Registration Dossier

Administrative data

Endpoint:
toxicity to reproduction
Remarks:
other: screening study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19th August 2011 to 16th February 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: A GLP study performed in compliance with appropriate guidelines.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
not specified
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Physical state: solid
- Storage condition of test material: room temperature in the dark

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories UK Ltd, Oxon, UK
- Age at study initiation: 12 weeks
- Weight at study initiation: 313-374 g (males); 193-235 g (females)
- Housing: individually in polypropylene cages
- Diet (e.g. ad libitum): Rodent 218C Teklad Global Certified Diet ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2 ºC
- Humidity (%): 55 ± 15%
- Air changes (per hr): at least fifteen changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours dark

IN-LIFE DATES: From: 23rd August 2011 To: 7th October 2011

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on mating procedure:
On day 15, animals were paired on a one male:one female basis within each dose group for a maximum of 14 days and smearing will commence.
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
42 days
Frequency of treatment:
Daily
Details on study schedule:
1. Groups of ten males and ten females were treated daily at the appropriate dose level throughout the study (except for females during parturition where applicable). The first day of dosing was designated as day 1.
2. On day 15, animals were paired on a 1 male to 1 female basis within each dose for a maximum of 14 days.
3. Following evidence of mating (designated as day 0 post coitum) animals were returned to their original cages.
4. Pregnant females were allowed to give birth and maintain their offspring until day 5 post partum. Evaluation of each litter size, litter weight, mean offspring weight by sex, clinical observations and landmark developmental signs were also performed during this period.
5. The male dose groups were killed and examined macroscopically on day 43.
6. At day 5 post partum, all pregnant females and surviving offspring were killed and examined macroscopically. Non-pregnant females were killed and examined macroscopically on day 26 post coitum.
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 300, 1000 mg/kg bw/day
Basis:
nominal conc.
No. of animals per sex per dose:
Ten
Control animals:
yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:
MORBIDITY/MORTALITY: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: immediately before dosing, up to 30 minutes, one hour and five hours after dosing during the normal working week; immediately before dosing, soon after dosing and at one hour after dosing during weekends and public holidays.

BODY WEIGHT: Yes
- Time schedule for examinations: on day 1 for males and then weekly until termination. For females on day 1 and then weekly until mating was evident; then on days 0, 7, 14 and 20 post coitum and on days 1 and 4 post partum.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- during the maturation period, weekly food consumption was recorded for each cage of adults until pairing; this was continued for males after the mating phase. For females showing evidence of mating, for periods days 0-7, 7-14 and 14-20 post coitum. For females with live litters, during the period of lactation covering days 1-4.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: daily
Litter observations:
On day 0 post partum, the number of live and dead offspring were recorded. For each litter the following were recorded:
1. Number of offspring born.
2. Number of offspring alive recorded daily and reported on days 1 and 4 post partum.
3. Sex of offspring on days 1 and 4 post partum.
4. Clinical conditions of offspring from birth to day 5 post partum.
5. Individual offspring weights on days 1 and 4 post partum (litter weights were calculated retrospectively from this data).
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals at termination (day 43).
- Maternal animals: All surviving animals at termination (day 5 post partum).

GROSS NECROPSY
- For all females, the uterus was examined for signs of implantation and the number of uterine implantation sites in each horn was recorded.
- All animals underwent a full external and internal examination and any macroscopic abnormalities were recorded.

HISTOPATHOLOGY / ORGAN WEIGHTS
- The epididymides and testes from all adult males were weighed.
- Samples of the following tissues were subject to microscopic examination: epididymides, ovaries, pituitary, prostate, seminal vesicles, coagulating gland, testes, mammary gland, uterus with cervix, vagina.
Postmortem examinations (offspring):
SACRIFICE
- All surviving animals at termination.

GROSS NECROPSY
- Full external and internal examination and any macroscopic abnormalities were recorded.
Statistics:
The following parameters were subject to statistical analysis:
Body weight and body weight change
Food consumption for females during gestation and lactation
Pre-coital interval and gestation length
Litter size and litter weights
Sex ratio
Corpora lutea and implantation sites
Implantation losses and viability indices
Offspring body weight and body weight change
Offspring surface righting
Adult absolute and body weight-relative organ weights (males)

Data for males and females prior to pairing and where appropriate, quantitative data were analysed by Provantis™ Tables and Statistical Module. For each variable, the most suitable transformation of the data was found, the use of possible covariates checked and the homogeneity of means assessed using ANOVA and ANCOVA and Bartlett's test. The transformed data were analysed to find the lowest treatment level that showed a significant effects using the Williams Test for parametric data or the Shirley test for non-parametric data. If no dose response was found, but the data showed nonhomogeneity of means, the data were analysed by a stepwise Dunnett (parametric) or Steel (non-parametric) test to determine significant differences from the control group. Finally, if required, pair wise tests were performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).

Data for females during gestation and lactation, and offspring data, were assessed for dose response relationships by linear regression analysis, followed by one way ANOVA incorporating Levene's test for homogeneity of variance. Where variances were shown to be homogenous, pairwise comparisons were conducted using Dunnett's test. Where Levene's test showed unequal variances the data were analysed using non-parametric methods: Kruskal-Wallis ANOVA and Mann-Whitney U test.

Non-parametric methods were also used to analyse implantation loss, offspring sex ratio and landmark developmental markers.
Reproductive indices:
MATING PERFORMANCE AND FERTILITY
Pre-coital interval: calculated as the time elapsing between initial pairing and the observation of positive evidence of mating.

Fertility indices:

Mating Index (%) = (number of animals mated / number of animals paired ) x 100

Pregnancy Index (%) = (number of pregnant females / number of animals mated ) x 100

GESTATION AND PARTURITION DATA
Gestation length : calculated as the number of days of gestation including the day for observation of mating and the start of parturition

Parturition Index (%) = (number of females delivering live offspring / number of pregnant females) x 100

LITTER RESPONSES
Implantation Losses
% pre-implantation loss = (number of corpora lutea - number of implantation sites / number of corpora lutea ) x 100

% post-implantation loss = (number of implantation sites - total number of offspring born / number of implantation sites ) x 100

Live Birth and Viability Indices
Live Birth Index (%) = (number of offspring alive on day 1 / number of offspring born) x 100

Viability Index (%) = (number of offspring alive on day 4 / number of offspring alive on day 1) x 100

Sex Ration (% males)
(Number of male offspring / total number of offspring) x 100

Results and discussion

Results: P0 (first parental generation)

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
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
no effects observed

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)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
There were no unscheduled deaths.

There were no clinical signs of treatment-related toxicity detected in any treated group. One 1000 mg/kg bw/day female had fur loss evidence between days 36 and 44. Observations of this nature are commonly observed during the lactation period and are not considered to be of toxicological importance.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
There were no toxicologically significant effects detected in body weight development. 1000 mg/kg bw/day females showed a statistically significant reduction in body weight gain during the first week of treatment. Recovery was evident thereafter and in the absence of any significant associated changes, the intergroup difference was considered not to be of toxicological significance.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
There were no treatment-related effects detected on mean food consumption or mean food efficiency during this study, when compared to the control. 1000 mg/kg bw/day females showed a slight reduction in food efficiency during the first week of treatment. Recovery was evident thereafter and in the absence of any associated changes, the intergroup difference was considered not to be of toxicological significance.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
No treatment-related effects on fertility were detected for treated animals when compared to the controls.

One 1000 mg/kg bw/day female did not achieve pregnancy following evidence of mating. No histopathological correlates were evident in the female reproductive organs. However, the male partner of this female showed moderate tubular atrophy in both testes and oligospermia in the epididymides that correlated macroscopically with small and flaccid testes and small epididymides, respectively. These lesions were considered to be the reason for the non-pregnancy but both findings are naturally occurring background changes in rats and therefore considered not to be of toxicological significance in this study.

One 300 mg/kg bw.day female did not achieve pregnancy following evidence of mating. No histopathological correlates were evident in the male or female reproductive organs. Thus the exact cause of this non-pregnancy was not established.

Once control female did not achieve pregnancy following evidence of mating. This female had slight inflammation evident in the uterus and vagina that may have contributed to the failed pregnancy.

No treatment-related effects were detected in the length of gestation for treated females when compared to controls. All animals showed gestation lengths of 22 to 24½ days.

ORGAN WEIGHTS (PARENTAL ANIMALS)
There were no treatment-related effects detected in the organ weights measured. Statistical analysis of the data did not reveal any significant intergroup differences.

GROSS PATHOLOGY (PARENTAL ANIMALS)
No toxicologically significant macroscopic abnormalities were detected. One 1000 mg/kg bw/day male had small and flaccid testes and small epididymides at necropsy. These findings correlated with microscopic findings evident as moderate tubular atrophy in both testes and oilgospermia in the epididymides. Both of these findings, however, are naturally occurring background changes that are evident in rats and in this study were not considered to be of toxicological importance. One 100 mg/kg bw/day male had an enlarged left kidney, filled with clear fluid and increased pelvic space. Hydronephrosis was evident microscopically for this male but in the absence of a true dose relate response, the intergroup difference was considered to be due to a congenital abnormality and not related to test substance toxicity. Once control male had small seminal vesicles; in the absence of treatment this was considered to be incidental.

HISTOPATHOLOGY (PARENTAL ANIMALS)
No treatment-related microscopic findings were detected.
OTHER FINDINGS (PARENTAL ANIMALS)
There were no adverse effects detected in water consumption for treated animals when compared to the control animals.

Effect levels (P0)

Dose descriptor:
NOEL
Effect level:
1 000 other: mg/kg/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: overall effects

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:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

In total, nine females from the control, 300 and 1000 mg/kg bw/day dose groups and all females from the 100 mg/kg bw/day dose group gave birth to a live litter and successfully reared to day 5 of age.

VIABILITY (OFFSPRING)
There were no toxicologically significant effects detected. Litter viability on day 4 was statistically significantly reduced in 100 mg/kg bw/day females. In the absence of any associated changes or a true dose related response, the intergroup difference was not considered to be of toxicological importance. Statistical analysis of the remaining data did not reveal any significant intergroup differences.

CLINICAL SIGNS (OFFSPRING)
No obvious signs of clinical toxicity were detected for offspring from treated females when compared to controls. The incidental clinical signs detected throughout the control and treated groups, consisting of small size, offspring found dead or missing, cold, weak, pale and physical injury were considered to be low incidence findings observed in offspring in studies of this type and were considered to be unrelated to test substance toxicity.

BODY WEIGHT (OFFSPRING)
Statistical analysis of the litter, offspring weights or surface righting reflex data did not reveal any significant intergroup differences.

GROSS PATHOLOGY (OFFSPRING)
No treatment-related effects were detected for interim death or terminated offspring. The incidental findings observed were those occasionally observed in reproductive studies of this type and were considered to be unrelated to the toxicity of the test substance.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Under the conditions of the test, the oral administration of the test substance to rats via oral gavage at 100, 300 and 1000 mg/kg bw/day did not result in any treatment-related reproductive/developmental effects. The NOEL for reproductive/developmental toxicity was determined to be 1000 mg/kg/day.
Executive summary:

In a GLP compliant reproduction/developmental toxicity study conducted in accordance with standardised guideline OECD 421, the reproductive/developmental toxicity of the test substance was determined in a screening study.

The test substance was administered orally by gavage to three groups of ten male and ten female rats for up to eight weeks (including a two week maturation phase, pairing, gestation and early lactation for females) at dose levels of 100, 300 or 1000 mg/kg bw/day. A control group of ten males and ten females was dosed with vehicle alone. Clinical signs, body weight changes, dietary intake and water consumption were assessed. Pairing of animals within each dose group was undertaken on a one male to one female basis on day 15. Females were subsequently allowed to litter and rear their offspring to day 5 of lactation. During the lactation phase, daily clinical observations were performed on all surviving offspring. Litter size, individual offspring body weights and assessment of offspring surface righting reflex were recorded. Adult males were terminated on day 43 followed by the termination of all pregnant females and surviving offspring on day 5 post partum or on day 26 post coitum for non-pregnant females. All animals were subject to a gross necropsy examination and histopathological evaluation of reproductive tissues was performed.

There were no unscheduled deaths or clinical signs of toxicity in parents and offspring. There were no toxicologically significant effects on body weights or adverse effects on food and water consumption. There were no treatment-related effects noted during mating or on conception rates and gestation lengths. Litter size at birth, offspring body weight gain and litter weights at birth and subsequently on days 1 and 4 post partum were comparable to controls. No toxicologically significant macroscopic abnormalities, treatment-related effects on body weights and treatment-related microscopic findings were detected.

The oral administration of the test substance to rats by gavage, at dose levels of 100, 300 and 1000 mg/kg bw/day, did not result in any treatment-related reproductive/developmental effects. The NOEL for the test substance was determined to be 1000 mg/kg/day.