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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
Study period:
Between 06 May 2009 and 30 September 2009.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study; well documented study report.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
One animal was weighed incorrectly and dosed wrongly for 4 times (0.4 ml off); no observations made for control and 15 mg/kg/day females at 5h after 24th dosing; surface righting wasn’t recorded for one offspring. No effect the integrity of the study.
GLP compliance:
yes (incl. certificate)
Remarks:
Date of inspection 19/08/2008. Date of signature 04/03/2009.
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Physical state: pale yellow solid block (frozen)
- Analytical purity: 100%
- Lot/batch No.:
- Storage condition of test material: in the dark at approximately 4°C, under Nitrogen, over silica gel.

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female

Administration / exposure

Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
For the purpose of the study, the test material was prepared at the appropriate concentrations as a solution in Arachis oil BP (dried). The stability and homogeneity of the test material formulations were determined by Harlan Laboratories Ltd. Results show the formulations to be stable for at least fourteen days. Formulations were therefore prepared daily and stored at approximately +4ºC in the dark, under nitrogen, over silica gel.
Samples were taken of each test material formulation and were analyzed for concentration of test material at Harlan Laboratories Ltd. The results indicate that the prepared formulations were within ± 9% of the nominal concentration.

DIET PREPARATION
- Rate of preparation of diet (frequency): not applicable.
- Mixing appropriate amounts with (Type of food): not applicable.
- Storage temperature of food: not applicable.

VEHICLE: Arachis oil BP
- Justification for use and choice of vehicle (if other than water): no data available.
- Concentration in vehicle: 3.75, 37.5, 188/125 mg/ml
- Amount of vehicle (if gavage): 4 ml/kg
- Lot/batch no. (if required): no data available
- Purity: no data available
Details on mating procedure:
- M/F ratio per cage: 1 male: 1 female
- Length of cohabitation: for a period of up to fourteen days.
- Proof of pregnancy: cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of the oestrous cycle or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation).
- Further mating after two unsuccessful attempts: no data.
- After successful mating each pregnant female was caged (how): the males were returned to their original cages and females were transferred to individual cages.
- Any other deviations from standard protocol: none.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of test material in the test material formulations was determined by gas chromatography (GC) using an external standard technique.
Sample:
The test material formulations were diluted with tetrahydrofuran to give a final, theoretical test material concentration of approximately 1 mg/ml. Procedural recoveries were performed at each level on all occasions and results were corrected for recovery.

Homogeneity Determinations:
The test material formulations were deemed to be homogeneous by visual inspection.
Sampling for homogeneity determinations was performed in triplicate.

Stability Determinations
The test material formulations were sampled and analyzed initially and then after storage at ambient conditions for four hours.

Results:
Concentration expressed as % of nominal range from 91% to 103% from week 1 to 7;
The analytical method has been satisfactorily validated in terms of linearity and specificity for the purposes of the study.
The test solution preparation was proved to be stable under test conditions.
Duration of treatment / exposure:
The test material was administered to three groups each of ten male and ten female rats, for up to 56 consecutive days
Frequency of treatment:
Daily
Details on study schedule:
- Chronological Sequence of Study
i) Groups of ten male and ten female animals 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 of the study.
ii) On Day 15, all animals were paired on a 1 male:1 female basis within each dose group for a maximum of fourteen days (age at mating of the mated animals in the study: 14 weeks).
iii) Following evidence of mating (designated as Day 0 post coitum), the males were returned to their original cages and females were transferred to individual cages.
iv) Pregnant females were allowed to give birth and maintain their offspring until Day5 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.
v) The male dose groups were killed and examined macroscopically on Day 43.
vi) At Day 5 post partum, all females and surviving offspring were killed and examined macroscopically.

- Age at mating of the mated animals in the study: [~14] weeks
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
15 mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
150 mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
750 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
10 animals per sex per dose (including a control group).
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: no data available.

- Rationale for animal assignment (if not random): the animals were allocated to dose groups using a randomisation procedure based on stratified bodyweights and the group mean bodyweights were then determined to ensure similarity between the dose groups. The animals were uniquely identified within the study, by an ear punching system routinely used in these laboratories.
Positive control:
Not applicable.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: no data available.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were examined for overt signs of toxicity, ill-health and behavioral change immediately before dosing, soon after dosing and one and five hours after dosing, during the working week. Animals were observed immediately before dosing, soon after dosing and one hour after dosing at weekends (except for females during parturition where applicable).

BODY WEIGHT: Yes
- Time schedule for examinations: Individual bodyweights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until mating was evident. Bodyweights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum.

FOOD EFFICIENCY: Yes.
Weekly food efficiency (bodyweight gain/food intake) was calculated retrospectively for males and for females during the pre-mating phase and during the first two weeks of gestation. Due to offspring growth and milk production, food efficiency could not be accurately calculated during the final week of gestation and during lactation.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes.
- Time schedule for examinations: Water intake was observed daily by visual inspection of water bottles for any overt changes. A possible treatment-related effect was detected on Day 3 therefore gravimetric measurements were initiated from Day 4 through to study termination.
Oestrous cyclicity (parental animals):
No data available
Sperm parameters (parental animals):
No data available
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: [no]

PARAMETERS EXAMINED
The following parameters were examined in [F1] offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, sex of offspring on Day 1 and 4 post partum, clinical condition of offspring from birth to Day 5 post partum;
Physical Development
All live offspring were assessed for surface righting reflex on Day 1 post partum

GROSS EXAMINATION OF DEAD PUPS: yes, for external and internal abnormalities; possible cause of death was not determined for pups born or found dead.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: Adult males were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination on Day 43.
- Maternal animals: Adult females were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination on Day 5 post partum. Any females that failed to achieve pregnancy or produce a litter were killed on or after Day 26 post coitum.

GROSS NECROPSY
All adult animals including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.
For all females the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded. This procedure was enhanced as necessary, by staining the uteri with a 0.5% ammonium polysulphide solution.

HISTOPATHOLOGY / ORGAN WEIGHTS
Samples of the following tissues were preserved from all animals from each dose group,
in buffered 10% formalin, except where indicated:
Coagulation gland
Epididymides
Ovaries
Mammary gland
Pituitary
Prostate
Seminal vesicles
Testes
Uterus/Cervix
Vagina
Since there were indications of treatment-related changes in the ovary, examination was subsequently extended to include similarly prepared sections of ovary tissue from females from the 15 and 150 mg/kg/day dose groups.
Microscopic examination was conducted by the Study Pathologist. All findings were entered into the ROELEE Pathology computerisation system for tabulation and report production.
Postmortem examinations (offspring):
SACRIFICE
- Surviving offspring were terminated via intracardiac overdose of sodium pentobarbitone.

GROSS NECROPSY
- All offspring including those dying during the study were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

HISTOPATHOLOGY / ORGAN WEIGTHS
Not examined.
Statistics:
The following parameters were subjected to statistical analysis:
Bodyweight and bodyweight change, Food consumption for females during gestation and lactation, Litter data, Sex ratio, Implantation losses and viability indices, Offspring bodyweight, and bodyweight change, Offspring surface righting, Adult absolute and bodyweight relative organ, weights (males).
The following statistical procedures were used:
Data were assessed for dose response relationships by linear regression analysis, followed by one way analysis of variance (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.
The non-parametric methods were also used to analyse implantation loss, offspring sex ratio and landmark developmental markers. Probability values (P) are presented as follows:
P < 0.001 ***
P < 0.01 **
P < 0.05 *
p ≥0.05 (not significant)
In addition, histopathological findings were analysed (excluding any decedents, nonmated females and females not producing a pregnancy/litter) using the following methods:
1. Chi-squared analysis for differences in the incidence of lesions occurring with an
overall frequency of 1 or greater.
2. Kruskal-Wallis one-way non-parametric analysis of variance for the comparison of
severity grades for the more frequently observed graded conditions.
Probability values (P) were calculated as follows:
P<0.001 +++ --- ***
P<0.01 ++ -- **
P<0.05 + - *
P<0.1 (+) (-) (*)
p>0.01 N.S. (not significant)
Plus (+) signs indicate positive differences from the control group and minus (-) signs indicate negative differences. Asterisks refer to overall differences between group variation which is non-directional.
Reproductive indices:
Mating Performance and Fertility
i) Pre-coital Interval
ii) Fertility Indices
Gestation and Parturition Data
i) Gestation Length
ii) Parturition Index
Litter Responses
i) Implantation Losses (%)
ii) Live Birth and Viability Indices
iii) Sex Ratio (% males)
Offspring viability indices:
Of the litters born, litter size at birth and subsequently on Day 1 and 4 post partum were recorded.
Viability index (%) = (No. of offspring alive on day 4/No. of offspring alive on day 1) x 100

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
See "Details on results (parental animals)"
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
See "Details on results (parental animals)"
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
See "Details on results (parental animals)"
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:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
See "Details on results (parental animals)"

Details on results (P0)

Mortality. There were no unscheduled deaths.
Clinical Signs. Three females treated with 750 mg/kg/day had occasional tremors on Day 3. Following the reduction of the high dose level episodes of yellow fur staining, yellow staining around the ano-genital region, red/brown stained mouth/snout/ano-genital region, increased salivation and noisy respiration was evident in animals of either sex throughout the treatment period. Females from this treatment group also showed incidents of diuresis. Animals of either sex treated with 150 mg/kg/day showed episodes of red/brown stained mouth/snout and increased salivation throughout the treatment period. Females treated with 150 mg/kg/day also showed yellow stained fur, yellow staining around the ano-genital region and diuresis. Observations detected at 15 mg/kg/day were confined to increased salivation, yellow stained fur and yellow staining around the ano-genital region.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Bodyweights. Males treated at the high dose level showed a statistically significant reduction in cumulative bodyweight gain during the first two weeks of treatment. Females from this treatment group showed a statistically significant reduction in bodyweight gain during the final week of gestation. No such effects were detected in animals of either sex treated with 150 or 15 mg/kg/day.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
No data available.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
No data available.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
No data available.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Mating. There were no toxicologically significant effects on mating.
Fertility. There were no treatment-related effects detected in conception rates. Females treated at the high dose group however showed an increase in pre and post implantation losses. Post implantation losses were also increased in females treated with
150 mg/kg/day. No such effects were detected in females treated with 15 mg/kg/day.
Gestation Length. There were no differences in gestation lengths. The distribution for treated females was comparable to controls.

ORGAN WEIGHTS (PARENTAL ANIMALS)
Organ Weights: No treatment-related effects were detected in the organ weights measured.

GROSS PATHOLOGY (PARENTAL ANIMALS)
The following treatment-related changes were observed:
OVARY: A higher incidence of hypertrophy/vacuolation of interstitial gland cells was
seen in relation to treatment for females treated at the high dose level, but not at any other dose level. Although the incidence or severity of the condition did not attain statistical significance it was nonetheless considered to have been marginally influenced by treatment. In addition, a higher incidence of hypertrophy/vacuolation of corpora luteal cells was seen for a few animals at this treatment level (P <0.05).

HISTOPATHOLOGY (PARENTAL ANIMALS)
No data available.

Effect levels (P0)

Dose descriptor:
NOEL
Remarks:
Reproduction toxicity
Effect level:
15 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
See "Details on results (Offspring)"
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
See "Details on results (Offspring)"
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
See "Details on results (Offspring)"
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined

Details on results (F1)

Offspring Litter Size and Viability. Females treated at the high dose group showed a reduction in litter size at Day 1 and Day 4 of lactation. Live birth index for these litters was statistically significantly reduced and post natal survival was also reduced. Litter size at 150 mg/kg/day was also reduced at Day 1 and Day 4 of lactation. No such effects were detected in litters from females treated with 15 mg/kg/day.

Offspring Growth and Development. Offspring bodyweight gain between Days 1 and 4 of lactation was statistically significantly reduced in litters at the high dose level and subsequent mean litter weights were reduced on Days 1 and 4 of lactation. At the high dose level the percentage of offspring who successfully showed surface righting reflex on Day 1 was reduced. No such effects were detected in litters from females treated with 150 or 15 mg/kg/day.

Effect levels (F1)

Key result
Dose descriptor:
NOEL
Generation:
F1
Effect level:
15 mg/kg bw/day (nominal)
Sex:
not specified
Basis for effect level:
viability
clinical signs

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 15 mg/kg/day.
Executive summary:

Introduction.The study was performed to screen for potential adverse effects of the test material on reproduction including offspring development and provides an initial hazard assessment for effect on reproduction. The study complies with the recommendations of the OECD Guidelines for Testing of Chemicals No. 421 “Reproduction/Developmental Toxicity Screening Test” (adopted 27 July 1995).

Methods.The test material was administered by gavage to three groups each of ten male and ten female Wistar Han™ :HsdRccHan™ :WIST strain rats, for up to fifty six consecutive days, (including a two week maturation phase, pairing, gestation and early lactation for females) at dose levels of 15, 150 and 750 mg/kg/day. Due to the clinical observations detected in 750 mg/kg/day females on Day 3, the high dose animals were not dosed on Day 4 and the high dose level was reduced to 500 mg/kg/day from Day 5 onwards. A control group of ten males and ten females was dosed with vehicle alone (Dried Arachis oil BP).

Clinical signs, bodyweight development, dietary intake and water consumption were monitored during the study.

Pairing of animals within each dose group was undertaken on a one male: one female basis within each treatment group on Day 15 of the study, with females subsequently being 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, together with litter size and offspring weights and assessment of surface righting reflex.

Adult males were terminated on Day 43, and all females and surviving offspring on Day 5 post partum. All animals were subjected to a gross necropsy examination and histopathological evaluation of reproductive tissues was performed.

 

Results.

Mortality.There were no unscheduled deaths.

Clinical Observations.Three females treated with 750 mg/kg/day had occasional tremors on Day 3. Following the reduction of the high dose level episodes of yellow fur staining, yellow staining around the ano-genital region, red/brown stained mouth/snout/ano-genital region, increased salivation and noisy respiration was evident in animals of either sex throughout the treatment period. Females from this treatment group also showed incidents of diuresis. Animals of either sex treated with 150 mg/kg/day showed episodes of red/brown stained mouth/snout and increased salivation throughout the treatment period. Females treated with 150 mg/kg/day also showed yellow stained fur, yellow staining around the ano-genital region and diuresis. Observations detected at 15 mg/kg/day were confined to increased salivation, yellow stained fur and yellow staining around the ano-genital region.

Bodyweight.Males treated at the high dose level showed a statistically significant reduction in cumulative bodyweight gain during the first two weeks of treatment. Females from this treatment group showed a statistically significant reduction in bodyweight gain during the final week of gestation. No such effects were detected in animals of either sex treated with 150 or 15 mg/kg/day.

Food Consumption and Food Efficiency.Females treated at the high dose group

showed a statistically significant reduction in food consumption during the final week of gestation. No such effects were detected in females treated with 150 or 15 mg/kg/day.

No adverse effects in food consumption were detected in treated males however high dose males showed a reduction in food efficiency during the first week of treatment.

Water Consumption. Animals of either sex treated at the high dose group showed a significant increase in water consumption from Day 4 onwards. No such effects were detected in animals of either sex treated with 150 or 15 mg/kg/day.

Reproductive Performance:

Mating.There were no toxicologically significant effects on mating.

Fertility.There were no treatment-related effects detected in conception rates. Females

treated at the high dose group however showed an increase in pre and post implantation losses. Post implantation losses were also increased in females treated with

150 mg/kg/day. No such effects were detected in females treated with 15 mg/kg/day.

Gestation Length.There were no differences in gestation lengths. The distribution for treated females was comparable to controls.

 

Pathology:

Necropsy.No toxicologically significant macroscopic abnormalities were detected.

Organ Weights. No treatment-related effects were detected in the organ weights measured.

Histopathology.The following treatment-related changes were observed:

OVARY:A higher incidence of hypertrophy/vacuolation of interstitial gland cells was seen in relation to treatment for females treated at the high dose level, but not at any other dose level. Although the incidence or severity of the condition did not attain statistical significance it was nonetheless considered to have been marginally influenced by treatment. In addition, a higher incidence of hypertrophy/vacuolation of corpora luteal cells was seen for a few animals at this treatment level (P <0.05).

Discussion

This substance causes adverse effects on reproduction in rats only at maternally toxic doses when tested in accordance with OECD Guideline 421. Exposure of the parents to the high dose (750 mg/kg/day then reduced to 500 mg/kg/day on day 5) for fifty six consecutive days caused a statistically significant reduction in cumulative body weight gain in males during the first two weeks of treatment and a statistically significant reduction in body weight gain in females during the final week of gestation. A non-statistically significant increase in the incidence of hypertrophy/vacuolation of the interstitial glands of the ovaries also was evident at the high dose females. The only statistically significant effect was a decrease in the live birth index at the high dose level. There were no statistically significant increases in pre- and post implantation loss and no treatment-related effects on conception rates. The NOAEL for reproductive effects in the absence of maternal toxicity is 150 mg/kg/day. The no observed effect level (NOEL) for reproductive toxicity considering non-statistically significant changes is considered to be 15 mg/kg/day. In accordance to Directive 67/548/EEC and EU CLP (Regulation (EC) No. 1272/2008), classification is not required for reproductive toxicity occurring at maternally toxic doses.

 

Conclusion.The oral administration of test material to rats by gavage, for a period of up to fifty six consecutive days at dose levels of 15, 150 and 750 mg/kg/day (reduced to 500 mg/kg/day on Day 5) resulted in treatment-related reproductive effects at 500 and 150 mg/kg/day. The ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was therefore considered to be 15 mg/kg/day.