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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:
From 2012-05-09 to 2012-06-25
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in accordance with current test methods and in compliance with GLP regulations

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD 422
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): METH-E (Methyl-(endo)-5-norbornene-2,3-dicarboxylic anhydride)
- Physical state: Liquid
- Analytical purity: 99.6%
- Lot/batch No.: T120211293
- Expiration date of the lot/batch: 2012-10-20
- Storage condition of test material: Cool, dry, well ventilated place, protected from humidity

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Toxi-Coop Zrt., Hungary
- Age at study initiation: 68 - 74 days (males and females)
- Weight at study initiation: 287 - 345 g (males); 159 - 196 g (females)
- Fasting period before study: No
- Housing: Before mating - 2 animals of the same sex/cage; During mating - 1 male and 1 female/cage; Pregnant females – individually; Males after mating - 2 animals/cage
- Diet (e.g. ad libitum): Ssniff SM R/M-Z+H complete rodent diet, ad libitum
- Water (e.g. ad libitum): Municipal supply, ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25 deg C
- Humidity (%): 30 - 70% RH
- Air changes (per hr): 8 - 12
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 2012-05-09 To: 2012-06-25

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
vegetable oil
Remarks:
Sunflower oiul
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water): Substance unstable in aqueous media
- Concentration in vehicle: 1.4, 4.0 and 10.0 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg body weight
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Performed twice during the study. Concentration of the test item in the dosing formulations varied in the range of 95% to 105% relative to nominal values.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: Until mating (within 6 days except for 1 pair which took 11 days for mating to be confirmed)
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: Individually
- Any other deviations from standard protocol: No
Duration of treatment / exposure:
Males - 42 days; starting 2 weeks before mating
Females - 41 - 47 days, depending on date of mating; Those with living pups dosed for 14 days pre-mating, during mating period, through gestation and up to lactation days 3-10. Non-pregnant animals were treated up to and including the day before necropsy (for 42 days)
Frequency of treatment:
Daily, 7 days/week
Duration of test:
Males - 42 days; starting 2 weeks before mating
Females - 41 - 47 days, depending on date of mating; Those with living pups dosed for 14 days pre-mating, during mating period, through gestation and up to lactation days 3-10. Non-pregnant animals were treated up to and including the day before necropsy (for 42 days)
No. of animals per sex per dose:
12 males / 12 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on outcome of 14-day preliminary study
- Rationale for animal assignment: Random

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily, after treatment
- Cage side observations included: General clinical observations including behavioural changes.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Males - weekly; Females - weekly with additional measurements on gestation days 10 and 17 and post-partum days 0 and 4

FOOD CONSUMPTION AND COMPOUND INTAKE):
- Food consumption determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule for examinations: Males - weekly; Females - weekly with additional measurements on post-partum days 0 and 4

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on post-partum Day 4
- Organs examined: Examination of external appearance, appearance of the tissues and organs examined after opening of the cranial, thoracic and abdominal cavities. Abnormalities recorded with details of location, color, shape and size. Special attention was paid to the organs of the reproductive system. The number of implantation sites and of corpora lutea was recorded.
- Organ weights: All males - testes, epididymides, brain. 5 males/5 females/group - Adrenal glands, Brain, Heart, Kidneys, Liver, Spleen, Thymus
- Tissues fixed and preserved: (from all animals) - Uterus with cervix, vagina, testes, epididymides (total and cauda), prostate, seminal vesicles with coagulating glands, ovaries, pituitary and all organs showing macroscopic lesions. (from 5 males/5 females/group) – Abnormalities, Adrenal glands, Aorta, Bone marrow (from femur), Brain (cerebrum, cerebellum, pons and medulla oblongata), Caecum, Colon, Duodenum, Epididymides, Eyes, Mammary area, Heart, Ileum, Jejunum (including Peyer’s patches), Kidneys, Liver, Lungs (including mainstem bronchi), Lymph nodes - submandibular, Lymph nodes - mesenteric, Oesophagus, Ovaries, Pancreas, Pituitary gland, Prostate gland, Rectum, Salivary glands, Sciatic nerve, Seminal vesicles with coagulating gland, Skeletal muscle, Skin, Spinal cord (cervical, mid-thoracic, lumbar), Spleen, Sternum, Stomach, Testes, Thymus, Thyroid, Trachea, Urinary bladder, Uterus – cervix, Vagina

HISTOPATHOLOGY: Yes
Tissues examined - Ovaries, uterus, vagina, pituitary, testes and epididymides (with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure) of the animals in the control and high dose groups and non-pregnant females and corresponding cohabited males. Examination of the ovaries covered the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma. Full examinations undertaken on the preserved organs and tissues of the randomly selected animals in the control and high dose groups and in dead animals. Examination of kidneys was extended to 5 animals/sex from the low- and mid-dose groups.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes / No / No data
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
SACRIFICE
- The F1 offspring were sacrificed at 4 days of age.
- These animals were subjected to postmortem examinations (macroscopic examination) as follows:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: No
- Skeletal examinations: No
- Head examinations: No
Statistics:
Homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test.
Where no significant heterogeneity was detected a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant Duncan Multiple Range test was used to assess the significance of inter-group differences.
Where the result of the Bartlett’s test was significant the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test.
Chi2 test was performed if feasible.
The frequency of clinical signs, pathology and histopathology findings were calculated.
Indices:
Gestation index, pre-implantation mortality, post-implantation mortality, intra-uterine mortality, post-natal mortality, sex ratio, survival index

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Salivation post-dose
Mortality:
mortality observed, treatment-related
Description (incidence):
Mortaliy in 2 females dosed at 50 mg/kg/day
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Reduced body weight in females at 20 and 50 mg/kg/day duruing the last week of gestation
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Reduced mean daily food consumption was observed in female animals at 50 mg/kg/day during the gestation and lactation periods and at 20 mg/kg/day between lactation days 0 and 4.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Kidney weight slightly increased in femaels treated at 50 mg/kg/day
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Effects in kidney at 50 mg/kg/day - focal or multifocal tubular basophilia with flattened tubular epithelial cells, slight inter-tubular lymphocytic infiltration in the cortical region, affecting the proximal convoluted tubules

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
no effects observed
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
At 50 mg/kg/day - changes in body weight during gestation and food consumption during gestation and lactation. Changes in organ pathology (slightly higher kidney weights), segmental tubular basophilia accompanied with flattened tubular epithelial cells and occasionally with slight inter-tubular lymphocytic infiltration affecting the proximal convoluted tubules of the kidneys
At 20 mg/kg /day - slight, but toxicologically not relevant, changes in body weight during gestation and food consumption during lactation.
At 7 mg/kg/day - No treatment related adverse effect. Slight change in body weight gain during last week of gestation was not considered to be toxicologically significant.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
20 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic

Maternal abnormalities

Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Skeletal malformations:
not specified
Visceral malformations:
not specified
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment related effects

Fetal abnormalities

Abnormalities:
no effects observed

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
In a repeat-dose toxicity combined with a screening study of reproductive toxicity conducted in accordance with OECD test methods, the substance caused no changes in male and female reproductive performance (gonad function, mating behaviour, conception, pregnancy, parturition) and dam’s delivery data. There were no test item related effects on offspring’s development. Based on these observations the No Observed (Adverse) Effect Level for reproductive performance of the male and female rats was regarded as 50 mg/kg body weight/day and the NO(A)EL for F1 Offspring was 50 mg/kg body weight/day
Executive summary:

In a repeat-dose toxicity combined with a screening study of reproductive toxicity conducted in accordance with OECD test methods, the substance caused no changes in male and female reproductive performance (gonad function, mating behaviour, conception, pregnancy, parturition) and dam’s delivery data. There were no test item related effects on offspring’s development. Based on these observations the No Observed (Adverse) Effect Level for reproductive performance of the male and female rats was regarded as 50 mg/kg body weight/day and the NO(A)EL for F1 Offspring was 50 mg/kg body weight/day