Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
From January 11 to October 30, 2000.
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted according to internationally accepted testing guidelines and performed according to GLP. Justification for Read Across is reported in the endpoint summary and in the Category Justification Report attached to the Section 13 of this dossier.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report Date:
2001

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EPA OPPTS 870.3800 (Reproduction and Fertility Effects)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Portage, Michigan.
- Age at study initiation: approx 4 weeks of age.
- Weight at study initiation: 52 to 78 g.
- Housing: individually in suspended, stainless steel, wire-mesh type cages except during mating where females were individually housed in plastic cages containing wood chip bedding.
- Fasting period before study: none.
- Diet: certified meal Rodent chow #5002 from PMI Nutritionals International Inc. St. Louis, Missouri, available ad libitum.
- Water: tap water, available ad libitum.
- Acclimation period: during the 2-week acclimation period, all rats were observed daily for any clinical signs of disease, and all animals were given a detailed clinical examination prior to selection fur study.

ENVIRONMENTAL CONDITIONS
- Temperature: 18.89 to 24.44 °C
- Humidity: 34 to 77 %
- Photoperiod: fluorescent lighting was provided for approx. 12 hours per day.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS
The required amount of test article was weighed and suspended in vehicle and mixed using a Polytron tissue homogenizer. The solutions were stirred with a magnetic stir bar, and then dispensed into amber glass containers using a syringe. Fresh suspensions were prepared for each dose group weekly, and stored refrigerated and protected from light for approximately 1 week.

ADMINISTRATION
- Volume: the test article was administered at a constant volume of 10 ml/kg/day.

VEHICLE
- Concentration: 0.5 % aqueous carboxymethylcellulose.
- Storage: the contents of the container were mixed thoroughly and stored refrigerated when not in use.
- Lot/batch no.: 108H0070.
Details on mating procedure:
Alter a minimum of 10 weeks of treatment, the males were cohabited, 1 male to 1 female, with the corresponding females from the same group. The pairing was not random. Instead, the first male in the group was paired with the tlrst female in the same group. The pairings were assigned in sequential fashion until all animals in each group were cohabited. Mating took place in the cage of the male.

Vaginal smears (lavage) were performed daily on females during the mating period, and the presence or absence of sperm or vaginal plug was recorded. The day on which evidence of mating was observed was designated as Day 0 of gestation.
The rats were paired for a maximum of 14 consecutive days. When evidence of mating was noted, the female was removed from the cage of the male and individually housed. After the mating period, females with no positive evidence of mating were individually housed in a plastic cage with wood chip bedding.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples were diluted with methanol and analyzed by HPLC (UV detection) for verification of concentration.
Samples of vehicle and test article suspensions at each concentration were collected weekly for the first 4 weeks and every 4 wceks, thereaher, for analysis of test article concentration. The assayed concentrations ranged from 90 to 109 %.
Duration of treatment / exposure:
Treatment of the parental (P) generation began 10 weeks prior to mating and continued until euthanasia.
F1 and F2 generation offspring were potentially exposed in uterus and during lactation.
The F1 offspring selected to comprise the F1 parental group were exposed for ≥70 days prior to mating (beginning at age ≥ 28 days), until euthanasia.
Frequency of treatment:
Once daily.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 100, 300, 1000 mg/kg body weight/day
Basis:
nominal conc.
No. of animals per sex per dose:
26 males and 26 females.
Control animals:
yes, concurrent vehicle
Details on study design:
SELECTION OF THE F1 GENERATION AND TERMINATION OF THE P GENERATION
At weaning (Day 21 of lactation), a minimum of l male and 1 female from each litter in each group was randomly selected to become parents oft he next generation (F1, parents). A total of 26 males and 26 females per group were selected, but due to deaths, 24 to 26/sex/group were paired.
All pups were available for selection except those that were not expected to survive because of physical abnormalities. Records were maintained on any pup excluded from the selection process. The parentage of each weanling was ascertained to avoid sibling pairings.

After selection, the remaining offspring from each litter were euthanized and subjected to a necropsy.

All P females, those that delivered litters and those that did not deliver, were continued on treatment until the F2, pups were weaned and following assessment as to whether a second mating was warranted. After the Sponsor, in consultation with the Study Director, decided not to conduct a second mating of the P parental animals, the P females were euthanized and subjected to a necropsy.

F1 GENERATION OBSERVATIONS
The selected F1, rats were individually housed and treated for a minimum of 10 weeks prior to mating. Dosing initiated when all rats were at least 28 days of age. During the premating period, the following indices of sexual maturity were assessed:
- Vaginal Opening: beginning on Day 28 ofage, each female pup selected for the next generation was examined daily for the presence of vaginal opening until this developmental landmark was observed.
- Preputial separation: beginning on Day 35 of age, each male pup selected for the next generation was examined for preputial separation. Pups mat did not show complete retraction of the penile prepuce on Day 35 were examined daily until this developmental landmark was achieved.
- Mating, Gestatinn, and Lactation: following the procedures described in the P Breeding Procedures, the F1 parents were mated to produce the F2 generation. Care was taken to avoid sibling matings. Treatment continued through mating until termination.
After the mating period, the F1 males were individually housed and continued on treatment until euthanasia. All F1 females were continued on treatment until the F2 pups were weaned and following assessment as to whether a second mating was warranted. At weaning, the F2 offspring were euthanized and subjected to a necropsy.
Positive control:
Not used

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS
All rats were observed twice a day, 7 days a week, for morbidity, mortality, and signs of injury. Any positive findings were recorded. Mortality or other signs of toxicity were recorded on the day they were observed.

DETAILED CLINICAL OBSERVATIONS
A detailed clinical examination of each rat was performed once during each study week. The examination included, but was not limited to, observations of the general condition, skin, fur, eyw, ears, nose, oral cavity, thorax, abdomen, external genitalia, limbs and feet, as well as evaluation of respiration. Modifiers were included in the clinical sign when necessary to describe the location, size, shape, color, or other characteristics.

BODY WEIGHT
Individual body weights were recorded on the day of arrival, prior to randomization, the first day of dosing, and weekly during the study, with the following exceptions. Mated female rats were weighed cm Days 0, 7, 14, and 20 of gestation. Rats that delivered litters were weighed on Days O, 4, 7, 14, and 21 of lactation.

FOOD CONSUMPTION AND COMPOUND INTAKE
Individual food consumption was measured and recorded weekly during the study, with the following exception. During the mating period, food consumption was not measured because the animals were cohoused. Food consumption for mated female mts was recorded on Days 0, 7, 14, and 20 of gestation. Food consumption for rats that delivered litters was recorded on Days 0, 4, 7, 14, and 21 of lactation.
Oestrous cyclicity (parental animals):
Vaginal smears (lavage) were performed daily beginning 3 weeks prior to initiation of mating in all parental females to establish estrous cyclicity.
Sperm parameters (parental animals):
Sperm production, testes weighting, motility, morphology and analysis epididymal sperm count were performed following dissection.
Litter observations:
GESTATION PERIOD
Toward the end of the gestation period, females were examined twice daily for signs of parturllion. The females were allowed to give birth (F1).
The duration of gestation was calculated.

STANDARDISATION OF LITTERS
For each lilter, the day on which all pups were delivered was designated as Day 0 of lactation. ln 1 P and 1 F additional pups were found after the date initially designated as Day 0 of lactation. Day 0 of lactation was changed to the date these pups were found.

PARAMETERS EXAMINED
The litters were examined as soon as possible alter delivery, and the following parameters were recorded: litter size, nutnber of stillbom pups, number of live born pups, gross abnormalities ofthe pups, and pup weight by sex.
Litters were housed with their dams for 3 weeks alter birth. The dams and litters were observed twice daily for survival and behavioral alterations in nesting and nursing, and the presence of dead pups was recorded.
Pups were individually weighed by sex on Days 0, 4, 7, 14, and 21 of lactation. Detailed clinical observations were recorded at the same intervals.
Postmortem examinations (parental animals):
MORIBUNDITY
Any animal that showed signs of severe debility or toxicity, particularly if death appeared imminent, was euthanized for humane reasons and to prevent the loss of tissues through autolysis. All animals euthanized in exrremis or found dead were subjected to a necropsy.

SACRIFICE
Performed on:
- P, F1, and F2 animals dying spontaneously or euthanized in extremis
- P and F1 females that showed evidence of mating but failed to deliver
- P and F1 females that showed no evidence of mating and failed to deliver
- P and F1 animals surviving to scheduled termination
- F1 and F2 pups culled on day 4 of lactation
- three F1 pups not chosen to continue on study per sex from each litter at weaning
- all F2 pups at weaning

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations.

HISTOPATHOLOGY / ORGAN WEIGHTS
The following organs and tissues were prepared for microscopic examination: brain, adrenal glands, epididymis, kidney, liver, pituitary gland, prostate, seminal vesicles with coagulating glands, spleen, testis, and ovaries.
Postmortem examinations (offspring):
SACRIFICE
On Day 4 of lactation after weighing of each pup, litter size was reduced by random selection to 8 pups of equal sex distribution, whenever possible. The culled pups were euthanized and subjected to a necropsy. Any intact, dead pups were subjected to a necropsy.
The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at 21 days of age were subjected to postmortem examinations macroscopic and microscopic examination.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations
Statistics:
The following parameters were analyzed statistically for significant differences: body weights, change in body weights, food consumption, fertility indices, reproductive organ weights, sperm parameters, follicle counts, number of uterine implantations, litter parameters (size, weight, and viability), and developmental indices.

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
all observations noted are commonly seen in eaged rats of this age and strain and were not attributed to test article administration (P1 and F1 parent)
Body weight and weight changes:
no effects observed
Description (incidence and severity):
sporadic, statistically signiticant, but not considered to be test article-related (P1 and F1 parent)
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
sporadic, statistically signiticant, but not considered to be test article-related (P1 and F1 parent)
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
change in kidney weight was considered to be test article related (P1 and F1 parent)
Gross pathological findings:
no effects observed
Description (incidence and severity):
the findings were not considered to be test article related (P1 and F1 parent)
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
findings comparable to the control (P1 and F1 parent)
Other effects:
no effects observed
Description (incidence and severity):
Test substance intake: sporadic, statistically signiticant, but no dose-related changes were evident (P1 and F1 parent)

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
(P1 and F1 parent)
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
findings were not considered to be test article related (P1 and F1 parent)
Reproductive performance:
no effects observed
Description (incidence and severity):
no test article-related effects on reproductive performance noted (P1 and F1 parent)

Details on results (P0)

P GENERATION
MORTALITY
Three females died or were euthanized in exrremis during the P generation.
One female from the 1000 mg/kg/day group was found dead on Day 118 of the study. This female had red fluid and severe redness of the lungs at necropsy, and moderate congestion ofthe lungs at microscopic evaluation. Based on the macroscopic and microscopic evidence, technical (gavage) error may have been the cause of moribundity in this female.
One female from the 300 mg/kg/day group was euthanized in exzremis on Day 87 ofthe study and had distonded intestines, small spleen, enlarged mesenteric lymph nodes, and an ulceration of the palate. Microscopic evaluation did not reveal a cause of moribundity.
A second female from the 1000 mg/kg/day group was euthanized in eztremilr on Day 90 ofthe study. Other than distended intestines, no other findings were noted at necropsy and a cause of moribundity could not be ascenained from microscopic evaluation.
In the absence of any evidence of any toxicity at 1000 mg/kg/day, it is unlikely that these deaths were test article related.

CLINICAL SIGNS
No test article-related effects on survival or clinical observations were noted during the P parental generation.
Few clinical observations were noted during the prernating, gestation, or lactation periods in P generation animals and the incidences were similar in all groups or occurred sporadically in 1 to several animals within a single group with no dose-related patterns discemable. All observations noted are commonly seen in eaged rats of this age and strain and were not attributed to test article administration.

BODY WEIGHT and BODY WEIGHT CHANGES
No test article-related changes in body weight or body weight gain were noted during the premating, postmating (males), gestation, or lactation periods.
Sporadic, statistically signiticant increases or decreases were noted in the treatment groups when compared with controls, but there were no corresponding changes in food consumption during these intervals and a dose-related pattem was not evident. Consequently, these changes were not considered to be test article-related.

FOOD CONSUMPTION
No test article-related changes in food consumption were noted during the premating, postmating (males), gestation, or lactation periods.
Sporadic, statistically signiticant increases or decreases were noted in the treatment groups when compared with controls, but there were no corresponding changes in body weight and no dose-related pattems were evident. Therefore, these changes were not considered to be a result of test article administration.

MACROSCOPIC
No test article-related changes in macroscopic observations were noted during the P generation.
Very few abnormalities were noted at necropsy ofthe P generation animals. Since observations were only noted in 1 or 2 animals in any group, the observations noted were those commonly seen in rats of this age and strain and no dose-related pattems were evident, the findings were not considered to be test article related.

ORGAN WEIGHTS
A test article-related increase in absolute and relative kidney weight was noted in females at 1000 mg/kg/day. There were statistically significant increases in absolute and relative (to brain and body weight) kidney weight in females at 1000 mg/kg/day when compared with control values. Since similar changes in kidney weight were noted in F1 parents at 1000 mg/kg/day, the change in kidney weight was considered to be test article related.
Statistically significant decreases in absolute and relative (to brain weight) epididymides weight were noted at 300 and 1000 mg/kg/day when compared with controls. These changes were not correlated with any microscopic changes in the epididymis and there was no evidence of test article-related change in epididymal sperm concentrations. Since there was no microscopic correlation or a dose-related change in epididymal sperm concentrations, the decreases in epididymides weights were not considered to be test article related.
Also noted were statistically signilicant increases in absolute and relative (to brain weight) pituitary weight in females at 1000 mg/kg/day when compared with control values. Microscopic evaluation cf the pituitary did not reveal any changes, and consequently, these increases were not considered to be test article related.

MICROSCOPIC
No test article-related changes in microscopic incidences were noted following microscopic evaluation of tissues.
There were very few microscopic findings noted in either male or female parental animals.
Three males had abnormalities of the reproductive organs. One male from the 300 mg/kg/day group had small testes noted at necropsy with microscopic evidence of mild hypospermia noted in the left epididymis. Evaluation of sperm parameters in this animal revealed a reduced sperm count, 0 % motility, and 94 % abnormal sperm. This animal exhibited positive evidence of mating, but the female was not pregnant.
One male from the 1000 mg/kg/day group had small testes bilateral at necropsy and was diagnosed with aspem1ia ofthe epididymides. No sperm were found in this animal following sperm evaluation, and mating data indicated that this male did not mate or sire a litter.
A second male from the 1000 mg/kg/day group had small epididymides and llaccid left testis at necropsy. Microscopically, this animal had spermatid giant cells in the epididymides. Sperm evaluation of this animal revealed no abnormalities, and this animal sired a litter.
Occasionally, male rats have morphological problems and low sperm counts, as well as other changes in sperm parameters. Furthermore, there was 1 F1 control animal in this study that had no sperm. Since only 1 animal at 300 mg/kg/day and 1 animal at 1000 mg/kg/day were affected, these findings were not considered to be test article related.
These 2 animals contributed to the low fertility and mating indices noted during this generation.
The microscopic findings noted in females were similar in incidence among all groups, including controls, and none were considered to related to lest article administration.

REPRODUCTIVE PERFORMANCE
There were no test article-related effects on reproductive performance noted during the P generation.
There were slight decreases in the male and female fertility indices in all treatment groups when compared with controls. Historical control ranges for rnale and female fertility indices are 65.4 to 90.0 and 65.4 to 100.0 percent, respectively. Historical control ranges for both male and female mating indices are 88.5 to 100.0 percent. These values were neither statistically different from concurrent controls nor out of the range of historical control data for this laboratory and, consequently, were not considered to be test article related.
Copulatory interval was statistically higher at 1000 mg/kg/day when compared with controls. This was due mainly to 2 females who did not become pregnant until 8 and 13 days after pairing. These females appeared to be cycling normally and the 2 males with whom these females had been paired appeared to have normal sperm parameters. Since no morphological abnomralities were found, it is questionable that the change in copulatory indexat 1000 mg/kg/day is toxieologically meaningful.
All other reproductive perfomiance parameters, including female and male mating indices. female and male fecundity indices, estrous cycle length and duration, in the treatment groups were comparable with controls, and no statistically sigtiticant or toxicologically relevant changes were obtained.
Slight, though statistically significant, decreases in the majority of sperm evaluation parameters were noted nt 300 mg/kg/dey when compared with controls. Since similar decreases were not noted at 1000 mg/kg/day, however, these changes were not considered to be toxicologically meaningful or test article related.

F1 GENERATION:
CLINICAL SIGNS AND MORTALITY
No test article-related changes in survival or clinical observations were noted during the F1 parental generation.
A total of 0, 2, 1, and 5 animals from the 0, 100, 300, and 1000 mg/kg/day groups died or were euthanized in extremis during the study.
Two females from the 100 mg/kg/day group died prior to initiation of dosing for the F1 parental generation. The macroscopic findings included distended intestines and stomach and/or red, tarry material on the stomach mucosa. Microseopically, immature ovaries and uteri were noted, but a cause of death was not ascertained. Since these animals were not dosed and were only potentially exposed to the test article via the milk during the lactation period and based on the lack of any test article-related tindings in pups necropsied during lactation and weaning, the deaths of these animals were considered to be incidental and unrelated to test article administration.
One male from the 300 mg/kg/day group died on Day 16 of study. Clinical signs included skin cold to touch, decreased activity, and material around eyes and nose. Macroscopic findings included distention and reddish brown fluid in the stomach and intestines, and microscopic evaluation revealed no cause of death.
Two males and l female from the 1000 mg/kg/day group were euthanized in extremis due to moderately to severely swollen feet/hind limbs. No abnormalities were noted microscopieally for these animals, although the feet (gross lesions) were not examined. The swollen limbs were the major reason for euthanasia of these animals and may have been a result of mechanical injury. There were no other animals at 1000 mg/kgjday exhibiting swollen limbs during the study. Based on this, the gross finding was not considered to be a result of test article administration, and consequently, the deaths of these animals were not attributed to test article administration.
An additional male and female from the high-dose group died during the study. Macroscopic and microscopic evaluation ofthe female suggested that the cause of death may have been technical error (gavage). The rna.le had no macroscopic or microscopic tindings to suggest a cause of death. ln the absence of any evidence of toxicity at 1000 mg/kg/day, it is unlikely that this death was test article related.
There were no changes in clinical observations between control and treatment groups during the premating, postmating (males), gestation, or lactation periods. The incidences were comparable between control and treatment groups or the iindings were those commonly seen in caged rats of this age and strain and were not attributed to test article administration.

BODY WEIGHT and BODY WEIGHT CHANGES
No test article-related changes in body weight or body weight gain were noted during the F1prernating, postrnating (males), gestation, or lactation intervals.
Statistically significant changes (increases and decreases) in body weight gain were noted very sporadically in the treatment groups when compared with controls. Since there was no dose-related pattern and the changes were not corroborated by statistically significant changes in food consumption, however, these changes were not considered to be test article related. Body weight was comparable between controls and treatment groups during all intervals, and no statistically signiticant changes were noted.

FOOD CONSUMPTION
No test article-related changes in food consumption were noted during the F1 premating, ppstmating (males), gestation, or lactation intervals.
A statistically significant decrease in food consumption was noted in males at 300 mykg/day during the first 2 weeks ofthe premating period, but since a similar change was not noted at 1000 mg/kg/day and no other statistically significant changes in food consumption were seen, these changes were not considered to be test article related. No other statistically significant or toxioologically relevant changes in food consumption were noted in males or females during the remainder ofthe F1 parental generation.

MACROSCOPIC
No test article-related changes in necropsy findings were noted in the F1 parental generation.
Very few findings were noted at neoropsy of the F1 parents. Since observations were only noted in 1 or 2 animals in any group, the observations noted were those commonly seen in rats of this age and strain, and no dose-related pattems were evident, the findings were not considered to be test article related.

ORGAN WEIGHT
Test article-related changes in kidney weight were noted at 300 and 1000 mg/kg/day.
There were statistically significant increases in absolute and relative kidney weight in both males-and females at 1000 mg/kg/day as well as relative kidney (to body weight) in females at 300 mg/kg/day. No microscopic evidence of kidney changes was noted. However, there was a clear change noted in both males and females at 1000 mg/kg/day, and this was considered to be test article related. The statistical change in 300 mg/kg/day females was considered to be spurious since no changes in absolute weight or the kidney weight relative to brain weight were affected, and similar decreases were not seen in 300 mg/kg/day males.
Absolute liver weight was statistically lower in males at 300 and 1000 mg/kg/day when compared with controls. Relative (to brain and body) liver weight was statistically lower than controls in males at 300 mg/kg/day, but this was not noted in males at 1000 mg/kg/day.
No changes in liver weight were noted in the females. Also noted were statistically significant increases in absolute and relative adrenal weight in females, but not males, at 1000 mg/kg/day. Therc were no corresponding macroscopic changes noted in these organs, and, consequently, the changes in the adrenal and liver weights were considered to be sporadic and unrelated to test article administration.

MICROSCOPIC
No test article-related microscopic changes were noted in parental males or females.
The incidences of all findings were either comparable between control and treatment groups or those commonly sccn in rats of this age and strain and considered to be unrelated to test article administration. One control male had epididymal aspermia and testicular atrophy and one male at 1000 mg/kg/day had trace degeneration of the seminiferous tubules of the testis. Neither of these males sired a litter.

REPRODUCTIVE PERFORMANCE
No test article-related changes in F, parental reproductive performance were noted.
Slight decreases in male and female mating and fertility indices were noted at 1000 mg/kg/day. Historical control ranges for male and female fertility indices are 6544 to 90.0 and 65.4 to 100.0 percent, respectively. Historical control ranges for both male and fcmale mating indices are 88.5 to 100.0 percent. These changes were not statistically different when compared with concurrent controls and/or out of the range of historical controls for this laboratory, howcvcr, and consequently were not considered to be toxicologically relevant or test article related. Fccundity index was slightly, but not statistically higher in males and females at 1000 mg/kg/day when comparcd with controls. A statistically signiticant increase in the number ofestrous cycles was noted in females at 300 mg/kg/day when compared with controls. Since no changes were noted in females from the high-dose group, this increase was not considered to be a result of test article administration. Length of estrous cycle was comparable between controls and all treatment groups.
No statistically significant or toxicologically relevant changes in primordial follicle counts were noted in the treatment groups when compared with controls. A slight decrease in follicle count was noted at 300 mg/kg/day, but since follicle count at 1000 mg/kg/day was comparable with controls, a dose response was not evident. Therefore, this decrease was considered to be spurious and unrelated to test article administration.
All spenn parameters, including motility, concentration, homogenization resistant sperm head count, daily sperm production, spermatogenic efticiency, and percent abnormal sperm, were comparable among all treatment groups when compared with controls, and no test article·related changes were noted. lt should be noted that 1 male in the control group had no sperm found at evaluation of sperm parameters. This male also had morphological evidence of infertility.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
Toxicity
Effect level:
300 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Dose descriptor:
NOAEL
Remarks:
Reproduction
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Dose descriptor:
NOAEL
Remarks:
Growth and Development
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: Generation: F1 and F2 (migrated information)

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
very few clinical observations were noted, but incidences were either comparable between control (F1 and F2)
Mortality / viability:
no mortality observed
Description (incidence and severity):
(F1 and F2)
Sexual maturation:
no effects observed
Description (incidence and severity):
comparable between the control and treatment groups (F1 and F2)
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
no statistically signiticant or toxicologically meaningful changes were noted (F1 and F2)
Gross pathological findings:
no effects observed
Description (incidence and severity):
few observations not attributed to test article administration (F1 and F2)

Details on results (F1)

F1 GENERATION - LITTER DATA
PARTURITION DATA
No test article·relnted changes in litter parameters were noted for the F1 litters.
There were no statistically significant or toxicologically relevant changes in gestation length, litter size on Day 0, numbers of live bum or stillborn pups/litter un Day 0, or sex ratio in the treatment groups when compared with controls during the F1 litter generation.

F1 OFFSPRING SURVIVAL
No test article-related change in survival was noted for the F1 litters. Survival at birth and during lactation was comparable in control and treatment groups.

CLINICAL OBSERVATIONS
No test artice-related changes in observations were noted during the F1 lactation period.
Very few clinical observations were noted, but incidences were either comparable between control and treatment groups or commonly seen in rats of this age and strain, and no dose-related pattems were evident. Therefore, none of the observations noted were considered to be a result oftest article administration.

OFFSPRING GROWTH
No test article-related changes in growth were noted for the F1 litters.
A statistically significant increase in body weight (males and total pups) was noted at birth in pups at 1000 mg/kg/day when compared with controls. The increase was not considered to be adverse or biologically meaningful, however, since the difference from controls was less than 10 %. Furthermore, pup weights at 1000 mg/kg/day were comparable with controls throughout the remainder of the lactation period and similar increases at birth were not noted in F1 pups. Consequently, no test article-related changes in growth were noted during the F1 lactation period.

MACROSCOPIC OBSERVATIONS
No test article-related changes in macroscopic observations were noted in F1 offspring euthanized after weaning.
Very few macroscopic observations were noted at necropsy. The findings observed were either seen in only 1 to 2 animals per group, were of similar incidence in control and treatment groups, or are commonly seen in rats of this age and strain and were not attributed to test article administration.

ORGAN WEIGHTS
No test article·related changes in organ weights were noted in F1 offspring euthanized after weaning.
No statistically signiticant or toxicologically meaningful changes in organ weights were noted in treatment groups when compared with controls.

SEXUAL MATURATION
No test article-related changes in sexual maturation were noted in F1 offspring.
The mean day on which vaginal opening and preputial separation occurred was comparable between the control and treatment groups.


F2 GENERATION:
PARTURITION
No test anticle·related changes were noted.
A statistically significant increase in the number of implant scars was noted at 100 mg/kg/day when compared with controls. Since a similar change was not noted at either 300 or 1000 mg/kg/day and consequently no dose response relationship was evident, this change was not considered to be test article related. Also noted was a statistically significantly higher sex ratio (% males) at birth and Day 4 of lactation for pups in the 300 mg/kg/day group. This statistical change appeared to be due to the fact that the % males/litter in the concurrent control group was low (42.58 % at Day 0 and 42.42 % at Day 4), rather than an increase in the % males/litter at 300 mg/kg/day. Furthermore, a similar change in sex ratio was not evident at 1000 mg/kg/day. Consequently, this statistical change in sex ratio at 300 mg/kg/day was not considered to be tcst article related. All other parturition parameters, including number of females delivering litters, litter size (total and live born pups) at birth, gestation, live birth, and stillbom indices, and gestation length were comparable between controls and treatment groups.

F2 OFFSPRING SURVIVAL
There were no test article-related changes in survival in F2 pups during lactation.
Pup survival during the first 4 days and throughout lactation was comparable between control and treatment groups, and no statistically significant changes in viability or lactation indices were noted.

CLINICAL OBSERVATIONS
Very few clinical observations were noted, and the incidences were either comparable between control and treatment groups or commonly seen in rats of this age and strain. Consequently, no clinical signs were considered to result from test article administration.

OFFSPRING GROWTH
No adverse, test article-related changes in offspring growth were noted in F2 pups during lactation.
Sporadic, statistically significant increases in pup body weights were noted in all the treatment groups when compared with controls during the lactation period. Based on the fact that all treatment groups were increased, this effect appeared to be test article related, but was not considered to be adverse.

MACROSCOPIC OBSERVATIONS
No test article-related changes were noted at necropsy.
As was noted in the F2 offspring, very few Endings were observed at necropsy. All observations noted were either seen with equal frequency in control and treatment groups or occurred in 1 or 2 animals within a group and were not considered to be toxicologically relevant. No dose related pattem in the incidence of findings was observed; thus, all findings were considered to be sporadic in nature or commonly seen in rats of this age and strain and unrelated to test article administration.

ORGAN WEIGHTS
No test article-related changw in brain, spleen or thymus weights were noted in the treatment groups when compared with controls.
As was seen during the lactation period, terminal body weight of pups in the treatment groups was higher than controls, and the changes were statistically significant in males at 300 and 1000 mg/kg/day when compared with controls. In these same groups, brain weight was also statistically increased when compared with controls. Although not statistically signiticant, body weights in female pups were also increased and brain weight in females at 1000 m g/kg/day was statistically higher when compared with controls. As stated earlier, the increases in body weight were not considered to be adverse, even though they may have been test article related, and the increase in brain weight was considered to be a consequence of the increased body weight. In all cases, the increases in both brain and body weight were less than 10 % when compared with controls, and consequently, the changes were not considered to he biologically relevant.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Based on the results, the No Observed Adverse Effect Level (NOAEL) for parental toxicity was 300 mg/kg day and for parental reproductive performance, the NOAEL was l000 mg/kg day. For offspring growth and development, the NOAEL was also 1000 mg/kg day.
Executive summary:

Method

The effects of the test article on the integrity and performance of male and female reproductive systems, including gonadal function, estrous cycle, mating behaviour, conception, gestation, parturition, lactation, weaning and growth and development of the spring were assessed testing rats, according to the EPA OPPTS 870.3800 (Reproduction and Fertility Effects) guideline.

Animals were exposed to the test articloe daily, via oral gavage at the dose levels of 0, 100, 300 and 1000 mg/kg bw, at a constant volume of 10 ml/kg. The duration of the whole study was approx. 9 months.

Results

Based on the results, the No Observed Adverse Effect Level (NOAEL) for parental toxicity was 300 mg/kg day and for parental reproductive performance, the NOAEL was l000 mg/kg day. For offspring growth and development, the NOAEL was also 1000 mg/kg day.