Registration Dossier

Administrative data

Endpoint:
one-generation reproductive toxicity
Remarks:
based on generations indicated in Effect levels (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and appropriate guidelines.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Principles of method if other than guideline:
n/a
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
E-AF098T (Lot # 474-0306-17), a white powder. Stored at room temp, dry, closed container.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
Source - Taconic Farms, Germantown, NY for use in study.
Number of animals - 60 males and 60 females
Age of start of treatment- Approximately 6-7 weeks old. Age at initiation of dosing: 8 weeks old.
Bodyweight: 148 to 206 g
Identification- Ear tag
Husbandary: Daily average animal room temperature and relative humidity: 18 to 26oC and 30-70% respectively.
Light: 12 hr artificial light 12 hr dark.
Accommodation: Upon arrival- individually in labelled cages. During the mating period, one female and one male rat were housed together.
Acclimatisation period was at least 5 days before start of the treatment
Diet- Harlan Teklad Certified Rodent Diet (W) 8728C ad libitum.
Water- City of Chicago water ad libitum by means of an automatic watering system.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: methyl cellulose (MC)
Details on exposure:
A dosing volume of 5 ml/kg was administered.
All rats were administered the test material formulations or vehicle alone by gavage, 7 days/week, for 2 weeks prior to mating and during the 2-week cohabitation period. Females were also treated through the gestation and lactation periods and until necropsy post mortum day 4, while males
received a minimum of 35 doses.
Details on mating procedure:
Rats from a given group were mated 1:1 for up to 2 weeks. Mating was determined by the presence of sperm in the vaginal smear.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of the test material formulations were collected from the first preparation (i.e., prior to dosing), mid-way through the study (approximately week 4),
and towards the end of the study, and analyzed for concentration. Samples for homogeneity (homogeneity was performed on duplicate samples collected from the top, middle and bottom of the test material formulation) were collected from the low and high dose formulations and analyzed concurrently with the first concentration analyses. Experimur criterion for acceptance of suspention formulations is that the analytically determined concentrations must be within 15% of target concentrations. For homogeneity, the mean of each set of values must be within 10% of each other and within 15% of the target of concentration.
Duration of treatment / exposure:
All parental males were dosed for a minimum of 28 consecutive days prior to sacrifice.
The recovery animals (5 additional rats/sex in the control and high dose groups) were held for 14 days after treatment termination. Parental dams selected for F1 littering were administered the test article through gestation and lactation until post-partum day 4.
When calculating the duration of the treatment:
10 core parental animals/sex/group were euthanized on Day 36/males or 51/females and the remaining 5 animals/sex from Groups 1 and 4 were continued
on study without further dosing, and euthonized 14 days after the last dose. (males: 36+14= 50 days, females: 51+14=65 days)
Frequency of treatment:
Daily treatment
Details on study schedule:
1. The parental rats were administered with the test material by oral gavage for 14 days prior to mating.

2. Following the 14 days one male was housed with one female (from the same concentration group) and allowed to mate for up to 2 weeks. Administration of
the test substance continued during the mating period.

3. The control and high dose groups each contained 5 additional rats/ sex for the same duration but were not mated. These rats were designated as recovery
animals and were held for at least 14 days after treatment termination to evaluate the recovery potential from any adverse treatment related effects.

4. Daily vaginal smears, taken from female rats during the mating period, were evaluated for the presence of sperm, as well as the stage of the estrus cycle.

5. All parental males were dosed for a minimum of 35 consecutive days prior to sacrifice and were terminated when they were no longer needed for mating.

6. Females were treated through gestation and lactation periods, until necropsy.

7. At termination of the study, a complete gross necroscopy was performed on all animals. Protocol -specified necroscopy procedures included examination
of the external surface of the body, all orifices, the cranial, thoracic, and peritoneal cavities and their contents.

Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0 mg/kg/day
Basis:
other:
Remarks:
Doses / Concentrations:
50 mg/kg/day
Basis:
other:
Remarks:
Doses / Concentrations:
250 mg/kg/day
Basis:
other:
Remarks:
Doses / Concentrations:
1000 mg/kg/day
Basis:
other:
No. of animals per sex per dose:
Male: 10 animals at 0 mg/kg bw/day (Control- P) P= Parent
Male: 5 animals at 0 mg/kg bw/day (Were not mated)
Male: 10 animals at 50 mg/kg bw/day (Treatment- P)
Male: 10 animals at 250 mg/kg bw/day (Treatment- P)
Male: 10 animals at 1000 mg/kg bw/day (Treatment- P)
Male: 5 animals at 1000 mg/kg bw/day (Were not mated)
Female: 10 animals at 0 mg/kg bw/day (Control- P)
Female: 5 animals at 0 mg/kg bw/day (Were not mated)
Female: 10 animals at 50 mg/kg bw/day (Treatment- P)
Female: 10 animals at 250 mg/kg bw/day (P treatment)
Female: 10 animals at 1000 mg/kg bw/day (P treatment)
Female: 10 animals at 50 mg/kg bw/day (Treatment- P)
Female: 10 animals at 250 mg/kg bw/day (Treatment- P)
Female: 10 animals at 1000 mg/kg bw/day (Treatment- P)
Female: 5 animals at 1000 mg/kg bw/day (Were not mated)
Control animals:
yes, concurrent vehicle
Details on study design:
Dose formulations of the test material were prepared by Experimur approximately every two weeks in a vehicle of 1%methyl cellulose (1% MC) at
graded concentrations of 10, 50 and 200 mg/ml. These concentrations coresponded to target doses of 50, 250 and 1000 mg/kg, respectively, when administered at a dosing volume of 5 ml/kg. The stability of test material in the vehicle, when stored at room temperature was established by the lab. Test material formulations were stored under refrigeration and were removed from the refrigerator approximately 1 hour prior to use and thoroughly mixed on a stir plate to and during dosing. Samples for homogenicity were collected from the low and high dose formulations and analyzed concurrently with the first concentration analysis.

Positive control:
no

Examinations

Parental animals: Observations and examinations:
Moribundity/Mortality observation was done at least once daily during the quarantine period and twice daily during the study. any abnormal clinical signs were recorded.
Clinical observations: A ditailed clinical examination was performed on all animals during the quarantine period and weekly throughout the study.
Neurotoxicity Assessment: A functional observational battery (F.O.B.) and automated motor activity assessment were performed toward the end of the study
on 5 rats/sex/group (males after 27 doses and females after parturition).
Body weights: All study animals were weighed during quarantine (pretest), and weekly prior to mating. Mated dams were weighed on gestation days 0, 7, 14 and 21. Dams and their litters were weghed on the day of observed partiturition (postnatal day 0), and again on postnatal day 4. Males were weighed weekly until
sacrifice. A fasted body weight was collected prior to terminal sacrifice. The fasted body weight was used to calculate organ-to-body weight ratios.
5 rats/sex/group were fasted overnight for blood collection for clinical chemistry and hematology.

A bone marrow smear was collected and fixed for future evaluation. Part of the tissues were weighed at scheduled necropsy and organ-to-body and organ-to-brain weighrt were calculated.
Oestrous cyclicity (parental animals):
Daily vaginal smears, taken from the female rats during the mating period, were evaluated for the presence of sperm, as well as the stage of the estrus cycle.
Sperm parameters (parental animals):
The presence of sperm in the vaginal smear taken daily or sperm-plug indicated a positive mating, and that day was designated as gestation day 0.
Litter observations:
Rat pups (F1 generation) were sexed and given a gross external physical examination on postnatal day 0. Dead or stilborn pups were examined for
gross external anomalies and, when feasible, a visceral exam was performed. Six fetuses (3, 1, and 2 in the vehicle control, 250 and 1000 mg/kg
groups, respectively) underwent visceral examination.
Postmortem examinations (parental animals):
Necroscopy: All parental animals received a complete necroscopy that included examination of the external surface of the body, all orifices, the
cranial, thoraic, and peritoneal cavities, and their contents. The uterus of each rat was examined for evidence of implantation sites and the number of sites was recorded. The corpora lutea were counted. In addition, a bone marrow smear was collected and fixed for possible future evaluation. Testes
and epididymides were fixed in Bouin's solution and the eyes and associated tissues (e.g., optic nerve) were fixed in Davidson's solution. All other
tissues were fixed in 10% neutral buffered formalin. Various tissues were weighed at scheduled necroscopy and organ-to-body and organ-to-brain weight ratios were calculated.

Histopathology: Various tissues were obtained from 5 rats/sex randomly selected from the vehicle control and high dose groups, were evaluated
microscopically by a broad-certified veterinary pathologist.
Postmortem examinations (offspring):
Hematology, Organ weight, Neurotoxicity, Gross pathology and Histopathology.
Statistics:
Data were manually collected, entered into Excel spreadsheets, and means and standard deviations were calculated using the same software.
Continuous data were analyzed for homogenity of variance using Levene's test. When the variancees were homogeneous (p> 0.001), the data were further
analyzed by one-way analysis of variance (ANOVA). When a significant F value was observed (p ≤ 0.05), each treatment group was compared to the vehicle
control group using Dunnett's two-tailed t-test. Statistical significance was decleared at p ≤ 0.05 for Dunnett's test.
Additional information regarding the statistics is attached.
Reproductive indices:
Offsprings number was compared.
Offspring viability indices:
No difference in litter viability among the groups.

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

Mortalities: No death occurred during the study.

Clinical observations: Alopecia, scabs, lacrimation, red material around the eye. All considered not to be treatment related effects.

Food consumption: No adverse effects

Body weights: No adverse treatment related changes.

Clinical Chemistry: No adverse treatment related changes.

Hematology: No adverse treatment related changes.

Organ weight: No adverse treatment related changes. Reproductive organs checked: ovaries, testes, epididymdes.

Neurotoxicty assessment: No adverse treatment related changes in motor activity or the functional observational battery were observed.

Gross pathology: Unrelated findings

Histopathology:
All histopathology findings were considered to be unrelated to treatment and typically seen in animals of similar age and environment. Preserved tissues taken for reproductive endpoints: Epididymides, testes, uterus, vagina, ovaries.
Microscopic evaluation of testicle and epididymis stained sections with special emphasis on stages of spermatogenesis and interstitial testicular cell structure, revealed no abnormalities.

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed

Details on results (F1)

Overall, mating, pregnancy, litter survival, and pup body weights were unaffected by treatment with E-AF98T. The number of sperm-positive breeding pairs was similar across groups (10/10 in all groups except for 250mg/kg group 9/10).
Successful pregnancy was 100% in all groups except for 50mg/kg group). The length of gestation was similar in all groups and averaged 22 days. The number of dams undergoing successful parturition was similar. However, one dam treated with 50mg/kg delivered a non-viable litter. Litter survival was similar across all groups on postnatal days 0 and 4.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
> 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
No treatment- related deaths or adverse clinical of toxicity were observed during the study.
No adverse treatment- related alteration in food consumption, body weights, organ weights, clinical pathology, motor activity or functional observational
battery were noted. Additionally, no effects of mating, pregnancy, litter viability or pup body weights were observed in groups treated with E-AF098T.
No treatment-related gross lesions or microscopic findings were noted. Additionally, no effects on mating, pregnancy, litter viability or pup body weights were observed in groups treated with the test substance.
Executive summary:

No treatment- related deaths or adverse clinical of toxicity were observed during the study. No adverse treatment- related alteration in food consumption, body weights, organ weights, clinical pathology, motor activity or functional observational battery were noted. Additionally, no effects of mating, pregnancy, litter viability or pup body weights were observed in groups treated with E-AF098T. No treatment-related gross lesions or microscopic findings were noted. Additionally, no effects on mating, pregnancy, litter viability or pup body weights were observed in groups treated with the test substance.