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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Between 02 June 2009 and 27 November 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:
other: OECD Guideline 422 (combined repeated dose toxicity study with the reproduction/developmental screening test)
Deviations:
no
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 amber coloured liquid
- Analytical purity: 100%
- Lot/batch No.:
- Storage condition of test material: room temperature in the dark

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Wistar Han™:HsdRccHan™:WIST strain rats from Harlan UK, Ltd, Oxon, UK
- Age at study initiation: Approximately 12 weeks old
- Weight at study initiation: the males weighed 301 to 352 g; the females weighed 184 to 219 g
- Fasting period before study: Not applicable
- Housing: Initially, all animals were housed in groups of five in solid floor polypropylene cages withstainless steel mesh lids and softwood flake bedding (Harlan UK Ltd). During the mating phase, animals were transferred to polypropylene grid floor cages suspended over trayslined with absorbent paper on a one male: one female basis within each dose group. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation, in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.
- Diet: The animals were allowed free access to food. A pelleted diet (Rodent 2018CTeklad Global Certified Diet Harlan UK Ltd, Oxon, UK) was used throughout the study period. The diet was considered not to contain any contaminant at a level that mighthave affected the purpose or integrity of the study.
- Water: The animals were allowed free access to water. Mains drinking water was supplied from polycarbonate bottles attached to the cage. The drinking water was considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.
- Acclimation period: 12 days

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

IN-LIFE DATES: between 28 July 2009 (first day of treatment) and 16 September 2009 (final necropsy).

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: Arachis oil BP
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. 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 21 days. Formulations were therefore prepared fortnightly and stored at approximately 4ºC in the dark.
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 ± 8% 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): Test material was poorly water soluble, so Arachis oil BP was used as vehicle to prepare test material solution.
- Concentration in vehicle: 12.5, 87.5, 250 mg/ml
- Amount of vehicle (if gavage): 4 ml/kg
- Lot/batch no. (if required): No data available.
- Purity: No data available.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration, homogeneity and stability of test material in the test material formulations was determined by high performance liquid chromatography (HPLC) using an external standard technique.
Homogeneity Determinations
- The test material formulations were mixed thoroughly and samples were taken from the top, middle and bottom of the container, shaking between sampling. Sampling for homogeneity determinations was performed in triplicate.

Stability Determinations
- The test formulation were sampled and analyzed initially and then after storage at approximately 4 oC in the dark for 21 days.

Verification of Test Material Formulation Concentrations
- The test material formulations were sampled and analyzed within 2 days of preparation.

Method Validation
- The analytical method has been satisfactorily validated in terms of linearity, specificity and accuracy for the purposes of the study.
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 matings after two unsuccessful attempts: not applicable, all paired animals mated within the first four days of pairing.
- After successful mating each pregnant female was caged (how): the females were transferred to individual cages.
- Any other deviations from standard protocol: none.
Duration of treatment / exposure:
The test material was administered to four groups each of ten male and ten female rats, for up to fifty-four consecutive days
Frequency of treatment:
Daily
Duration of test:
Between 02 June 2009 and 27 November 2009.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
50 mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
350mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1000 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
10 animals per sex per dose (including control).
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were chosen based on the results of a preliminary range-finder performed as part of the study.

- Rationale for animal assignment (if not random): The animals were randomly allocated to treatment groups using a stratified bodyweight randomisation procedure and the group mean bodyweights were then determined to ensure similarity between the treatment groups. The cage distribution within the holding rack was also randomised. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Immediately before dosing, up to thirty minutes after dosing, and one and five hours afterdosing, during the working week. Animals were observed immediately before dosing, soon after dosing, and one hour after dosing at weekends and public holidays (except forfemales during parturition where applicable).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Immediately before dosing, up to thirty minutes after dosing, and one and five hours afterdosing, during the working week. Animals were observed immediately before dosing,soon after dosing, and one hour after dosing at weekends and public holidays (except forfemales 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 CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes.
During the maturation period, weekly food consumption was recorded for each cage of adults. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded for the periods covering Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded on Days 1 and 4 post partum.

- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Group mean weekly food consumptions
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data


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.

POST-MORTEM EXAMINATIONS: No.
- Sacrifice on gestation day #: no data available.
- Organs examined: no data available.
Ovaries and uterine content:
The uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: No
- Skeletal examinations: Yes: No
- Head examinations: Yes: No
Statistics:
For males and females during the pre-mating phase, quantitative data were analysed by the Provantis™ Tables and Statistics 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 Barletts’s test. The transformed data were analysed to find the lowest treatment level that showed a significant effect, 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 used to analyse implantation loss, offspring sex ratio and landmark developmental markers.
Histopathology data were analysed (excluding any decedents, nonmated females and females not producing a pregnancy/litter) using the following methods to determine significant differences between control and treatment groups for the individual sexes:
1. Chi-squared analysis for differences in the incidence of lesions occurring with an overall frequency ≥ 1.
2. Kruskal-Wallis one-way non-parametric analysis of variance for the comparison of severity grades for the more frequently observed graded conditions.
Indices:
Litter Data
On completion of parturition (Day 0 of post partum), the number of live and dead offspring was recorded. Offspring were individually identified within each litter by tattoo on Day 1 post partum. For each litter the following was recorded:
i) Number of offspring born
ii) Number of offspring alive recorded daily and reported on Day 1 and 4 post partum
iii) Sex of offspring on Day 1 and 4 post partum
iv) Clinical condition of offspring from birth to Day 5 post partum
v) Individual offspring and litter weights on Day 1 and 4 post partum

Physical Development
All live offspring were assessed for surface righting reflex on Day 1 post partum.
Historical control data:
Not available.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Mortality.
One female from this treatment group was also terminated early due to an unscheduled mating on Day 18. There were no further unscheduled deaths during the study.

Clinical Signs.
No clinical signs of toxicity were detected.

Bodyweights.
No adverse effects on bodyweight development were detected.

Food Consumption and Food Efficiency.
No adverse effects on food consumption or food efficiency were detected.

Water Consumptions. No significant intergroup differences were detected.
No intergroup differences were detected.

Haematology.
No toxicologically significant effects were detected in the haematological parameters measured.

Blood Chemistry.
No toxicologically significant effects were detected in the blood chemical parameters measured.

Behavioural Assessment.
There were no treatment-related changes in the behavioural parameters measured.

Functional Performance Tests.
There were no treatment-related changes in functional performance.

Sensory Reactivity Assessments.
There were no treatment-related changes in sensory reactivity.

Necropsy.
No toxicologically significant effects were detected

Organ Weights.
No toxicologically significant effects were detected in the organ weights measured.

Histopathology - non-neoplastic.
The following treatment-related effects were detected:
KIDNEY: A higher incidence of globular accumulations of eosinophilic material was observed in the tubular epithelium of males treated with 1000 or 350 mg/kg/day.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: other:

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Offspring Growth and Development. Offspring bodyweight gain and litter weights at birth and subsequently on Day 1 and 4 post partum were comparable to controls.

Litter observations. No clinically observable signs of toxicity were detected for offspring from all treatment groups.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The oral administration of test material to rats by gavage, at dose levels of 1000, 350 and 50 mg/kg/day, resulted in treatment-related effects at 1000 and 350 mg/kg/day. These effects were considered not to represent an adverse effect of treatment, hence the NOAEL for systemic and reproductive/developmental toxicity was considered to be 1000 mg/kg/day.
No treatment-related effects were observed for reproduction, therefore, a NOEL for reproductive toxicity was considered to be 1000 mg/kg/day.
Executive summary:

Introduction.The study was designed to investigate the systemic toxicity and potential adverse effects of the test material on reproduction (including offspring development) and complies with the recommendations of the OECD Guidelines for Testing of Chemicals No. 422 “Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test” .

 

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-four consecutive days (including a two week maturation phase, pairing, gestation and early lactation for females), at dose levels of 50, 350 and 1000 mg/kg/day. A control group of ten males and ten females was dosed with vehicle alone (Arachis oil BP).

Clinical signs, behavioural assessments, bodyweight development, food and water consumption were monitored during the study. Haematology and blood chemistry were evaluated prior to mating and at termination on five selected males and females from each dose group.

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.

Extensive functional observations were performed on five selected males from each dose group after the completion of the mating phase, and for five selected parental females from each dose group on Day 4post partum.

Males were terminated on Day 43, followed by the termination of all surviving females and offspring on Day 5post partum. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

Results.

Mortality.One male treated with 350 mg/kg/day was found dead on Day 35. One female from this treatment group was also terminated early due to an unscheduled mating on Day 18.

There were no further unscheduled deaths during the study.

Clinical Observations.No clinical signs of toxicity were detected.

Behavioural Assessment.There were no treatment-related changes in the behavioural parameters measured.

Functional Performance Tests.There were no treatment-related changes in functional performance.

Sensory Reactivity Assessments.There were no treatment-related changes in sensory reactivity.

Bodyweight.No adverse effects on bodyweight development were detected.

Food Consumption.No adverse effects on food consumption or food efficiency were detected.

Water Consumption.No intergroup differences were detected.

Haematology.No toxicologically significant effects were detected in the haematological parameters measured.

Blood Chemistry.No toxicologically significant effects were detected in the blood

chemical parameters measured.

Reproductive Performance

Mating.There were no treatment-related effects on mating or conception rates.

One female treated with 350 mg/kg/day was non pregnant.

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

Litter Size and Viability.Of the litters born, litter size at birth and subsequently on Day 1 and 4post partumwere comparable to controls.

Offspring Growth and Development.Offspring bodyweight gain and litter weights at birth and subsequently on Day 1 and 4post partumwere comparable to controls.

Litter observations.No clinically observable signs of toxicity were detected for offspring from all treatment groups.

Organ Weights.No toxicologically significant effects were detected in the organ weights measured.

Necropsy.The male treated with 350 mg/kg/day that was found dead on Day 35 had reddened lungs and fluid in the thoracic cavity.

Histopathology.The following treatment-related effects were detected:

LIVER: Centrilobular hepatocyte enlargement was seen in relation to treatment for males only treated with 1000 mg/kg/day.

Hepatocyte enlargement is commonly observed in the rodent liver following the administration of xenobiotics and, in the absence of associated inflammatory or degenerative changes, is generally considered to be adaptive in nature.

KIDNEY: A higher incidence of globular accumulations of eosinophilic material was observed in the tubular epithelium of males treated with 1000 or 350 mg/kg/day. A slightly higher incidence of the condition was also seen for males treated with

50 mg/kg/day compared with the control group, but this condition is seen occasionally as a spontaneous change and an effect of treatment at the low dose was not convincing.

This finding is consistent with the presence of hydrocarbon nephropathy, which results from the excessive accumulation ofα2-microglobulin in renal proximal tubular epithelial cells.α2-Microglobulin is found only in the proximal tubular epithelium of adult male rats.

Conclusion.The oral administration of test material to rats by gavage, at dose levels of 1000, 350 and 50 mg/kg/day, resulted in treatment-related effects at 1000 and 350 mg/kg/day. These effects were considered not to represent an adverse effect of treatment, hence the 'No Observed Adverse Effect Level' (NOAEL) for systemic and developmental toxicity was considered to be 1000 mg/kg/day.