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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
December 2012 - May 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study in accordance with OECD guidelines
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Specific details on test material used for the study:
EXP1200078
Lot no. E00048-256
Exp. date: 31 December 2014
Dark brown, opaque, viscous liquid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC
- Age at study initiation: At the initiation of dose administration (study day 0 for females and study day 14 for males), the males and females were approximately 12.5 and 10.5 weeks old, respectively.
- Weight at study initiation: Male body weights 397-482 g on study day 14 and female body weights 199-281 g on study day 0
- Fasting period before study: no
- Housing: Following receipt and until pairing, all F0 animals were housed individually in clean, stainless steel wire mesh cages suspended above cage board. Following positive evidence of mating, the males were housed in suspended wire mesh cages until the scheduled necropsy, and the females were transferred to plastic maternity cages with nesting material, ground corncob bedding.
- Diet: ad libitum PMI Nutrition International, LLC Certified Rodent LabDiet® 5002
- Water: ad libitum reverse osmosis-purified (on site) drinking water
- Acclimation period: at least 14 days

ENVIRONMENTAL CONDITIONS
- Temperature: 21.4°C to 21.6°C
- Humidity: 32.5% to 46.2%
- Air changes: a minimum of 10 fresh air changes per hour
- Photoperiod: 12 hour light (0600 hours to 1800 hours)/12 hour dark photoperiod

IN-LIFE DATES: From: 29 January 2013 To: 27 March 2013

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: Mineral oil, USP
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance formulations were prepared approximately weekly as single formulations for each dosage level, divided into aliquots for daily dispensation, and stored at room temperature (18°C to 24°C). The test substance formulations were stirred continuously throughout the preparation, sampling, and dose administration procedures. The dosage volume for all groups was 5 mL/kg.

The vehicle and test substance formulations were administered orally by gavage once daily via an appropriately sized flexible, Teflon®-shafted, stainless steel ball-tipped dosing cannula (Natume, Japan).

VEHICLE
- Concentration in vehicle: 0, 20, 60 and 200 mg/ml
- Amount of vehicle (if gavage): 5 ml/kg
- Lot/batch no.: lot nos. 1AL0557 and 2BC0539
Details on mating procedure:
- M/F ratio per cage: 1/1
- Proof of pregnancy: Positive evidence of mating was confirmed by the presence of a vaginal copulatory plug or the presence of sperm following a vaginal lavage and verified by a second biologist
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples for concentration analysis were collected from the middle stratum of each dosing formulation (including the control group) prepared during the in-life phase of the study. The analyzed dosing formulations were within WIL Research’s SOP range for suspensions (85% to 115%). The test substance was not detected in the analyzed vehicle formulation that was administered to the control group (Group 1).
Duration of treatment / exposure:
The males were dosed during study days 14-41 (14 days prior to pairing through 1 day prior to scheduled euthanasia) for a total of 28 doses. The females were dosed during study days 0 through the day prior to euthanasia (28 days prior to pairing through lactation day 3) for a total of 53-57 doses. Females with no evidence of mating or that failed to deliver were dosed through the day prior to euthanasia (post-mating or post-cohabitation day 25) for a total of 53-66 doses.
Frequency of treatment:
Daily.
Details on study schedule:
No further details.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Vehicle control
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
Dosage levels were determined based on the results of previous studies, including a 14 day (Haas, 2013, WIL-537025) and a 28-day (Haas, 2013, WIL-537026) oral gavage toxicity study in male and female Sprague Dawley rats. In the 14-day study (Haas, 2013, WIL-537025), the test substance in the vehicle (mineral oil) was administered orally by gavage once daily for 14 consecutive days to 3 groups consisting of 5 rats/sex at dosage levels of 100, 300, and 1000 mg/kg/day. All animals survived to the scheduled necropsy. Test substance-related effects were limited to low incidence of clear material around the mouth noted in males at 1000 mg/kg/day. Based on these results, the maximum tolerated dose (MTD) was considered to be 1000 mg/kg/day. In the 28-day study (Haas, 2013, WIL-537026), the test substance in the vehicle (mineral oil) was administered orally by gavage once daily for 28 consecutive days to 3 groups consisting of 5 rats/sex at dosage levels of 100, 300, and 1000 mg/kg/day. There were no test substance-related deaths. Test substance-related effects were limited to clear material around the mouth and higher liver weights in the 1000 mg/kg/day group males and females and a lower seminal vesicles weight in the 1000 mg/kg/day group males. However, none of these effects were considered to be adverse. Therefore, the no-observed-adverse-effect level (NOAEL) was considered to be 1000 mg/kg/day, the highest dosage level administered.
The selected route of administration for this study was oral (gavage) because this is a potential route of human exposure. Historically, this route has been used extensively for studies of this nature.
Positive control:
No, not required and not relevant.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily. Each male and female was also observed for signs of toxicity 1-2 hours following dose administration.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly. Individual male body weights were recorded weekly throughout the study, beginning on the first day of dose administration, and prior to the scheduled euthanasia. Individual female body weights were recorded weekly until evidence of copulation was observed. Mean weekly body weights and body weight changes are presented for each interval. In addition, cumulative mean body weight changes are presented for the pre-mating period (males and females) and for the entire generation (males only). Once evidence of mating was observed, female body weights were recorded on gestation days 0, 4, 7, 11, 14, 17, and 20 and on lactation days 0 (when possible), 1, and 4.

FOOD CONSUMPTION: Yes
- Time schedule: Individual male and female food consumption was recorded on the corresponding weekly body weight days until pairing. Food intake was not recorded during the breeding period. Once evidence of mating was observed, female food consumption was recorded on gestation days 0, 4, 7, 11, 14, 17, and 20 and on lactation days 1 and 4. Following the breeding period, food consumption for females with no evidence of mating was measured on a weekly basis until the scheduled euthanasia.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
A complete necropsy was conducted on all F0 parental animals at the scheduled termination. All surviving F0 adults were euthanized by carbon dioxide inhalation. Males were euthanized following completion of the mating period. Females that delivered were euthanized on lactation day 4; the number of former implantation sites and corpora lutea were recorded. Females that failed to deliver were euthanized on post-mating day 25 (females with evidence of mating) or post-cohabitation day 25 (females with no evidence of mating).

Necropsy included examination of the external surface, all orifices, the cranial cavity, the external surface of the brain, and the thoracic, abdominal, and pelvic cavities, including viscera. At the time of necropsy, the following tissues and organs were placed in 10% neutral-buffered
formalin (except as noted):
Brain
Pituitary gland
Coagulating glands
Prostate gland
Kidneys (2)
Seminal vesicles (2)
Liver (sections of 2 lobes)
Testes with epididymidesa (2)
Mammary glands and vas deferens
Ovaries and oviducts (2)
Uterus with cervix and vagina
All gross lesions

Microscopic examination was performed on the following tissues from all animals in the
control and 1000 mg/kg/day groups, and gross lesions from all groups, at the scheduled necropsies:
Cervix
Seminal vesicles
Coagulating glands
Testes
Epididymides
Uterus
Mammary glands (female only)
Vagina
Ovaries
Vas deferens
Pituitary gland
All gross lesions (internal)
Prostate gland
Oestrous cyclicity (parental animals):
Not examined.
Sperm parameters (parental animals):
Not examined.
Litter observations:
- Each litter was examined daily for survival, and all deaths were recorded.
- Litters were examined daily for survival and any adverse changes in appearance or behavior. Each pup received a detailed physical examination on PND 1 and 4. Any abnormalities in nursing behavior were recorded.
- Pups were individually weighed on PND 1 and 4.
- Mean pup weights were presented by sex for each litter and by dose group.
- Pups were individually sexed on PND 0 and 4.
Postmortem examinations (parental animals):
A complete necropsy was conducted on all F0 parental animals at the scheduled termination.
The following organs were weighed from all F0 animals at the scheduled necropsies: Brain, Ovaries with oviducts, Epididymides, Pituitary gland, Kidneys, Testes, Liver.
Microscopic examination was performed on the following tissues from all animals in the control and 1000 mg/kg/day groups, and gross lesions from all groups, at the scheduled necropsies:
Cervix, Seminal vesicles, Coagulating glands, Testes, Epididymides, Uterus, Mammary glands (female only), Vagina, Ovaries, Vas deferens, Pituitary gland, All gross lesions (internal), Prostate gland.
Tissues and organs from all animals in the 100 and 300 mg/kg/day groups (except for gross lesions) were maintained in the appropriate fixatives for possible future microscopic examination.
Postmortem examinations (offspring):
On PND 4, surviving F1 rats were euthanized via an intraperitoneal injection of sodium pentobarbital and discarded without examination.
Statistics:
Each mean was presented with the standard deviation (S.D.), standard error (S.E.), and the number of animals (N) used to calculate the mean. All statistical tests were performed using WTDMS™ unless otherwise noted. Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test substance-treated group to the control group by sex. Parental mating, fertility, conception, and copulation indices were analyzed using the Chi-square test with Yates’ correction factor (Hollander and Wolfe, 1999). Mean parental body weights (weekly, gestation, and lactation), body weight changes, and food consumption, offspring body weights and body weight changes, gestation length, numbers of former implantation sites, number of pups born, live litter size on PND 0, corpora lutea, unaccounted-for sites, absolute and relative organ weights, and pre-coital intervals were subjected to a parametric one-way ANOVA (Snedecor and Cochran, 1980) to determine intergroup differences. If the ANOVA revealed significant (p<0.05) intergroup variance, Dunnett's test (Dunnett, 1964) was used to compare the test substance-treated groups to the control group. Mean litter proportions (percent per litter) of males at birth and postnatal survival were subjected to the Kruskal-Wallis nonparametric ANOVA (Kruskal and Wallis, 1952) to determine intergroup differences. If the ANOVA revealed significant (p<0.05) intergroup variance, Dunn’s test (Dunn, 1964) was used to compare the test substance-treated groups to the control group. Histopathological findings of each treated group were compared to those of the control group by Fisher’s Exact test (Steel and Torrie, 1980).
Reproductive indices:
Male (Female) Mating Index (%) = No. of Males (Females) with Evidence of Mating (or Confirmed Pregnant) / Total No. of Males (Females) Used for Mating x 100

Male Fertility Index (%) = Total No. of Malese Siring a Litter / Total No. of Males Used for Mating x 100

Male Copulation Index (%) = No. of Males Siring a Litter / No. of Males with Evidence of Mating (or Females with Confirmed Pregnancy) x 100

Female Fertility Index (%) = No. of Females with Confirmed Pregnancy / Total No. of Females Used for Mating x 100

Female Conception Index (%) = No. of Females with Confirmed Pregnancy / No. of Females with Evidence of Mating (or Confirmed Pregnancy) x 100
Offspring viability indices:
- Mean live litter size
- Postnatal survival between births and PND 0 and PND 4
- Postnatal survial for all other intervals

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
Clinical findings noted for males and females in the 300 and 1000 mg/kg/day groups (red material around the mouth) were considered non adverse.
Mortality:
no mortality 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:
effects observed, treatment-related
Description (incidence and severity):
higher mean liver weights were noted in the 1000 mg/kg bw/day F0 group males and females.
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed

Details on results (P0)

ORGAN WEIGHTS (PARENTAL ANIMALS):
Slightly higher mean liver weights (absolute, relative to final body weight, and relative to brain weight) were noted in the 1000 mg/kg/day F0 group males [5.9%, 5.9%, and 3.8% (values not statistically significant)] and females [14.2% (p<0.05), 13.8% (p<0.01), and 13.5% (p<0.05)]. These changes were of a minimal magnitude. Slightly higher mean liver weights were also noted at 1000 mg/kg/day in a previous 28-day study (Haas, 2013, WIL-537026) (approximately 12% higher in males and females) and in a concurrent 90-day study (Haas, Draft, WIL-537030) (9.5% and 8.1% higher in males and females, respectively) of the same test substance with no correlating adverse clinical pathology or histologic changes. Based on the lack of any correlating adverse clinical pathology or histologic changes in two additional studies on the same test substance with similar minimal liver weight elevations, microscopic examination of the liver was not performed in the current study and the slightly higher mean liver weights were considered and toxicologically irrelevant.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
parental toxicity
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse systemic or local toxicity was observed up to and including the highest dose level tested (1000 mg/kg bw/day)
Key result
Dose descriptor:
NOAEL
Remarks:
reproductive toxicity
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No reproductive toxicity was observed up to the highest dose level tested (1000 mg/kg bw/day)

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:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined

Effect levels (F1)

Key result
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:
viability
mortality
body weight and weight gain
Remarks on result:
not determinable due to adverse toxic effects at highest dose / concentration tested
Remarks:
Exposure via F0 female

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Under the conditions of this screening study, no test substance-related effects were noted on F0 reproductive endpoints, including male and female mating and fertility, male copulation, and female conception indices, mean number of days between pairing and coitus, gestation length, and the process of parturition at any dosage level. Therefore, the no-observed-adverse-effect level (NOAEL) for F0 male and female reproductive toxicity was considered to be 1000 mg/kg/day (the highest dosage level evaluated) when the test substance was administered orally by gavage to Crl:CD(SD) rats. There were no adverse test substance-related clinical findings and no effects on mean body weights, body weight changes, and food consumption noted at any dosage level. Furthermore, there were no adverse effects on organ weights or macroscopic/microscopic alterations in F0 males and females at any dosage level. Therefore, the NOAEL for F0 male and female systemic toxicity was considered to be 1000 mg/kg/day. The NOAEL for neonatal toxicity was considered to be 1000 mg/kg/day based on the absence of effects on F1 offspring at all dosage levels.
Executive summary:

The test substance was administered by daily oral gavage to male and female Sprague Dawley rats at dose levels of 0, 100, 300 or 1000 mg/kg bw/day according to the OECD 421 guideline. Males were exposed for 2 weeks prior to mating, during mating and up to termination (total of 29 doses). The females were exposed for 2 weeks prior to mating, during mating, during post-coitum, and during 3 days of lactation (53-57 doses).

All F0 males and females in the control, 100, 300, and 1000 mg/kg/day groups survived to the scheduled necropsies. Test substance-related clinical findings were noted for males and females in the 300 and 1000 mg/kg/day groups. Clear material around the mouth was noted for 11 of 12 males and all females at 1000 mg/kg/day and to a lesser extent (5 males and 6 females) at 300 mg/kg/day at 1-2 hours following dose administration generally throughout the respective treatment periods. The clinical findings noted for males and females in the 300 and 1000 mg/kg/day groups were considered non-adverse. Body weight and food consumption were unaffected by test substance administration.

Slightly higher mean liver weights (absolute, relative to final body weight, and relative to brain weight) were noted in the 1000 mg/kg/day F0 group males [5.9%, 5.9%, and 3.8% (values not statistically significant)] and females [14.2% (p<0.05), 13.8% (p<0.01), and 13.5% (p<0.05)]. These changes were of a minimal magnitude. Slightly higher mean liver weights were also noted at 1000 mg/kg/day in a previous 28-day study (Haas, 2013, WIL-537026) (approximately 12% higher in males and females) and in a concurrent 90-day study (Haas, Draft, WIL-537030) (9.5% and 8.1% higher in males and females, respectively) of the same test substance with no correlating adverse clinical pathology or histologic changes. Based on the lack of any correlating adverse clinical pathology or histologic changes in two additional studies on the same test substance with similar minimal liver weight elevations, microscopic examination of the liver was not performed in the current study and the slightly higher mean liver weights were considered toxicologically irrelevant. Therefore, the NOAEL for F0 male and female systemic toxicity was considered to be 1000 mg/kg/day.

No test substance-related effects were noted on F0 reproductive endpoints, including male and female mating and fertility, male copulation, and female conception indices, mean number of days between pairing and coitus, gestation length, and the process of parturition at any dosage level. Therefore, the no-observed-adverse-effect level (NOAEL) for F0 male and female reproductive toxicity was considered to be 1000 mg/kg/day (the highest dosage level evaluated) when the test substance was administered orally by gavage to Crl:CD(SD) rats.