Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: the male rats were received from Charles River Laboratories, Inc., Raleigh, North Carolina, the female rats were received from Charles River Laboratories, Inc., St. Constant, Quebec.
- Animal Husbandry: animal housing and care were based on the standards established by the Associatio n for Assessment and Accreditation of Laboratory Animal Care, international (AAALAC) and the guidelines set forth in the Guide for the Care and Use of Laboratory Animals, NIH Publication No. 96-03, 1996.
- Weight at study initiation: ranging from 162 to 201 grams.
- Age at study initiation: approximately 8 weeks.
- Housing: animals were housed individually during acclimation and while on study (except during cohabitation) in suspended stainless steel cages.
- Diet: PMI Cerified Rodent Meal #5002 (Purina Mills), ad libitum. The feed was analyzed by the supplier for nutritional components and environmental contaminants.
- Water: municipal tap water, ad libitum. The tap water was purifief by reversed osmosis and UV irradiation and supplied to the animals by an automatic watering system or water bottles. Water supplying the facility is analyzed for contaminants according to SLI Standard Operating Procedures.
- Acclimation period: the animals were acclimated to the laboratory conditions for a period of 36 days prior to initiation of dosing.
- Health check: the animals were examined upon receipt and daily thereafter during acclimation for signs of physical or behavioral abnormalities. General health/mortality checks were performed twice daily, in the morning and afternoon. individual body weights were recorded on the day following receipt and priorto randomization on day -1.

ENVIRONMENTAL CONDITIONS
- Temperature: 18-26 °C
- Humidity: 30-70 %
- Air changes: 10-15 air changes per hr.
- Photoperiod: 12 hrs light/12 hours dark cycle.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS
For each dose group, a specified amount of test item was weighed into a pre-calibrated beaker. A sufficient quantity of corn oil was added to the beaker to achieve the desired concentration and the mixture wasstirred for 30 minutes. Each test article solution was prepared fresh weekly, dispensed into daily aliquots and stored in amber glass containers at ambient conditions. During each preparation, corn oil was dispensed into daily aliquots for administration to control animals.
The physical state of the control and each dosing solution was recorded during each preparation.
The vehicle was a clear yellow liquid and each test article preparation was a clear yellow solution.
All dosing solutions were prepared and dispensed under a fume hood. The dosing solutions were stirred continuously prior to and during dosing.

ADMINISTRATION
- Dosage Volume: 2 ml/kg.
- Administration: all doses were administered using a plastic syringe with an attached gavage needle. Individual doses were based on the most recent body weight data.
Details on mating procedure:
- M/F ratio per cage: following a minimum of 14 days of treatment for the F0 females, each female designated for the mating phase was cohabited with a single male from the same treatment group (1:1 pairing) in the toxicity study.
- Proof of pregnancy: each mating pair was observed daily for evidence of copulation. Evidence of mating was determined by the presence of a copulatory plug in the vagina or a sperm positive vaginal smear. The day evidence of copulation was confirmed was designated as day 0 of gestation and thefemale was returned to her cage
- Unsuccessful attempts: if no evidence of copulation was observed after 14 days of mating, the female was separated from the male and the mating phase was concluded. F0 females with no evidence of mating were examined for signs of parturition beginning 19 days following initiation of the mating phase.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Prior to initiation of the study,homogeneity and stability analyses were performed on concentrations of the test article in the vehicle which encompassed the low- and high-dose concentrations.Homogeneity analyses were performed on duplicate samples taken from the top, middle and bottom of the two mixtures.Stability of the test article in the vehicle was evaluated on duplicate samples on day 0 and at 3 days and 10 days of room temperature storage.Concentration verification analysis was performed on the vehicle and each test article dosing solution prepared for study weeks 0, 2, 4 and 6.
Duration of treatment / exposure:
The animals were dosed for a minimum of 14 daysn prior to mating and continuing through lactation day 3.
Frequency of treatment:
Single dose, daily.
Details on study schedule:
Following a minimum of 14 days of treatment for the F0 females, each female designated for the mating phase was cohabited with a single male from the same treatment group (1 :1 pairing) in the toxicity study. Each mating pair was observed daily for evidence of copulation. Evidence of mating was determined by the presence of a copulatory plug in the vagina or a sperm positive vaginal smear. The day evidence of copulation was confirmed was designated as day 0 of gestation and the female was returned to her cage. If no evidence of copulation was observed after 14 days of mating, the female was separated from the male and the mating phase was concluded.
On approximately gestation day 18, females with confirmed copulation were transferred to individual plastic boxes containing nesting material. The day on which parturition was complete was designated as lactation day 0. The females and their offspring remained together until in-life conclusion on lactation day 4.
Doses / concentrationsopen allclose all
Remarks:
Dose level: 0, 75, 225, 750 mg/kg bw/day (nominal)
Remarks:
Dose concentration: 0, 37.5, 112.5, 375 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10 female rats per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: the high-dose level was expected to produce some toxic effects, but not excessive lethality. The mid-dose level was expected to produce no to minimal observable effects and the low-dose level was expected to produce no observable effects. Dosage levels were selected in an attempt to produce graded responses to the test article.
- Preliminary study: prior to initiation of the toxicity and reproduction/developmental screening studies, a 14-day range-finding study was conducted in rats to aid in selection of dosage levels.
- Rationale for animal assignment: animals determined to be suitable test subjects were randonly assigned to groups using a computer randomization program. The program ranked the animals according to day -1 body weights and randomly assigned the animals to study groups in a stratified block design.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS
Cage-side observations for overt signs of toxicity were performed once daily within approximately one-half hour to two hours following dosing.

DETAILED CLINICAL OBSERVATIONS
Mortality/general health checks were performed twice daily, in the morning and afternoon.Detailed clinical observations were performed a minimum of weekly and on the day of scheduled euthanasia.

BODY WEIGHT
Individual body weights were recorded on days 0, 3, 7 and 12. When positive evidence of mating was detected, the females were weighed on gestation days 0, 7, 14 and 20. Following parturition, the females were weighed on lactation days 1 and 4. Females without evidence of mating were weighed twice weekly until euthanasia.

FOOD CONSUMPTION
Individual food consumption was recorded on the same clays as body weights (except during cohabitation).

PARTURITION
Each F0 female was observed for signs of parturition a minimum of twice daily. The time parturition was first detected and the time parturition was completed were recorded, when possible. Signs of difficult or prolonged delivery were recorded, if observed. Abnormal nursing and nesting behaviors were recorded, if observed. F0 females with no evidence of mating were examined for signs of parturition beginning 19 days following initiation of the mating phase.
Litter observations:
On lactation day 0, pups in each litter were individually identified using a toe marking (tattooing) system.
The following parameters were recorded foreach pupduring lactation:
- Viability: daily from days 0 to 4
- External Examinations: days 0 and 4
- Sex Determinations: days 0 and 4
- Body Weights: days 1 and 4
Postmortem examinations (parental animals):
SACRIFICE
F0 females were euthanized by carbon dioxide inhalation followed by exsanguination and subjected to a complete gross necropsy examination. F0 females that delivered were euthanized on lactation day 4. Females that failed to deliver were euthanized 25 days after evidence of mating was first detected (post breeding day 25). F0 females with no evidence of mating were euthanized 25 days after completion of the mating period (post breeding period day 25). Females with total litter loss were euthanized and necropsied on the day that no surviving pups remained.

GROSS NECROPSY
The necropsy examination included examination of the external surfaces of the body, all orifices, and the cranial, thoracic, abdominal and pelvic cavities and their contents. Uterine contents were examined and the number of implantation sites and number of corpora lutea (per ovary) were recorded. Uteri with no macroscopic evidence of implantation were opened and placed in 10% aqueous ammonium sulfide solution for detection of implantation sites. All abnormalities were recorded.

HISTOPATHOLOGY / ORGAN WEIGHTS
The following organs and tissues were preserved in 10 % neutral buffered formalin for possible future histopathological examinination: accessory genital organs (uterus and vagina), Adrenals, All gross lesions, Aorta, Brain (including sections of medulla/pons, cerebellar cortex and cerebral cortex), Cecum, Colon, Duodenum, Esophagus, Exorbital lachrymal glands, Eyes with optic nerve, Femur (including articular surface) and bone marrow, Heart, Ileum, Jejunum, Kidneys, Liver (three sections collected), Lungs (infused with formalin) with bronchi, Mammary gland, Mandibular lymph node, Mediastinal lymph node, Mesenteric lymph node, Ovaries, Pancreas, Peripheral nerve (sciatic), Pituitary, Rectum, Skeletal muscle (thigh), Skin, Spinal cord (cervical, midthoracic and lumbar), Spleen, Sternum with bone marrow, Stomach (glandular/nonglandular), Submaxillary salivary gland, Thymus, Thyroid/parathyroid, Tongue, Trachea, Urinary bladder.
Postmortem examinations (offspring):
SACRIFICE
All surviving pups were euthanized on lactation day 4 by an intraperitoneal injection of sodium pentobarbital and examined macroscopically for structural abnormalities or other pathological changes.

GROSS NECROPSY
During the examination, special attention was paid to the organs of the reproductive system. All gross lesions were preserved in 10 % neutral buffered formalin for possible future histopathological examination.
F1 pups that were stillborn or died were subjected to a gross necropsy examination, with emphasis on developmental morphology. All internl gross lesions (except atelectasis; and scabbing, subcutaneous hemorrhage or other lesions resulting from apparent cannibalism) were preserved in 10 % neutral buffered formalin for possible future histopathological examination.
Statistics:
Statistical analysis were performed using the SU Alpha ReproTox computer system. Data, including body weights, body weight changes, food consumption and mean live litter size were analyzed by ANOVA. lf significance was observed with ANOVA, contral to treatment group comparisons were performed using Dunnett's test. Count data were analyzed using R x C Chi-Square test followed by Fisher's Exact Test for copulation and fertility indices, pup sex ratios, the number of live and dead pups per group (an lactation day 0) and pup survival (after lactation day 0). All analyses were two-tailed with a minimum significance level of 5 % (p<0.05).

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
clinical signs were observed primarily for females in the 225 and 750 mg/kg/day groups
Mortality:
no mortality observed
Description (incidence):
no mortality occurred in the F0 females
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
mean body weight change in the 750 mg/kg/day group was statistically lower than controls during gestation days 0-7
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
mean food consumption in the 225 and 750 mg/kg/day groups was statistically lower than controls during days 0-3
Histopathological findings: non-neoplastic:
not specified

Reproductive function / performance (P0)

Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
the live birth index was statistically lower in the 750 mg/kg/day group compared to controls, and the number of stillborn pups in the 750 mg/kg/day group was statistically higher than controls

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No mortality occurred in the F0 females. One F0 female in the control group with no evidence of mating was euthanized on post breeding period day 25. Two F0 females in the control group, one F0 female in the 75 mg/kg/day group and one F0 female in the 750 mg/kg/day with evidence of mating failed to deliver and were euthanized on post breeding day 25. Six F0 females in the 750 mg/kg/day group were euthanized due to total litter loss. These females were euthanized on lactation days 1-4, the day all pups in the litter were found dead. Ali other F0 females in the control, 75, 225 and 750 mg/kg/day groups survived to scheduled euthanasia on lactation day 4. Clinical signs were observed primarily for females in the 225 and 750 mg/kg/day groups. The clinical signs included a low incidence of urine stain and pre dose salivation, a low incidence of post dose feces small in size and post dose salivation in the 225 mg/kg/day group, and a low incidence of decreased activity, skin pale in color, unkempt appearance, rough coat, post dose dark material around nose, post dose few feces, and pre dose and post dose salivation and post dose wobbly gait in the 750 mg/kg/day group. In addition, one female in the 750 mg/kg/day group appeared to have prolonged parturition. Remarkable clinical signs for females in the 75 mg/kg/day group were limited to a single incidence of urine stain in two females.

BODY WEIGHT AND BODY WEIGHT CHANGES (PARENTAL ANIMALS)
Mean body weight change of F0 females in the 750 mg/kg/day group was statistically lower than controls during gestation days 0-7. No other statistically significant differences in mean body weight change were noted in the F0 females prior to mating or during gestation and lactation. However, F0 females in the 225 and 750 mg/kg/day groups lost slightly more body weight than controls during days 0-3 (prior to mating) and mean body weight change of F0 femalesin the 750 mg/kg/day group appeared to be slightly lower than controls during lactation days 1-4.There were no statistically significant differences in mean body weights between F0 females in the control, 75, 225 and 750 mg/kg/day groups. However, mean body weight of F0 females in the 750 mg/kg/day group was slightly (approximately 6 %) lower than controls on gestation day 7. At all other intervals, mean body weights of F0 females in the 750 mg/kg/day group were within approximately 4 % of controls. Mean body weights of F0 females in the 75 and 225mg/kg/day groups were within 4 % of controls prior to mating and during gestation and lactation.

FOOD CONSUMPTION (PARENTAL ANIMALS)
Mean food consumption (grams/animal/day) of F0 females in the 225 and 750 mg/kg/day groups was statistically lower than controls during days 0-3. In addition, mean food consumption appeared to be slightly, but not statistically lower than controls for F0 females in the 750 mg/kg/day group during gestation days 0-7 and lactation days 1-4. No other toxicologically meaningful differences in mean food consumption were noted between the control, 75, 225 and 750 mg/kg/day groups.

REPRODUCTIVE FUNCTION (PARENTAL ANIMALS)
The F0 female mating index was 100 % in the control, 75, 225 and 750 mg/kg/day groups. The F0 female fertility index was 77.8 % in the control group, 90 % in the 75 and 750 mg/kg/day groups and 100 % in the 225 mg/kg/day group. The F0 mean gestation length was 21.9 days in the control group and 22.0 days in the 75, 225 and 750 mg/kg/day groups. A total of seven females in the control group, nine females each in the 75 and 750 mg/kg/day groups and ten females in the 225 mg/kg/day groups completed delivery. However, two females in the control group, four females in the 75 mg/kg/day group, three females in the 225 mg/kg/day group and seven females in the 750 mg/kg/day group had stillborn pups.

GROSS PATHOLOGY (PARENTAL ANIMALS)
Two F0 females in the 750 mg/kg/day group with total litter loss had pup tissue mixed with ingest in the stomach. No other remarkable findings were noted at necropsy far F0 females in the control, 75, 225 or 750 mg/kg/day groups.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
750 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
reproductive performance

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
in the 225 and 750 mg/kg/day groups
Description (incidence and severity):
desquamation in the 225 and 750 mg/kg/day groups and a low incidence of gasping and skin pale in color, cool to the touch, skin with shiny appearance and skin appears tight
Description (incidence and severity):
number of pups dying, missing and/or cannibalized during lactation days 1-4 and the number of litters at 750 mg/kg/day were statistically higher than controls
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
mean weights in the 750 mg/kg/day group were statistically lower than controls on lactation days 1 and 4
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
no remarkable gross necropsy findings in the 75 and 225 mg/kg/day groups

Details on results (F1)

VIABILITY
The mean number of F1 pups delivered was comparable in the control, 75, 225 and 750 mg/kg/day groups. However, the live birth index (number of live born pups/total number of pups delivered) was statistically lower in the 750 mg/kg/day group compared to controls. In addition, the number of stillborn pups in the 750 mg/kg/day group was statistically higher than controls. A total of five F1 pups in the 750 mg/kg/day group was born alive and died later on lactation day 0. In addition, 79 pups in the 750 mg/kg/day group died or were missing and/or cannibalized during lactation days 1-4 compared to 6 pups in the control group, 5 pups in the 75 mg/kg/day group and 8 pups in the 225 mg/kg/day group. A total of six litters with live born pups had no live pups remaining on lactation day 4 (total litter loss) in the750 mg/kg/day group. The number of pups surviving to lactation day 4 was 99 in the control group, 131 in the 75 mg/kg/day group, 137 in the 225 mg/kg/day group and 25 in the 750 mg/kg/day group. The viability index on lactation day 4 was 94.3% in the control group, 96.3 % in the 75 mg/kg/day group, 94.5 % in the 225 mg/kg/day group and 22.9 % in the 750 mg/kg/day group. The number of F1 pups dying, missing and/or cannibalized during lactation days 1-4, the number of litters with total litter loss and the number of surviving pups on lactation day 4 in the 750 mg/kg/day group were statistically different from controlsThe mean live pups/litter in the 750 mg/kg/day group was slightly, but not statistically lower than controls on lactation day 0 and statistically lower than controls on lactation days 1-4. F1 pup viability in the 75 and 225 mg/kg/day groups was comparable to controls on lactation days 0-4. The F1 pup sex ratio was comparable between the control, 75, 225 and 750 mg/kg/day groups on lactation days 0 and 4.

CLINICAL SIGNS
Remarkable F1 pup observations during lactation days 0-4 included desquamation in the 225 and 750 mg/kg/day groups and a low incidence of gasping and skin pale in color, cool to the touch, skin with shiny appearance and skin appears tight- restricts movement in the 750 mg/kg/day group. No remarkable F1 pup observations were noted in the 75 mg/kg/day group.

BODY WEIGHT
Mean F1 pup weights in the 750 mg/kg/day group were statistically lower than controls on lactation days 1 and 4, and outside the range of SU historical control data. Mean F1 pup weights in the 75 and 225 mg/kg/day groups were comparable to controls on lactation days 1 and 4.

GROSS PATHOLOGY
Remarkable gross necropsy observations for F1 pups stillborn included a single incidence of cleft palate, a single incidence of edema, increased incidences of atelectasis, milk not present in the stomach, and dermal hypoplasia in the 750 mg/kg/day group. No remarkable gross necropsy findings were noted for F1 pups stillborn in the 75 and 225 mg/kg/day groups.
Remarkable gross necropsy findings for F1 pups born alive but found dead during lactation days 0-4 included scabbing, desquamation, dermal hypoplasia and an increased incidence of milk not present in the stomach in the 750 mg/kg/day group. In addition, autolysis was observed for 22 F1 pups found dead in the 750 mg/kg/day group. No remarkable gross necropsy findings were noted for F1 pups found dead in the 75 and 225 mg/kg/day groups.Remarkable gross necropsy findings for F1 pups at scheduled euthanasia on lactation day 4 included scabbing and desquamation in the 225 and 750 mg/kg/day groups. No remarkable gross necropsy findings were noted for surviving F1 pups in the 75 mg/kg/day group.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
225 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: significantly decreased live birth, decreased pup survival during the lactation phase and a decreased mean pup body weight in the high-dose group

Overall reproductive toxicity

Reproductive effects observed:
no
Lowest effective dose / conc.:
750 mg/kg bw/day (nominal)

Any other information on results incl. tables

Results of the homogeneity analyses revealed that the average recoveries of the 37.5 and 375 mg/ml dosing formulations were within 4.9 % of the nominal concentration, indicating that the formulations were homogeneous. Results of the stability analyses revealed that the 37.5 and 375 mg/ml dosing formulations were stable for 10 days following room temperature storage. The average results of the concentration verification analyses were within 5.3 % of the nominal concentrations. No substance was detected in the vehicle control samples analyzed during the course of the study.

Applicant's summary and conclusion

Conclusions:
NOAEL rat, reproductive toxicity: 225 mg/kg bw/day
NOAEL rat, fertility ≥ 750 mg/kg bw/day
Executive summary:

The study was conducted to provide initial information on the repeated-dose systemic toxicity of test item, with emphasis on potential neurological effects, and serve as a screening study for potential reproductive and developmental effects in male and female rats.

The test article was administered once daily (seven days a week) by oral gavage for a minimum of 14 days prior to mating and continuing through lactation day 3. Detailed clinical observations were performed a minimum of weekly until evidence of mating was detected, daily during gestation and lactation and on the day of scheduled euthanasia. After a minimum of 14 days of treatment, females were cohabited with males from the same treatment group in the toxicity study. Following parturition, pup viability, sex determinations, observations and body weights were recorded at specified intervals during lactation days 0 and 4. Surviving females and their pups were euthanized and necropsied on lactation day 4. Selected tissues and organs were collected from the females at necropsy.

No mortality occurred for the F0 females in the reproduction/developmental screening study. Clinical signs were observed for F0 females in the 225 and 750 mg/kg/day groups. The clinical signs included urine stain, predose salivation, postdose feces small in size and postdose salivation in the 225 mg/kg/day group, and decreased activity, skin pale in color, unkempt appearance, rough coat, postdose dark material around nose, postdose few feces, predose and postdose salivation and postdose wobbly gait in the 750 mg/kg/day group. ln addition, one female in the 750 mg/kg/day group appeared to have prolonged parturition. Remarkable clinical signs for females in the 75 mg/kg/day group were limited to a single incidence of urine stain in two females.

Mean body weight change of F0 females in the 750 mg/kg/day group was statistically lower than controls during gestation days 0-7. There were no statistically significant differences ir mean body weights; however, mean body weight of F0 females in the 750 mg/kg/day group was slightly (approximately 6 %) lower than controls on gestation day 7. No other toxicologically meaningful differences in mean body weights or body weight change were noted during the study.

Mean food consumption of F0 females in the 225 and 750 mg/kg/day groups was statistically lower than controls during days 0-3. ln addition, mean food consumption appeared to be slightly, but not statistically lower than controls for F0 females in the 750 mg/kg/day group during gestation days 0-7 and lactation days 1-4. No other toxicologically meaningful differences in mean food consumption were noted between the control, 75, 225 and 750 mg/kg/day groups.

The F0 female mating and fertility indices and mean gestation lengths were comparable between the control group, 75, 225 and 750 mg/kg/day groups. The mean number of F1 pups delivered was comparable in the control, 75, 225 and 750 mg/kg/day groups. However, the live birth index (number of liveborn pups/total number of pups delivered) was statistically lower in the 750 mg/kg/day group compared to controls, and the number of stillborn pups in the 750 mg/kg/day group was statistically higher than controls. The number of F1 pups dying, missing and/or cannibalized during lactation days 1-4 and the number of litters with total litter loss in the 750 mg/kg/day group were statistically higher than controls and the number of surviving pups on lactation day 4 in the 750 mg/kg/day group was statistically lower than controls. The mean live pups/litter in the 750 mg/kg/day group was slightly, but not statistically lower than controls on lactation day 0 and statistically lower than controls on lactation days 1-4.

Mean F1 pup weights in the 750 mg/kg/day group were statistically lower than controls on lactation days 1 and 4. F1 pup viability and mean F1 pup weights in the 75 and 225 mg/kg/day groups was comparable to controls during lactation days 0-4. The F1 pup sex ratio was comparable between the control, 75, 225 and 750 mg/kg/day groups on lactation days 0 and 4. Remarkable F1 pup observations included desquamation in the 225 and 750 mg/kg/day groups and a low incidence of gasping and skin pale in color, cool to the touch, skin with shiny appearance and skin appears tight — restricts movement in the 750 mg/kg/day group. No remarkable F1 pup observations were noted in the 75 mg/kg/day group.

Remarkable gross necropsy observations for F1 pups stillborn included a single incidence each of cleft palate and edema, increased incidences of atelectasis and milk not present in the stomach, and dermal hypoplasia in the 750 mg/kg/day group.

Remarkable gross necropsy findings for F1 pups born alive but found dead during lactation days 0-4 included scabbing, desquamation, dermal hypoplasia and an increased incidence of milk not present in the stomach in the 750 mg/kg/day group. ln addition, autolysis was observed for 22 F1 pups found dead in the 750 mg/kg/day. No remarkable gross necropsy findings were noted for F1 pups stillborn or found dead in the 75 and 225 mg/kg/day groups. Remarkable gross necropsy findings for F1 pups at scheduled euthanasia (lactation day 4) included scabbing and desquamation in the 225 and 750 mg/kg/day groups. No remarkable gross necropsy findings were noted for surviving F1 pups in the 75 mg/kg/day group. Remarkable gross necropsy findings in F0 females were limited to two females in the 750 mg/kg/day group with total litter loss that had pup tissue mixed with ingesta in the stomach. No other remarkable findings were noted at necropsy for F0 females in the control, 75, 225 or 750 mg/kg/day groups.

As a result of a significantly decreased live birth index (increased rate of stillbirthsl), as well as decreased pup survival during the lactation phase and a decreased mean pup body weight in the high-dose group, a dosage level of 225 mg/kg/day was considered a NOAEL for reproductive effects. However, the mating and fertility indices and mean gestation length for the test article-treated females appeared unaffected at dosage levels up to 750 mg/kg/day.

Conclusion

NOAEL rat, reproductive toxicity: 225 mg/kg bw/day

NOAEL rat, fertility 750 mg/kg bw/day