 of the postpartum period.

F1 Generation: Feed consumption values for male rats were recorded weekly
during the dosage period. Feed consumption values were recorded for female rats
weekly during the dosage period, on days 0, 7, 10, 14, 18, 21 and 25 of the gestation period and on days 1, 5, 8, 11 and 15 of the postpartum period. Because pups begin to consume maternal feed on or about DL 15, feed consumption values were not tabulated after DL 15.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not specified

FOOD EFFICIENCY: Not specified

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
Oestrous cyclicity (parental animals):
Estrous cycling was evaluated by examination of vaginal cytology for 21 days before the scheduled cohabitation period and then until spermatozoa were observed in a smear of the vaginal contents and/or a copulatory plug was observed in situ during the cohabitation period. The estrous cycle was also evaluated by examination of vaginal cytology prior to sacrifice on DL 22.
Sperm parameters (parental animals):
Sexual maturation, sperm motility, and concentration were evaluated for all P1 and F1 generation male rats.
To assess the potential toxicity of the test substance on the male reproductive system, reproductive organs were weighed and retained for possible histopathological evaluation and sperm evaluations were conducted. The following organs were individually weighed: right testis, left testis, left epididymis (whole and cauda), right epididymis, seminal vesicles (with and without fluid) and prostate. Sperm concentration and motility were evaluated using computerassisted
sperm analysis (CASA).
Motility was evaluated by the Hamilton Thorne IVOS by collection of a sample from the left vas deferens. A homogenate was prepared from the left cauda epididymis for evaluation by the Hamilton Thorne IVOS to determine sperm concentration (sperm per gram of tissue weight). Testicular and epididymal sperm counts were included in sperm evaluations. The remaining portion of the left cauda epididymis was used to manually evaluate sperm morphology. Sperm morphology evaluations included the following: 1) determination of the percentage of normal sperm in a sample of at least 200; and 2) qualitative evaluation of abnormal sperm, including such categories as abnormal head, abnormal tail, and abnormal head and tail. The testes were fixed in Bouin’s solution for 48 to 96 hours and then retained in neutral buffered 10% formalin for possible histopathological evaluation. The remaining portion of the left epididymis (corpus and caput), as well as the right epididymis, prostate and seminal vesicles were fixed in neutral buffered 10% formalin for histopathological evaluation.
Litter observations:
Litter sizes, litter sex ratio, pup birth weights were determined.
Postmortem examinations (parental animals):
P1 and F1 Generation Male Rats:
After completion of the cohabitation period, all male rats were sacrificed by carbon dioxide
asphyxiation, and a gross necropsy of the thoracic, abdominal and pelvic viscera was performed. Rats that died or were sacrificed before scheduled termination was examined for the cause of death or condition as soon as possible after the observation was made. The rat was examined for gross lesions. The urinary bladder and lungs were perfused with neutral buffered 10% formalin. When not precluded by autolysis, the heart, lungs, liver, kidneys, stomach and spleen were retained in neutral buffered 10% formalin for possible histological evaluation. To assess the potential toxicity of the test substance on the male reproductive system, reproductive organs were weighed and retained for possible histopathological evaluation and sperm evaluations were conducted. The following organs were individually weighed: right testis, left testis, left epididymis (whole and cauda), right epididymis, seminal vesicles (with and without fluid) and prostate. Sperm concentration and motility were evaluated using computer assisted sperm analysis (CASA). The testes were
fixed in Bouin’s solution for 48 to 96 hours and then retained in neutral buffered 10% formalin for possible histopathological evaluation. The remaining portion of the left epididymis (corpus and caput), as well as the right epididymis, prostate and seminal vesicles were fixed in neutral buffered 10% formalin for histopathological evaluation.

P1 and F1 Generation Male Rats:

Surviving female rats were sacrificed after completion of the 22-day postpartum period and a
gross necropsy of the thoracic, abdominal and pelvic viscera was performed. The number and
distribution of implantation sites was recorded. Rats that did not deliver a litter were sacrificed on day 25 of presumed gestation and uteri were stained with 10% ammonium sulfide to confirm the absence of implantation sites. A smear of the vaginal contents was collected and evaluated prior to sacrifice. The stage of estrous was confirmed at the time of histopathological evaluation by a Board Certified Veterinary Pathologist. Rats that died before scheduled termination were examined for the cause of death or condition as
soon as possible after the observation was made. The rats were examined for gross lesions. The urinary bladder and lungs were perfused with neutral buffered 10% formalin. When not
precluded by autolysis, the heart, lungs, liver, kidneys, stomach and spleen were retained in
neutral buffered 10% formalin for possible histological evaluation. Pregnancy status and uterine contents of female rats were recorded (when appropriate). Aborted fetuses and/or delivered pups were examined to the extent possible. Uteri of apparently non pregnant rats were stained with 10% ammonium sulfide to confirm the absence of implantation sites. On day 22 postpartum, the brain, spleen, thyroid (fixed), adrenals and thymus were weighed and
retained in neutral buffered 10% formalin from one F1 generation pup/sex/litter for possible
histological examination.

The following tissues or representative samples were collected from all P1 and F1 generation
male and female rats at necropsy and retained in neutral buffered 10% formalin: brain, thyroid (thyroid lobes and parathyroid), adrenals, pituitary gland, liver, kidneys, spleen, thymus and gross lesions including tissue masses. The ovaries (with uterus), vagina, mammary gland and uterus (with cervix and oviducts) were collected and retained from all female rats and the testes, seminal vesicles with coagulating gland, epididymides and prostate were collected and retained from all male rats. Testes were fixed in Bouin’s solution for 48 to 96 hours before being transferred to and retained in neutral buffered 10% formalin. Organ-to-body weight and organ-to-brain weight ratios were calculated. The following
organs were individually weighed: brain, ovaries, testes, thyroid [thyroid lobes and
parathyroids (fixed)], liver, kidneys, spleen, pituitary gland, uterus (with oviducts and cervix), epididymides, prostate, adrenals, seminal vesicles with coagulating gland and thymus. A table of random units was used to select ten rats per sex from the control and high test substance concentration groups that histological examination was performed. Particular
emphasis was placed on evaluation of reproductive organs and target tissues. All gross lesions were examined histologically. Reproductive organs (testes, epididymis, prostate and seminal vesicles with coagulating gland for the male rats and vagina, uterus with cervix and oviducts, and ovaries for the female rats) were evaluated for all P1 and F1 generation rats that did not mate, conceive, sire, or deliver healthy offspring, or for which estrous cyclicity or sperm morphology was affected. Tissues examined histologically were processed, embedded in paraffin, sectioned at 5 microns and stained with hematoxylin and eosin.
Postmortem examinations (offspring):
Pups were sacrificed by an intraperitoneal injection of sodium pentobarbital (pups ≤14 days of age) or by carbon dioxide asphyxiation (pups ≥15 days of age). Pups that died before
examination of the litter for pup viability were evaluated for vital status at birth. The lungs were removed and immersed in water. Pups with lungs that sink were identified as stillborn; pups with lungs that float were identified as live born and to have died shortly after birth. Pups found dead on day 1 postpartum were preserved in Bouin's solution for possible future evaluation. Should postmortem autolysis preclude these evaluations, it was noted in the necropsy data. Pups that died or were sacrificed before scheduled termination were examined for gross lesions and the cause of death or condition as soon as possible after the observation was made. Pups found on days 1 to 5 postpartum were preserved in Bouin's solution for possible future evaluation; pups found on days 6 to 22 postpartum were preserved in neutral buffered 10% formalin. Should postmortem autolysis preclude these evaluations it was noted in the necropsy data. All pups culled on day 5 postpartum were sacrificed and examined for gross lesions; pups with gross lesions were preserved in Bouin's solution. Necropsy included a single cross-section of the head at the level of the frontal-parietal suture and examination of the cross-sectioned brain for apparent hydrocephaly. All pups not selected for continued observation on day 22 postpartum were sacrificed and examined for gross lesions; gross lesions were reserved in neutral buffered 10% formalin. Necropsy included a single cross-section of the head at the level of the frontal-parietal suture and examination of the cross-sectioned brain for apparent hydrocephaly. Tissues [spleen, thymus, thyroid (fixed), adrenals (weighed paired), uterus and brain] from one randomly selected pup per sex per litter when possible were weighed and retained in neutral buffered 10% formalin for possible histological evaluation. All remaining pups were discarded without further examination.
Statistics:
Clinical observations and other proportional data were analyzed using the Variance Test for Homogeneity of the Binomial Distribution.

Continuous data (e.g., maternal body weights, body weight changes, and feed consumption values) were analyzed using Bartlett’s Test of Homogeneity of Variances and the Analysis of Variance, when appropriate [i.e., Bartlett’s Test is not significant (p≤0.001)]. If the Analysis of Variance was significant (p≤0.05), Dunnett’s Test was used to identify the statistical significance of the individual groups. If the Analysis of Variance was not appropriate [i.e., Bartlett’s Test is significant (p≤0.001)], the Kruskal Wallis Test was used (≤75% ties). In cases where the Kruskal Wallis Test was statistically significant (p≤0.05), Dunn’s Method of Multiple Comparisons was used to identify the statistical significance of the individual groups. If there were greater than 75% ties, Fisher’s Exact Test was used to analyze the data.

Count data were evaluated using the procedures described above for the Kruskal-Wallis Test.

Sperm motility data were expressed as percentages and analyzed, as indicated above, by parametric methods.
Reproductive indices:
Fertility Index (percentage ofmatings that result in pregnancies). Reported for each sex.
Gestation Index (percentage of pregnancies that result in birth of live litters).
Number and sex of offspring per litter (live and dead pups).
Number of implantation sites.
General condition of dam and litter during the postpartum period.
Offspring viability indices:
Litter size and viability [tabulated at birth (day 1) and on days 5, 8, 15 and 22 postpartum]. Viability Indices (percentages of pups born that survive to days 1 and 5 postpartum).
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
All clinical observations were considered unrelated to exposure to the test substance because: 1) the incidences were not dosage-dependent; 2) the observations occurred in only one or two rats in any particular dosage group; and/or 3) the observations are common in this strain of rat. The observations included moderate dehydration; chromorhinorrhea; soft or liquid faeces; chromodacryorrhea; pale extremities; misaligned and/or missing/broken incisors; a scab on the left forelimb or back; scant faeces; ungroomed coat; an ulceration on the palate or mouth; red perioral substance; sparse hair coat on the limbs; swollen snout; lacrimation; red substance in the cage; vocalization; corneal opacity; exophthalmos; and enophthalmos.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 2500 ppm, mean body weights were significantly reduced (p≤0.01) at all intervals during the precohabitation, gestation and lactation periods beginning on day of study (DS) 8 and continuing through day of lactation/postpartum (DL) 15 in comparison with the carrier control group. Body weight gains were also significantly decreased (p≤0.05 or p≤0.01) in the 2500 ppm exposure group on DSs 1 to 8 and 50 to 57. Although the body weight gain had a brief, significant (p≤0.05) rebound in the 2500 ppm exposure group on DSs 43 to 50, the overall body weight gain for the precohabitation period (calculated as DSs 1 to 70) was 26% lower than controls and was significantly reduced (p≤0.01) in this group.

During the lactation period, maternal body weight gains were significantly increased (p≤0.05 or p≤0.01) in the 1000 ppm exposure group on DLs 5 to 8, in the 2500 ppm exposure group on DLs 15 to 22 and during the entire lactation period (DLs 1 to 22) for both exposure groups.

Body weights and body weight gains were unaffected by exposure to concentrations of 1000 ppm and lower of the test substance. Body weight gains were significantly increased (p≤0.05 or p≤0.01) on DSs 15 to 22 and DLs 5 to 8 in the 75 ppm exposure group and in the 1000 ppm exposure group on DLs 5 to 8 and in comparison with the carrier control group. Body weight gains were also significantly increased (p≤0.01) on DSs 8 to 15 in the 300 ppm exposure group, which resulted in a significant increase (p≤0.05) in mean body weights in this exposure group on DSs 22, 29, 36 and 43. Mean body weights were significantly increased (p≤0.05) in the 75 ppm exposure group on DGs 0 and 14. These increases were not considered to be test substance-related because they were not dosage-dependent. Significant decreases (p≤0.05) in body weight gain were observed in the 300 and 1000 ppm exposure on DSs 50 to 57. These decreases were not considered to be test substance-related because they were single occurrences and did not persist.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At 2500 ppm, absolute and relative feed consumption and feed efficiency values were significantly decreased (p≤0.01) during the first week of exposure in the precohabitation (DSs 1 to 8) period in comparison with the carrier control. The absolute feed consumption values remained decreased or significantly decreased (p≤0.05 or p≤0.01) in the 2500 ppm exposure group at all intervals until DSs 36 to 43 as well as for the entire precohabitation period (DSs 1 to 70). Relative feed consumption was significantly increased (p≤0.05) in the 2500 ppm exposure group on DSs 43 to 50 during the precohabitation period. Feed efficiency was significantly reduced (p≤0.05 or p≤0.01) in the 2500 ppm exposure group on DSs 50 to 57 and for the overall precohabitation period (calculated as DSs 1 to 70).

Absolute feed consumption values were decreased or significantly decreased (p≤0.05 or p≤0.01) on DGs 0 to 7 in the 1000 ppm exposure group and at all tabulated intervals during the gestation period in the 2500 ppm exposure group. Absolute and relative feed consumption values were also decreased or significantly decreased (p≤0.05 or p≤0.01) on DLs 1 to 5 in the 1000 ppm exposure group and at all tabulated intervals during the lactation period in the 2500 ppm exposure group.

Maternal absolute and relative feed consumption values were unaffected by exposure to concentrations of 300 ppm and lower of the test substance. The maternal absolute feed consumption value was significantly increased (p≤0.05) in the 300 ppm exposure group on DSs 15 to 22 during the precohabitation period in comparison with the carrier control group.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
At 2500 ppm, feed efficiency values were significantly decreased (p≤0.01) during the first week of exposure in the precohabitation (DSs 1 to 8) period in comparison with the carrier control. Feed efficiency was significantly reduced (p≤0.05 or p≤0.01) in the 2500 ppm exposure group on DSs 50 to 57 and for the overall precohabitation period (calculated as DSs 1 to 70).

Feed efficiency values were significantly increased (p≤0.01) in the 75 ppm exposure group on DSs 15 to 22 and in the 300 ppm exposure group on DSs 8 to 15. These increases were not considered to be test substance-related because they were not dosage-dependent. Feed efficiency values were significantly decreased (p≤0.05 or p≤0.01) in the 300 and 1000 ppm exposure groups on DSs 50 to 57. These decreases were not considered to be test substance-related because they were single occurrences and did not persist. Feed efficiency values were significantly reduced (p≤0.05) in the 300 ppm exposure group on DLs 1 to 5 and significantly increased (p≤0.05) in the 75 and 1000 ppm exposure groups on DLs 5 to 8. These differences were not considered to be test substance-related because they were not dosage-dependent.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
A test substance-related effect was observed in the thymus of 6 of the 10 female rats selected for evaluation in the 2500 ppm exposure group. Microscopically, the thymus was small due to a generalized decreased cellularity (atrophy) of cortical and medullary lymphoid tissue. Similar thymic atrophy was not observed in the male rats in this exposure group. When the thymuses from all of the P1 generation female rats were evaluated by the second pathologist, minimal to moderate lymphoid atrophy of the thymus was observed in 11 of the 30 female rats in the 2500 ppm exposure group. There were no gross or microscopic test substance-related changes observed in the thymuses of the 75, 300 or 1000 ppm exposure groups in the P1 generation female rats.

No test substance-related microscopic changes were observed in the male rats selected for evaluation in the 2500 ppm exposure group.

The testes, epididymides, prostate, seminal vesicles and coagulating gland (male rats) or ovaries, oviducts, uterus, cervix and vagina (female rats) were examined from rats designated as “reduced fertility” animals. There were no test substance-related microscopic changes in these animals for their failure to reproduce.

There were a few other microscopic changes observed in the other various organs and tissues and these lesions were typical of those that occur spontaneously in male and female rats of this age and strain used in reproductive toxicity studies. Exposure to the test substance had no effect on the type, incidence or severity of these changes.
Histopathological findings: neoplastic:
not examined
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
The precohabitation estrous cycling observations [mean estrous stages per 22 days, rats with six or more consecutive days in diestrus and rats with six or more consecutive days of estrus] were unaffected by exposure to the test substance at levels up to and including 2500 ppm.
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
Description (incidence and severity):
All mating and fertility parameters [numbers of days in cohabitation, rats that mated, fertility index (number of pregnancies per number of rats that mated), rats with confirmed mating dates during the first week of cohabitation and during the second week of cohabitation and number of
pregnancies per number of rats in cohabitation] were unaffected by exposure to the test substance at levels as high as 2500 ppm.
Key result
Dose descriptor:
NOAEL
Effect level:
300 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
organ weights and organ / body weight ratios
histopathology: non-neoplastic
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
2 500 ppm
System:
endocrine system
Organ:
thymus
Treatment related:
yes
Dose response relationship:
not specified
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
All clinical observations observed during the precohabitation, gestation, and lactation periods were considered unrelated to exposure to the test substance because: 1) the observations were not dosage-dependent; 2) the observation occurred in only one or two rats in any exposure group; and/or 3) the finding is commonly observed in this strain of rat. These observations included chromodacryorrhea; chromorhinorrhea; ataxia; decreased motor activity; both eyes appeared red and sunken in head; scant feces; pale extremities; coldness to the touch; severe dehydration; hunched posture; tachypnea; thin appearance; missing/broken or misaligned incisors; soft or liquid feces; snout bent to the left; swollen snout; scab on the neck; both ears swollen; gasping; dyspnea; mass on the left flank; red perivaginal substance; pale in appearance; sparse hair coat on the limbs or underside; incisor actively bleeding; red perioral or perinasal substance; red
substance on the cage walls; lacrimation; urine-stained abdominal fur; and localized alopecia on the limbs.

A significantly increased (p≤0.01) number of F1 generation male rats in the 2500 ppm exposure group were observed with mild dehydration during the exposure period, which resulted in a significant increase (p≤0.01) in the total number of rats observed with dehydration in comparison with the carrier control group.

All other clinical observations were considered unrelated to exposure to the test substance because: 1) the incidences were not dosage-dependent; 2) the observations occurred in only one or two rats in any particular dosage group; and/or 3) the observations are common in this strain of rat. The observations included moderate dehydration; chromorhinorrhea; soft or liquid feces; chromodacryorrhea; pale extremities; misaligned and/or missing/broken incisors; a scab on the left forelimb or back; scant feces; ungroomed coat; an ulceration on the palate or mouth; red perioral substance; sparse hair coat on the limbs; swollen snout; lacrimation; red substance in the cage; vocalization; corneal opacity; exophthalmos; and enophthalmos.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
No deaths related to exposure to the test substance occurred in the F1 generation female rats. One female rat at 2500 ppm was humanely euthanized due to adverse clinical signs during the early postweaning exposure period. This death was considered unrelated to the test substance because it was a single occurrence and did not persist. Clinical and necropsy observations and body weights values for this F1 generation female rat are described below. All other F1 generation female rats survived until scheduled sacrifice.

Rat 4678 in the 2500 ppm exposure group was humanely euthanized due to adverse clinical signs on PPD 27. This rat was exposed to the test substance for 4 consecutive days. The adverse clinical signs noted for this rat included ataxia, decreased motor activity, chromorhinorrhea, both eyes appearing red and sunken in the head, pale extremities, coldness to the touch, severe dehydration, thin appearance, hunched posture, tachypnea, and scant feces, which all occurred on PPD 27. The body weight value for this rat recorded on PPD 23 was unremarkable. All tissues examined appeared normal at necropsy. Due to the death occurring shortly after weaning and
this being the only F1 generation female pup that died, this death was considered to be a spurious incidence of failure to survive rather than test substance-related.

One male rat at 2500 ppm was found dead during the exposure period. This death was not considered to be test substance-related because the death occurred shortly after weaning and no other rats in this group were found dead during the exposure period. Clinical and necropsy observations and body weight for this male rat are described below. All other F1 generation male rats survived
until scheduled sacrifice.

Rat 4540 in the 2500 ppm exposure group was found dead on PPD 26. This rat was exposed to the test substance for 3 consecutive days. The adverse clinical signs noted for this rat included mild dehydration and pale extremities on PPD 23. The body weight for this rat on PPD 23 was generally comparable with the other rats in this exposure group. At necropsy, the lungs were mottled red and dark red; all other tissues examined appeared normal. Due to the death occurring shortly after weaning and this being the only F1 generation male pup that died, this death was considered to be a spurious incidence of failure to survive rather than test substance related.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In female rats, the mean body weights at 1000 ppm were significantly reduced (p≤0.05) on PPDs 23 and 30. At 2500 ppm, mean body weights were significantly reduced (p≤0.05 or p≤0.01) at most intervals during the precohabitation, gestation, and lactation periods beginning on PPDs 23 and continuing through DL 15 in comparison with the carrier control group. There was an 8% reduction in mean body weights during the precohabitation and gestation (GD 21) periods, with a slight recovery (1% reduction) during the lactation period (DL 22). Body weight gains were also decreased or significantly decreased (p≤0.01) in the 2500 ppm exposure group on PPDs 23 to 30 and 30 to 37 and significantly increased (p≤0.01) on PPDs 37 to 44.

During the lactation period, maternal body weight gains were significantly increased (p≤0.05 or p≤0.01) in the 1000 and 2500 ppm exposure groups on DLs 5 to 8 and during the entire lactation period (DLs 1 to 22) as well as DLs 1 to 15 and 15 to 22 in the 2500 ppm exposure group.

Body weights and body weight gains were unaffected by exposure to concentrations of 300 ppm and lower of the test substance. The mean body weights were significantly increased (p≤0.05) on PPD 23 in the 75 ppm exposure group and on DL 22 in the 300 ppm exposure group in comparison with the carrier control group. These increases were not considered to be test substance-related because they were not dosage-dependent and/or did not persist.

In male rats, body weight gains were significantly reduced (p≤0.05 or p≤0.01) in the 2500 ppm exposure group at all intervals up to and including PPDs 44 to 51, PPDs 23 to 86, PPDs 23 to precohabitation (PPDs 23 to ranging from PPDs 79 and 95), PPDs 23 to 135, and the entire exposure period (7% lower than controls from PPDs 23 to termination) in comparison with the carrier control. In addition, mean body weights were significantly decreased (p≤0.01) in the 2500 ppm exposure group at all intervals during the exposure period in comparison with the carrier control group.

Body weights and body weight gains were unaffected by exposure to concentrations of 1000 ppm and lower of the test substance. Body weight gains were significantly reduced (p≤0.05) in the 1000 ppm exposure group on PPDs 30 to 37 in comparison with the carrier control. This decrease was not considered to be test substance-related because it was a single occurrence and did not persist.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At 1000 ppm, absolute feed consumption values were significantly decreased (p≤0.05) during the first week of exposure in the precohabitation (PPDs 23 to 30) period in comparison with the carrier control. The absolute feed consumption values were significantly decreased (p≤0.05 or p≤0.01) in the 2500 ppm exposure group at all intervals from weaning until PPDs 51 to 58 as well as for the entire precohabitation period (PPDs 23 to 86). At 2500 ppm, relative feed consumption values were significantly increased (p≤0.05 or p≤0.01) PPDs 30 to 37, 37 to 44 and 58 to 65 in comparison with the carrier control group. There was also a significant decrease (p≤0.05) in relative feed consumption in the 2500 ppm exposure group on DLs 11 to 15 in comparison with the carrier control group.

Absolute feed consumption values were decreased or significantly decreased (p≤0.05) on DGs 0 to 7 in the 1000 ppm exposure group and at all tabulated intervals during the gestation period in the 2500 ppm exposure group. The absolute feed consumption value was also significantly decreased (p≤0.05) on DLs 11 to 15 in the 2500 ppm exposure group.

In female rats, maternal absolute and relative feed consumption values were unaffected by exposure to concentrations of 300 ppm and lower of the test substance. The relative feed consumption value was significantly decreased (p≤0.05) in the 75 ppm exposure group on PPDs 30 to 37 during the precohabitation period in comparison with the carrier control group. The absolute feed consumption value was significantly increased (p≤0.05) in the 300 ppm exposure group on DLs 8 to 11 in comparison with the carrier control group. These differences were not considered to be test substance-related because they were not dosage-dependent.

In male rats, absolute feed consumption values were significantly reduced (p≤0.05 or p≤0.01) at all intervals up to and including PPDs 44 to 51, during the precohabitation period and during the exposure period in the 2500 ppm exposure group in comparison with the carrier control group. Relative feed consumption values were also significantly increased (p≤0.01) beginning on PPDs 37 to 44 and continuing through the remainder of the precohabitation and exposure period.

Absolute and relative feed consumption values and feed efficiency were unaffected by exposure to concentrations of 1000 ppm and lower of the test substance. Relative feed consumption values in the 75, 300 and 1000 ppm exposure groups and the absolute feed consumption value in the 1000 ppm exposure group were significantly decreased (p≤0.05 or p≤0.01) on PPDs 30 to 37 in comparison with the carrier control group. These decreases were not considered to be test substance-related because they were single occurrences and/or they did not persist throughout the exposure period. At 75 ppm, feed efficiency was significantly decreased (p≤0.05) on PPDs 37 to 44 and the absolute and relative feed consumption values were significantly increased (p≤0.05) on PPDs 107 to 114 in comparison with the carrier control group. These differences were not considered to be test substance-related because they were not dosage-dependent.

Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
In female rats, feed efficiency values were significantly increased (p≤0.05 or p≤0.01) in the 2500 ppm exposure group on PPDs 23 to 30 through 37 to 44 and during the entire precohabitation period. Feed efficiency values were significantly increased (p≤0.05 or p≤0.01) on DLs 5 to 8 in the 1000 and 2500 ppm exposure groups as well as on DLs 1 to 15 in the 2500 ppm exposure group.

In male rats, feed efficiency values were significantly increased (p≤0.01) in the 2500 ppm exposure group on DSs 23 to 30 in comparison with the carrier control group.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The absolute and relative (% body weight and % brain weight) weights of the right and left ovaries were decreased or significantly decreased (p≤0.05 or p≤0.01) in the 2500 ppm exposure group in comparison with the carrier control group. The absolute and relative (% body weight) fixed thyroid weights were also significantly decreased (p≤0.05) in the 2500 ppm exposure groups in comparison with the carrier control group. There was a significant increase (p≤0.05) in the relative weight of the liver to terminal body weight and brain weight at 2500 ppm in comparison with the carrier control group.

All other organ weights (absolute and relative) were unaffected by exposure to the test substance at levels as high as 1000 ppm. There was a significant decrease (p≤0.05) in the ratio of the liver weight to terminal body weight in the 75 ppm exposure group in comparison with the carrier control group. The absolute weights in the right kidney were significantly increased (p≤0.05) in the 75 and 300 ppm exposure groups in comparison with the carrier control group. The terminal body weight was also significantly increased (p≤0.05) in the 300 ppm exposure group. These differences were not considered to be test substance-related because they were not dosagedependent.
There was a significant decrease (p≤0.05) in the relative weights of the right and left ovaries to terminal body weights at 300 ppm in comparison with the carrier control group. These decreases were not considered to be test substance-related because there were no significant decreases observed in the absolute weights of the right and left ovaries.

The ratio of the fixed thyroid weights to terminal body weights was significantly decreased (p≤0.05) in the 1000 ppm exposure group in comparison with the carrier control group. This decrease was not considered to be test substance-related because there was no
significant decrease observed in the absolute fixed thyroid weight at 1000 ppm. There was a significant decrease (p≤0.05) in the absolute and relative (% body weight) weights of the left ovaries at 1000 ppm in comparison with the carrier control group. This decrease was not
considered to be test substance-related because it was observed only in the left ovary without a similar effect in the right ovary. The ratio of the non-gravid uterus with cervix weight to brain weight was significantly increased (p≤0.05 or p≤0.01) in the 2500 ppm exposure group. This increase was not considered to be test substance-related because the absolute organ weight value for the non-gravid uterus with cervix weights was generally comparable to the weight from the carrier control group.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test substance-related necropsy observations. All necropsy observations were considered unrelated to exposure because: 1) the incidences were not dosage-dependent; and/or 2) the observations occurred in only one or two rats in any exposure group. These necropsy observations included numerous red or dark red areas on the thymus; thymus appeared edematous; missing left lobe of the thymus; all lobes of the liver mottled red and dark red; yellow mass in the abdominal adipose; constricted area of the spleen; spleen
misshapen and small; black area on the pyloric region of the mucosal surface of the stomach; slight dilation of the pelvis in the right kidney; and a clear fluid-filled cyst on the left ovary.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
A test substance-related effect was observed in the thymus of 6 of the 10 female rats selected for evaluation in the 2500 ppm exposure group. Microscopically, the thymus was small due to a generalized decreased cellularity (atrophy) of cortical and medullary lymphoid tissue. Similar thymic atrophy was not observed in the male rats in this exposure group. When the thymuses from all of the P1 generation female rats were evaluated by the second pathologist, minimal to moderate lymphoid atrophy of the thymus was observed in 11 of the 30 female rats in the 2500 ppm exposure group. There were no gross or microscopic test substance-related changes observed in the thymuses of the 75, 300 or 1000 ppm exposure groups in the P1 generation female rats.

No test substance-related microscopic changes were observed in the male rats selected for evaluation in the 2500 ppm exposure group.

The testes, epididymides, prostate, seminal vesicles and coagulating gland (male rats) or ovaries, oviducts, uterus, cervix and vagina (female rats) were examined from rats designated as “reduced fertility” animals. There were no test substance-related microscopic changes in these animals for their failure to reproduce.

There were a few other microscopic changes observed in the other various organs and tissues and these lesions were typical of those that occur spontaneously in male and female rats of this age and strain used in reproductive toxicity studies. Exposure to the test substance had no effect on the type, incidence or severity of these changes.
Histopathological findings: neoplastic:
not examined
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
The precohabitation estrous cycling observations [mean estrous stages per 21 days, rats with six or more consecutive days in diestrus and rats with six or more consecutive days of estrus] were unaffected by exposure to the test substance at levels up to and including 2500 ppm.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Values for number and percent motile sperm, number of nonmotile sperm and total sperm count from the vas deferens, cauda epididymal sperm count and density and testicular sperm count and density were comparable among the five exposure groups in the F1 generation male rats. Values for the number of normal sperm, the percent abnormal, and the number observed with detached heads, broken flagellum, amorphous and coiled flagellum were unaffected by exposure to the test substance.

All sperm and cauda epididymal sperm morphology parameters evaluated were unaffected by exposure to the test substance in the diet at levels as high as 2500 ppm. At 75, 300, 1000 and 2500 ppm, there was a significant increase (p≤0.01) in the number of sperm observed to be normal and a significant decrease (p≤0.01) in the percent abnormal sperm and those observed with detached heads in comparison with the carrier control group. There was also a significant decrease (p≤0.05 or p≤0.01) in the number of sperm observed with no head in the 75 ppm exposure group and broken flagellum in the 75, 300 and 1000 ppm exposure groups in comparison with the carrier control group. This was not considered to be test substance-related, but rather an incidental difference in one of the multiple abnormal findings in the sperm
morphology especially since there was an overall increase in the percent abnormal sperm observed in the carrier control group.
Reproductive performance:
no effects observed
Description (incidence and severity):
All mating and fertility parameters [numbers of days in cohabitation, rats that mated, fertility index (number of pregnancies per number of rats that mated), rats with confirmed mating dates during the first week of cohabitation and during the second week of cohabitation and number of
pregnancies per number of rats in cohabitation] were unaffected by exposure to the test substance at levels as high as 2500 ppm.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
A significantly increased (p≤0.01) number of F1 generation male rats in the 2500 ppm exposure group were observed with mild dehydration during the exposure period, which resulted in a significant increase (p≤0.01) in the total number of rats observed with dehydration in comparison with the control group.
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
One rat at 2500 ppm was found dead during the exposure period. This death was not considered to be test substance-related because the death occurred shortly after weaning and no other rats in this group were found dead during the exposure period. All other F1 generation male rats survived until scheduled sacrifice.

Rat 4540 in the 2500 ppm exposure group was found dead on PPD 26. This rat was exposed to the test substance for 3 consecutive days. The adverse clinical signs noted for this rat included mild dehydration and pale extremities on PPD 23. The body weight for this rat on PPD 23 was generally comparable with the other rats in this exposure group. At necropsy, the lungs were mottled red and dark red; all other tissues examined appeared normal. Due to the death occurring shortly after weaning and this being the only F1 generation male pup that died, this death was considered to be a spurious incidence of failure to survive rather than test substance related.
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
300 ppm
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
organ weights and organ / body weight ratios
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no
Conclusions:
On the basis of these data, the no-observed-adverse-effect level (NOAEL) for reproductive toxicity is 2500 ppm based on the absence of any test substance-related effects on mating, fertility, litter sizes, litter sex ratio, pup birth weights, and all other endpoints describing reproductive performance.

The NOAEL for systemic toxicity was 300 ppm and was based on the following findings. In P1 generation female rats and F1 generation offspring/adults at 1000 ppm, there were test substance-related effects on body weight parameters at the end oflactation and immediately
following weaning that were considered the result of the relatively high levels of direct exposure to the test substance. P1 generation female rats had increased body weight and body weight gains at the end of lactation and F I generation offspring/adults had reduced body weights at the end of lactation and immediately following weaning. The period of time encompassing the end of lactation and immediately following weaning represents the time during which the F1 generation exposure to the test substance is greatest and therefore, these body weight effects are consistent with dose-related systemic toxicity that was clearly evident at 2500 ppm, a dietary level that results in generally similar intake levels to those at 1000 ppm during this window of exposure. Decreased F1 generation spleen weights observed at 1000 ppm are considered secondary to and consistent with the decreased body weights observed at this level.

At 2500 ppm, there were significant reductions in body weight and food consumption parameters in P1 and F1 generation male and female rats throughout the study. These reductions were accompanied by increases in mild dehydration (FI males) and organ weight reductions that were
generally considered secondary to body weight reductions. Lastly, the test substance-related effects at this level also included increased incidences of minimal to mild thymic atrophy in the P1 generation female rats.
Executive summary:

One hundred and fifty male and 150 female rats were assigned to five exposure groups, thirty rats per sex per group. Diets containing the test substance were offered to the P1 generation rats beginning at approximately 11 to 12 weeks of age, for at least 70 days before cohabitation and continuing until sacrifice at concentrations of 0, 75, 300, 1000 and 2500 ppm. The mg/kg/day dosages consumed were calculated and presented for periods corresponding to body weight and feed consumption observations. F1 and F2 generation pups may have been exposed in utero during gestation and via maternal milk and maternal feed during the postpartum period. F1 generation rats were given continual access to the same test diet concentration as their respective P1 generation sires and dams beginning at weaning (approximately 70 days before cohabitation) and continuing through sacrifice.

 

The following parameters were evaluated for P1 and F1 generation male and female rats: viability, clinical observations, body weights and body-weight changes, feed consumption values and necropsy observations. Estrous cycling, mating performance and fertility of the female rats was determined. Sexual maturation, sperm motility, and concentration were evaluated for all P1 and F1 generation male rats.

 

On postnatal day (PND) 5, F1 and F2 generation litters were reduced to eight pups each (four male and four female pups, when possible). Culled pups with gross lesions were preserved in Bouin’s solution. All other culled pups were discarded. The following parameters were evaluated for F1 and F2 generation pups/rats: viability, clinical observations, body weights and body-weight changes, and necropsy observations.

 

After completion of the 22-day postpartum period or an estimated DG 25 (those with no confirmed date of mating), all surviving female rats were sacrificed by carbon dioxide asphyxiation and a gross necropsy of the thoracic, abdominal and pelvic viscera was performed. All surviving male rats were sacrificed by carbon dioxide asphyxiation after the completion of cohabitation and a gross necropsy of the thoracic, abdominal and pelvic viscera was performed.

 

A blood sample was collected for analysis from the vena cava after sacrifice from all P1 and F1 generation rats. Specific organs were weighed, and a full set of tissues were processed histologically from all P1 and F1 generation male and female rats and tissues were then examined microscopically from ten randomly-selected rats per sex from the control and high test substance concentration groups.

Rats that died or were sacrificed before scheduled termination were examined for the cause of death or condition as soon as possible after the observation is made. The rats were examined for gross lesions. When not precluded by autolysis, the heart, lungs, liver, kidneys, stomach and spleen were retained in neutral buffered 10% formalin for possible histological evaluation.

Pregnancy status and uterine contents of female rats were recorded (when appropriate). Delivered pups were examined to the extent possible.

Results

 

1000 ppm and higher

 

• F1 generation female rats had significantly reduced mean body weight and absolute feed consumption values during the first week of exposure, and significant reductions in body weight and feed consumption values were observed in the P1 and F1 generation male and female rats.

• P1 and F1 generation female rats had a significant increase in lactation body weight gains.

• Absolute and/or relative liver weights were increased in the 1000 ppm P1 generation male rats and the 2500 ppm P1 and F1 generation male and female rats. In the absence of any histopathological findings, the increased liver weights were most likely due to a pharmacological response to an increased metabolism.

• F1 generation pup weights per litter were significantly reduced on PND 22 at 1000 ppm and on PND 8, 15 and 22 at 2500 ppm.

• F1 generation pups had reduced or significantly reduced absolute and relative (% brain weight) spleen weights, which may correlate with the decreased pup body weights observed in these exposure groups.

 

2500 ppm

 

• Significant reductions in body weight and feed consumption values were observed in the P1 and F1 generation male and female rats.

• Absolute and/or relative weights of the brain, pituitary, thymus, right ovary and nongravid uterus with cervix were reduced or significantly reduced in the P1 generation female rats.

• The absolute and relative (% brain weight) weights of the thymus, adrenal glands and fixed thyroid glands and absolute brain weights were reduced or significantly reduced in the F1 generation pups.

• Minimal to moderate thymic atrophy was observed in the P1 generation female rats.

• A significantly increased number of F1 generation male rats were observed with mild dehydration during the exposure period.

• The absolute weights of the right and left testes, brain, spleen and the fixed thyroid weights and the terminal body weight were significantly reduced in the F1 generation male rats. The ratio of the left epididymis, left cauda epididymis and seminal vesicles

with fluid to terminal body weight were also significantly increased in the F1 generation male rats.

• There were reduced or significantly reduced absolute and/or relative organ weights in the right and left ovaries and fixed thyroid weights in the F1 generation female rats.

• The F2 generation pup weights per litter were significantly reduced on PND 15 and 22.

• The absolute and relative (% brain weight) thymus, spleen and fixed thyroid weights were significantly reduced in the F2 generation male and female pups.

• Minimal to moderate thymic atrophy was observed in the P1 generation female rats.

• A significantly increased number of F1 generation male rats were observed with mild dehydration during the exposure period.

• The absolute weights of the right and left testes, brain, spleen and the fixed thyroid weights and the terminal body weight were significantly reduced in the F1 generation male rats. The ratio of the left epididymis, left cauda epididymis and seminal vesicles

with fluid to terminal body weight were also significantly increased in the F1 generation male rats.

• There were reduced or significantly reduced absolute and/or relative organ weights in the right and left ovaries and fixed thyroid weights in the F1 generation female rats.

• The F2 generation pup weights per litter were significantly reduced on PND 15 and 22.

• The absolute and relative (% brain weight) thymus, spleen and fixed thyroid weights were significantly reduced in the F2 generation male and female pups.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Study duration:
subchronic
Species:
rat
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

In a multi-generation reproduction study, groups of 30 male and 30 female Crl:CD(SD) rats were fed diet containing 0 (control), 75, 300, 1000 or 2500 ppm test substance. After approximately 70 days, the animals were mated to generate F1 offspring. F1 generation rats were given continued access to the same test diet concentrations as their respective P1 generation sires and dames beginning at weaning and were subsequently mated to produce the F2 generation. No test-substance related mortality or clinical signs of toxicity were observed. There were no effects of treatment on mating, fertility, litter sizes, litter sex ratio, pup birth weights, and all other endpoints describing reproductive performance (oestrus cycling, sperm parameters, and onset of puberty). Reductions in body weight, body weight gain and food consumption were observed in the P1 and F1 generation 2500 ppm male and female rats. The 1000 ppm F1 females also had reduced mean body weight and food consumption during the first week of exposure. Increased body weight gains during lactation were observed at 1000 and 2500 ppm in P1 and F1 generation females. At 2500 ppm, F1 and F2 generation pup weights were significantly reduced on postnatal day 8 (PND 8) (F1 pups only), 15, and 22. F1 generation pup weights were also significantly reduced on PND 22 at 1000 ppm. Pups begin to eat diet at approximately postnatal day 11. At the end of lactation and just after weaning, F1 exposure (on a mg/kg bw/day dose) is greatest. Therefore, the weight effects in the F1 pups at 1000 ppm were considered consistent with dose-related systemic toxicity that was clearly evident at 2500 ppm, a dietary level that resulted in similar intake levels to those at 1000 ppm during this window of exposure. Absolute and/or relative liver weights were increased in 1000 ppm P1 males and 2500 ppm P1 and F1 males and females. In the absence of microscopic evidence of liver toxicity, the liver weight changes were considered mostly likely to be a pharmacological response to increased metabolism. A number of decreases or increases in absolute and/or relative organ weights were noted in 2500 ppm P1 and F1 male and female rats that are considered, in the absence of any histological findings, to have resulted from the reductions in body weight that occurred at this dose level. These included reductions in brain, pituitary, ovary, non-gravid uterus with cervix, testes, fixed thyroid, right and left testis, and spleen weights; increases in left epididymis, left cada epididymis, and seminal vesicals with fluid weights. Statistically significant reductions in thymus weight (absolute and relative % of body and % of brain) were observed in 2500 ppm P1 females which correlated with the presence of minimal to moderate lymphoid atrophy upon histological examination. Organ weight reductions were also observed in the F1 and F2 pups in this dose group. Reductions in absolute and relative spleen weights were observed in the 1000 pm F1 generation pups. Organ weight reductions at were accompanied by increases in mild dehydration (2500 ppm F1 males) and were considered secondary to body weight reductions. There was no evidence of adverse effects on reproduction by the test substance in this study at dose levels up to and including the top dose of 2500 ppm. The NOAEL for parental and offspring systemic toxicity was considered to be 300 ppm. 

Effects on developmental toxicity

Description of key information

Rat Developmental Diet: Not a unique developmental toxin. OECD 414; Reliability = 1

Rabbit Developmental Diet: Not a unique developmental toxin. OECD 414; Reliability = 1

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
93.3% purity
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at arrival: not reported
- Weight at study initiation: mean of 3679-3824 g
- Housing: The rabbits were housed individually in mobile rabbit units.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: not reported

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17±3°C
- Humidity (%): 30-70%
- Air changes (per hr): at lease 15 per hour
- Photoperiod (hrs dark / hrs light): 12 hour light and 12 hour dark
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples were extracted with water acetonitrile and portions of supernatant solutions were diluted with acetonitrile, as appropriate, to give sample solution concentrations within the range of the calibration standards. These were analyzed by High Performance Liquid Chromatography (HPLC).

Nominally 100 mg of the test substance was accurately weighed into a volumetric flask and diluted with 50 mL water, swirling gently to dissolve. The solution was made to volume with acetonitrile and was sonicated for 5 minutes (nominally 1.0 mg/mL). Further appropriate dilutions were made with acetonitrile in volumetric flasks to give a range of solutions, nominally within 2 μg/mL to 10 μg/mL. The purity of the test substance was not taken into account in the preparation of the standard solutions.

Appropriate portions of samples, 1 g ± 0.5 g, were transferred to volumetric flasks, diluted to volume with 50 mL of water, made to volume with acetonitrile and sonicated for 5 minutes. The samples were further diluted, as required, with acetonitrile to a known nominal concentration within the range of calibration standards.

High Performance Liquid Chromatography Conditions are as follows: Pump: 600 Series (Waters); Mobile phase: Acetonitrile (65% v/v), Water (35% v/v), and Phosphoric acid (0.1% v/v); Flow rate: 0.8 mL/min; Detector: 486 Series (Waters); Detector wavelength: 245 nm; Column: 15 cm x 3.9 mm id Symmetry Cl8 (Waters); Column temperature: Ambient; Sample introduction: 717 plus (Waters); Injection volume: 20 μL; and Data handling: Waters 860 (Waters).

The analysis system was calibrated using a range of standards to determine the linearity of response. An appropriate standard of known concentration was interspersed at intervals throughout the analysis.

The limit of detection was calculated to be approximately 0.1 μg/mL test substance in the analyzed solution, corresponding to a formulation concentration of 0.1 mg/mL.
Details on mating procedure:
Virgin female New Zealand White rabbits were paired (at the suppliers) with males of the same strain. The day when mating was observed was designated day 1 of gestation. The animals were delivered to the test facility on either day 2 or day 3 of gestation (within a period of 2 weeks).
Duration of treatment / exposure:
Days 8-20 of gestation
Frequency of treatment:
Daily on days 8-20 (inclusive) of gestation.
Duration of test:
30 days
Dose / conc.:
8 mg/kg bw/day (actual dose received)
Dose / conc.:
25 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
20
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels selected for this study were based on the results of dose range finding studies in the pregnant rabbit conducted at the test facility.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Within 1 hour of being dosed and towards the end of each working day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily and where appropriate, at the same time that the body weights were recorded

BODY WEIGHT: Yes
- Time schedule for examinations: Day 4, immediately prior to dosing on days 8-20 (inclusive) and on days 23, 26, and 30 of gestation

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Time schedule for examinations: Measured over 3 or 4 day intervals and calculated to give a daily value (g food/rabbit/day)

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 30
- Organs examined: External observation and an examination of the thoracic and abdominal viscera. The pregnancy status of each animal was determined.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
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 / No / No data
Fetal examinations:
- External examinations: Yes
- Soft tissue examinations: Yes
- Skeletal examinations: Yes
- Head examinations: Yes
Statistics:
See the "Any other information on materials and methods" section for information on the statistical methods used in this study.
Indices:
Percentage pre-implantation loss and percentage post-implantation loss were calculated as follows:

% pre-implantation loss number of corpora lutea - number of implantations x 100/number of corpora lutea

% post-implantation loss = number of implantations - number of live fetuses x 100/number of implantations
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
There was a slightly increased incidence of animals in the 25 and 100 mg/kg/day groups producing only few faeces and/or with signs of diarrhoea, in comparison with the control group.

There were no other clinical findings which were considered to be treatment-related.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
There was a statistically significant loss of bodyweight between days 8 and 11 following the onset of treatment with 100 mg/kg/day of the test substance. Bodyweight gain was observed thereafter.

Bodyweights (adjusted for day 8 bodyweight) of the animals in the 25 mg/kg/day were essentially comparable with the control group throughout the study. A small loss of bodyweight was observed on day 9 only. The bodyweights (adjusted for day 8 bodyweight) of the animals in the 8 mg/kg/day group were higher than in the control group throughout the dosing and post-dosing periods.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Following the onset of treatment on day 8 with 100 mg/kg/day of the test substance, a statistically significant reduction in food consumption was observed for days 8-11 and days 14-17. Food consumption was lower than that of the control animals throughout the dosing period although food consumption was slightly greater in the post-dosing period. Food consumption of the animals in the 25 mg/kg/day group was slightly lower than in the control group throughout the dosing period although a statistically significant difference was attained for days 8-11 only. Parity with controls was attained in the post-dosing period. The food consumption of the animals in the 8 mg/kg/day group was comparable with the animals in the control group throughout the dosing and post-dosing periods.
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Number of abortions:
no effects observed
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
The number of corpora lutea in the 100 mg/kg/day group was lower than in the other groups. This difference was established prior to treatment with the test substance as was the increase in pre-implantation loss observed in this group.
Total litter losses by resorption:
effects observed, non-treatment-related
Description (incidence and severity):
1 litter was totally resorbed at termination in the low-dose group (1/20). There were no other resorptions.
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
The number of pregnant animals with live fetuses in utero on day 30 of gestation was 17, 16, 18 and 16 in the 0, 8, 25 and 100 mg/kg/day groups, respectively.
Key result
Dose descriptor:
NOAEL
Effect level:
25 mg/kg bw/day
Basis for effect level:
clinical signs
body weight and weight gain
food consumption and compound intake
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
Mean foetal weights were 43.1 g, 42.3 g, 40.7 g, and 44.3 g in the control, 8, 25, and 100 mg/kg/day groups, respectively.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not examined
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
The mean number of live foetuses were 8.35, 9.00, 9.56, and 6.94 in the control, 8, 25, and 100 mg/kg/day groups, respectively.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The percentages of male foetuses were not different (49.5, 54.2, 51.9, and 51.6) in the control, 8, 25, and 100 mg/kg/day groups, respectively.
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The percentage of foetuses with minor external/visceral defects in the each of the test substance groups was either comparable with or lower than in the control group and provided no evidence for any treatment-related effect. Consideration of the specific defects provided no evidence for any effect of the test substance on external or visceral development of the foetuses.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The percentage of foetuses with minor skeletal defects in the test substance groups showed no relationship with dose and there were no statistically significant differences from control. Consideration of the specific defects revealed only one finding, incomplete ossification of the odontoid, the incidence of which was statistically significantly increased in the 100 mg/kg/day group in comparison with the control group. This increase is considered to be incidental to treatment in the absence of any other indication of reduced ossification of the foetal skeleton.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The percentage of foetuses with minor external/visceral defects in the each of the test substance groups was either comparable with or lower than in the control group and provided no evidence for any treatment-related effect. Consideration of the specific defects provided no evidence for any effect of the test substance on external or visceral development of the foetuses.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
The percentage of foetuses with skeletal variants was statistically significantly higher in the 100 mg/kg/day group in comparison with the control group. For the specific skeletal variants, the incidence of fetuses with 13 thoracolumbar ribs of long length was increased in the 25 and 100 mg/kg/day groups and, statistically significant in comparison with the control group. The incidence of this finding in both groups was within the range seen previously in control rabbits in the testing facility. At 100 mg/kg, the incidence of 13th throacolumbar ribs of long length, although within the range of historical control values, coincided with a slightly increased incidence of 27th pre-pelvic vertebrae and for this reason, an association with treatment at the maternally toxic dose of 100 mg/kg cannot entriely be dismissed . At 25 mg/kg, the statistically significant increase in incidence of 13th thoracolumbar ribs of long length was within historical control range, ws not associated with any other skeletal findings, and is considered not to reflect any effect of treatment. In summary, the increased incidence of foetuses with 27th pre-pelvic vertebrae and 13th thoracolumbar ribs of long length at 100 mg/kg represent small changes in the proportions of common variants, and as such are considered not to be of toxicological significance.
Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: highest dose tested
Key result
Developmental effects observed:
no
Conclusions:
100 mg/kg/day of the test substance produced clear evidence of maternal toxicity.
25 mg/kg/day is the dose level at which no maternal toxicologically significant findings were observed.
100 mg//kg/day is the dose level at which no toxicologically significant developmental effects were observed.
Executive summary:

Groups of 20 pregnant rabbits were administered by gavage, 0, 8, 25, or 100 mg/kg/day of the test substance dissolved in 1% carboxymethyl cellulose on days 8-20 of gestation.

 

Administration of 100 mg/kg/day of the test substance was associated with maternal toxicity manifest as a loss of bodyweight and reduced food consumption. Signs of diarrhoea and reduced faecal output were also noted. There were no maternal toxicologically significant findings attributable to 8 or 25 mg/kg/day of the test substance. There was no adverse effect of the test substance on the number, growth or survival of the foetuses in utero and no evidence of teratogenicity. Evaluation of the minor external/visceral foetal defects and variants provided no evidence for an adverse effect of the test substance on development. Evaluation of the foetal skeletal variants revealed only two findings which may be related to treatment, namely an increased incidence of foetuses with 27 pre-pelvic vertebrae and an increased incidence of foetuses with 13 thoracolumbar ribs of long length. However, these findings reflect small changes in the proportions of common variants and as such are considered not to be of toxicological significance.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
99% purity
Species:
rat
Strain:
other: Alpk:APrSD
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at arrival: approximately 10-12 weeks old
- Weight at study initiation: approximately 220-300 g
- Housing: The rats were housed singly, in cages, on multiple rat racks
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: not reported

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21±2°C
- Humidity (%): 40-70%
- Air changes (per hr): 25-30 per hour
- Photoperiod (hrs dark / hrs light): 12 hour light and 12 hour dark
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: For each dose level an appropriate amount of corn oil was added to a weighed amount of the test substance (adjusted for purity). Each preparation was thoroughly mixed and subdivided into aliquots. Fresh aliquots were used for each day of the study. One preparation was made per concentration (i.e., 0. 1, 2, and 10 mg/mL). The dose preparations were stored at room temperature.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
See the "Any other information on materials and methods" section for details of the analytical verification of doses.
Details on mating procedure:
Purchased time pregnant
Duration of treatment / exposure:
Days 7-16 of gestation
Frequency of treatment:
Daily
Duration of test:
22 days
Dose / conc.:
10 mg/kg bw/day (actual dose received)
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
24
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels selected for this study were based on the results of a dose ranging finding study in the pregnant rat carried out in the test facility.
Maternal examinations:
All rats were examined on arrival to ensure that they were physically normal and exhibited normal activity. Detailed clinical observations were recorded daily and, when appropriate, at the same time that the bodyweights were recorded. Cage-side observations were also made as soon as possible after dosing, and daily towards the end of the working day.

The bodyweight of each rat was recorded on arrival, on day 4, immediately prior to dosing on days 7-16 (inclusive) and on days 19 and 22 of gestation.

The amount of food consumed by each animal was measured by giving a weighed quantity of food on days 1, 4, 7, 10, 13, 16, and 19 and calculating the amount consumed (g food/rat/day) from the residue on days 4, 7, 10, 13, 16, 19, and 22 respectively.

All animals were subjected to an examination post mortem. This involved an external observation and an examination of the thoracic and abdominal viscera. Pregnancy status was determined. For the animal which died prior to scheduled termination the corpora lutea were counted, the uterine contents were assessed and the number and type of implantations were recorded. No uterus weight was recorded nor individual foetal details.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
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
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [all per litter]
- Skeletal examinations: Yes: [all per litter]
- Head examinations: Yes: [all per litter]
Statistics:
See the "Any other information on materials and methods" section for details on the Statistical methods used in this study.
Indices:
Percentage pre-implantation loss and percentage post-implantation loss were calculated as follows:

% pre-implantation loss = number of corpora lutea - number of implantations x 100/number of corpora lutea

% post-implantation loss = number of implantations - number of live foetuses x 100/number of implantations
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Administration of 100 mg/kg/day was associated with an increased incidence of signs of diarrhoea and with urine staining during the dosing period.

Two and 3 animals in the 30 and 100 mg/kg/day groups respectively, showed salivation. The 3 animals in the 100 mg/kg/day group showed salivation between days 15 and 17. Only 1 of the 2 animals in the 30 mg/kg/day group showed salivation during this period whilst the other showed salivation on day 22 only. Salivation following dosing is considered to reflect a minor pharmacological response to the test substance of no toxicological significance.

None of the other changes in clinical condition were considered to be treatment-related.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One animal given 30 mg/kg/day was killed on day 10 following an accidental injury.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Administration of 100 mg/kg/day was associated with a reduction in bodyweight gain from the onset of dosing through to termination. There was no effect of 10 or 30 mg/kg/day on maternal bodyweight
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Administration of 100 mg/kg/day was associated with a reduction in maternal food consumption during the dosing period. There was no effect of 10 or 30 mg/kg/day on maternal food consumption.
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Number of abortions:
no effects observed
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
Occasional statistically significant differences from control were due, primarily, to a greater number of foetuses in the 10 and 100 mg/kg/day groups and to a reduction in pre-implantation loss.
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
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
no effects observed
Key result
Dose descriptor:
NOEL
Effect level:
30 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
clinical signs
body weight and weight gain
food consumption and compound intake
Fetal body weight changes:
no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The number of foetuses with major defects was 0/309, 3/352, 0/278 and 2/331 in the control, 10, 30 and 100 mg/kg/day groups respectively. Neither the type nor incidence of major defects provided evidence for an adverse effect of the test substance.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The proportion of foetuses with minor skeletal defects was similar for all groups, including the control group. However, consideration of the specific defects revealed an increased incidence of foetuses in the 100 mg/kg/day group with an unossified 3rd cervical centrum and an increased incidence of litters in the same group with an extremely misaligned 5th sternebra. In the absence of other significant changes in the foetal skeleton, these minor changes are considered not to reflect an adverse effect of the test substance. None of the specific minor defects were statistically significantly increased in the 10 or 30 mg/kg/day groups.
Visceral malformations:
no effects observed
Description (incidence and severity):
The proportion of foetuses with minor external/visceral defects was similar for all groups, including the control. Consideration of the specific defects provided no evidence for an adverse effect of the test substance.
Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects at highest dose tested
Key result
Developmental effects observed:
no
Conclusions:
No developmental toxicity was observed in this study. The no effect level for maternal toxicity was 30 mg/kg/day.
Executive summary:

Groups of 24 time-mated, female Alpk:APfSD (Wistar-derived) rats were dosed orally, by gavage, with 0 (control), 10, 30 or 100 mg/kg/day of the test substance (using corn oil as a vehicle) on days 7-16 (inclusive) of gestation (the day of mating being designated day 1). The rats were killed on day 22 of gestation.

Administration of 100 mg/kg/day was associated with maternal toxicity; a reduction in maternal bodyweight gain, a reduction in food consumption and an increased incidence of animals with diarrhea and urine staining. There was no evidence for maternal toxicity in the 10 or 30 mg/kg/day groups.

 There was no evidence of a test substance-related adverse effect of on the number, growth or survival of the fetuses in utero. There was no evidence for an adverse effect on foetal development. Consideration of the incidence and type of major and minor defects and variants revealed no treatment-related effects.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

Groups of 24 time-mated female Alpk:APfSD (Wistar derived) rats were dosed orally by gavage with 0 (control), 10, 30 or 100 mg/kg bw/day in corn oil (10 mL/kg) on Days 7-16 (inclusive) of gestation. The day of mating was designated as Day 1. One female given 30 mg/kg bodyweight/day was terminated on Day 10 of the study for humane reasons, unrelated to treatment, following an accidental injury. Clinical observations of toxicity related to treatment with the test substance were an increased incidence of diarrhoea/signs of diarrhoea and urine staining at 100 mg/kg bw/day and a slight increase in the incidence of salivation at 100 and 30 mg/kg bw/day. Administration of 100 mg/kg bw/day was associated with a reduction in bodyweight gain from the onset of dosing to termination. There was no effect of 10 or 30 mg/kg bw/day on maternal bodyweight. Food consumption was reduced in animals given 100 mg kg bw/day throughout the dosing period. There was no effect of 10 or 30 mg/kg bw/day on maternal food consumption. There were no macroscopic findings which were considered to be related to treatment with the test substance. There were no adverse effects of the test substance on number, growth or survival of foetuses in utero. There was no treatment related effect on the type or incidence of foetal malformations. A statistically significant difference was seen in the number of foetuses in the 100 mg/kg bw/day group with the finding 3rd cervical centrum - unossified and in the incidence of litters in the same group with the finding 5th sternebra - misaligned extremely. When considered relative to the historical control data, these changes were minor and, in the absence of other significant changes in the foetal skeleton, are considered not to reflect an adverse effect of the test substance. The maternal NOAEL was 30 mg/kg and foetal NOAEL was 100 mg/kg.

 

Groups of 20 time-mated, female New Zealand White rabbits were dosed orally by gavage with 0 (control), 8, 25 or 100 mg/kg bw/day, using 1% carboxymethylcellulose (1 mL/kg), on Days 8-20 (inclusive) of gestation. The day of mating was designated Day 1 of gestation. All animals survived the duration of the study. The number of pregnant animals with live foetuses in utero on Day 30 of gestation was 17, 16, 18 and 16 in the 0, 8, 25 and 100 mg/kg bw/day groups respectively. Administration of 100 mg/kg bw/day was associated with maternal toxicity manifested as reduced bodyweight and food consumption. Incidences of “signs of diarrhoea” and “reduced faecal output” were also slightly increased. There were no maternal toxicologically significant findings attributable to 8 or 25 mg/kg bw/day. Macroscopic examination of the animals post mortem revealed no treatment-related findings. There was no adverse effect of the test substance on the number, growth or survival of the foetuses in utero and no evidence of teratogenicity. Evaluation of the minor external/visceral foetal defects and variants provided no evidence of an adverse effect of the test substance on development. Evaluation of the minor skeletal defects and variants revealed three findings. One of these, “odontoid incompletely ossified”, is a low incidence finding in the New Zealand White rabbit (historical foetal incidence 0.6-8.0%, litter incidence 0-33.3%). The incidence of foetuses with an incompletely ossified odontoid in the 100 mg /kg bw/day group was slightly increased as compared to controls based on foetal incidence (9%), but not on litter incidence (18.8%). This small increase is concluded to be incidental to treatment in the absence of any other indication of reduced ossification of the foetal skeleton. The two other skeletal findings, 27th pre-pelvic vertebrae and 13th thoracolumbar ribs of long length (more accurately termed “not short” or “full rib”), are common, high incidence findings in the New Zealand White rabbit. The incidence of foetuses with 27th pre-pelvic vertebrae (combined to include unilateral and bilateral) was increased in the 100 mg/kg bw/day group only (foetal incidence 44.1%, litter incidence 93.8%) and was of a slightly higher incidence than seen in control rabbits in the testing laboratory (foetal incidence 14.6-36.5%, litter incidence 52.9-89.5%, based on HCD contained in study report). The incidence of foetuses with 13th thoracolumbar ribs of long length was apparently increased in the 100 mg/kg bw/day group and also in the 25 mg/kg bw/day group. The incidence of this finding in both groups was within the range seen previously in control rabbits in the laboratory (foetal incidence 17.1-55.2%, litter incidence 50-100%), was not associated with any other skeletal findings and is concluded to not reflect an effect of treatment with the test substance. The maternal NOAEL was 25 mg/kg and the foetal NOAEL was 100 mg/kg.

Justification for classification or non-classification

There is no evidence to suggest that the test substance causes adverse effects on reproduction or development of the offspring. Therefore, the test substance is not classified for reproductive or developmental toxicityaccording to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.