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Administrative data

Endpoint:
one-generation reproductive toxicity
Remarks:
based on generations indicated in Effect levels (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From December 02, 2014 to January 25, 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to OECD Guideline 422, in compliance with GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2015

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
2-Propenenitrile, reaction products with 3-amino-1,5,5-trimethylcyclohexanemethanamine
EC Number:
292-053-3
EC Name:
2-Propenenitrile, reaction products with 3-amino-1,5,5-trimethylcyclohexanemethanamine
Cas Number:
90530-15-7
Molecular formula:
C10 H22 N2 . C3 H3 N1
IUPAC Name:
2-Propenenitrile, reaction products with 3-amino-1,5,5-trimethylcyclohexanemethanamine
Test material form:
other: liquid

Test animals

Species:
rat
Strain:
other: Crl:WI rats
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, D-97633, from SPF colony.
- Age at study initiation: Young adult rats, approximately 11 weeks old at starting and 13 weeks at mating.
- Weight at study initiation: Males: 344-415 g, Females: 209-259 g; did not exceed ±20% of the mean weight for each sex at onset of treatment.
- Housing: Type II polycarbonate with Laboratory bedding, GRADE 5 (produced by Johannes Brandenburg GmbH & Co. KG; Arkeburger Str. 31; DE-49424 Goldenstedt) were available to animals during the study.
- Diet: Ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and Maintenance" produced by Ssniff Spezialdiäten GmbH, D-59494 Soest, Germany ad libitum
- Water: Tap water from municipal supply, as for human consumption from 500 mL bottle ad libitum
- Acclimation period: 5 d

ENVIRONMENTAL CONDITIONS
- Temperature: 20.3–24.8°C
- Humidity: 30–58%
- Air changes: 15-20/h
- Photoperiod: 12 h dark/12 h light

IN-LIFE DATES: From: December 02, 2014 To: January 25, 2015

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: Phosphate Buffered Saline (pH=7.2)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance was formulated in the vehicle, as a visibly stable homogenous formulation at the appropriate concentrations according to the selected dose level and volume, in the Central Dispensary of CiToxLAB Hungary Ltd. Formulations were prepared in up to 7 d intervals and stored at room temperature, based on the stability assessment results.

VEHICLE
- Lot/batch number: SLBG8294
- Manufacturer: Sigma-Aldrich Co.
- Expiry/retest Date: August 2015
- Storage: Room temperature
Details on mating procedure:
- Mating procedure:
Mating began after the animals had attained full sexual maturity, 2 weeks after the initiation of treatment, with one female and one male from the same dose group (1:1 mating) in a single cage. Females remained with the same male until copulation occurred, for up to 14 d.

A vaginal smear was prepared daily during the mating period and stained with 1% aqueous methylene blue solution. The smears were examined with a light microscope. The presence of a vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (Day 0 of pregnancy as defined by the relevant guidelines). Sperm positive females were housed individually.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability of the test substance in the vehicle was assessed in the conditions employed on the study. Analysis of formulation samples of 0.1–50 mg/mL concentration range showed no decrease of concentration and can be considered as stable for 7 d in room temperature.
Duration of treatment / exposure:
Males were dosed for 28 d (14 d pre-mating and 14 d mating/post-mating) and then euthanized and subjected to necropsy examination.
Females were dosed for 14 d pre-mating, during the mating period, through gestation and until the day before the necropsy (at least 4 d post-partum dosing).
Frequency of treatment:
daily once
Details on study schedule:
- Dosing procedure:
Test substance or negative control treated animals were administered the dosing formulations daily on a 7 d/week basis by oral gavage using a tipped gavage needle attached to a syringe. A constant volume of 10 mL/kg bw was administered to all animals. The actual volume administered was calculated and adjusted based on most recent individual body weights.

Dosing of both sexes began after 5 d of acclimation and 2 weeks before mating and continued up to the day before necropsy. The mating began after the animals had attained full sexual maturity and continued in both sexes during the mating period.

Males were dosed for 28 d (14 d pre-mating and 14 d mating/post-mating) and then euthanized and subjected to necropsy examination.

Females were dosed for 14 d pre-mating, during the mating period, through gestation and until the day before the necropsy (at least 4 d post-partum dosing). The day of birth (when parturition was complete) was defined as Day 0 post-partum. Females showing no-evidence of copulation were sacrificed 25 d after the end of the mating period (one in the control and one low dose, with no delivery).

All F1 offspring were terminated on Day 4 post-partum. In order to allow for overnight fasting of dams prior to urine collection on PPD5, the offspring were euthanized on PPD/PND 4 and the dams on PPD/PND 5.
Doses / concentrations
Remarks:
Doses / Concentrations:
10, 30, or 100 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
48 male, 48 female rats, 12 animals/sex/group, 4 groups
Control animals:
yes, concurrent vehicle
Details on study design:
- Rationale for dose selection and route of administration:
The dose levels were selected based on available data and information from previous experimental work, including the results of a repeated dose range finding study in the rat. The aim was to induce toxic effects but ideally no death or suffering at the highest dose and a NOAEL at the lowest dose. During the Dose Range Finding study, treatment-related mortality was observed at dose levels 1,000, 500 and 300 mg/kg bw/day. There was no mortality in the lower dose groups (100 and 30 mg/kg bw/day). The oral route was selected as it is a possible route of exposure to the test substance in humans.

- Rationale for animal assignment:
All parental (P) animals were sorted according to body weight by computer and divided into weight ranges. There were an equal number of animals from each weight group in each of the experimental groups assigned by randomization to ensure that animals of all test groups were as close as practicable to a uniform weight. The grouping was controlled by SPSS/PC software according to the actual body weight, thereby verifying the homogeneity/variability between/within the groups and cages. Males and females were randomized separately.

Examinations

Oestrous cyclicity (parental animals):
During the mating period, until a sperm positive vaginal smear or a vaginal plug is identified.
Statistics:
The statistical evaluation of appropriate data (marked † below) was performed with the statistical program package SPSS PC+4.0 (SPSS Hungary Kft, Budapest). The 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 access the significance of inter-group differences. For a significant result at Bartlett’s test, the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test. Chi2 test was performed as feasible. For SMART evaluation, T-test was used.

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:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
(Test substance related microvacuolation of the epithelial cells in the body segment of the epididymis in the high dose males (100 mg/kg bw/day) was noted, in correlation with the organ weight changes)

Reproductive function / performance (P0)

Reproductive performance:
no effects observed

Details on results (P0)

DOSE FORMULATION ANALYSIS
The measured concentrations of test substance evaluated for each test substance-dose group varied between 94% and 110% of the nominal contents. No test substance was detected in the control samples. These results were within acceptable ranges (85% - 115%) and are acceptable for the study purposes.

PARENTAL/ADULT EVALUATION
Mortality
There was no mortality during the study.

Clinical observation
No test substance related adverse effects or systemic clinical signs were noted during the study.

Neurological assessment
There were no treatment related effects.

There were no toxicologically significant changes in animal behavior, general physical condition or in the reactions to different type of stimuli in the control or test groups.

There was no effect of treatment noted during the assessment of foot splay, grip strength or motor activity. The total travelled distance in the test groups was comparable to the control groups for both sexes.

Body weight and body weight gain
No test substance related effects were noted on the mean body weight and body weight gain values following daily administration of test substance at dose levels up to and including 100 mg/kg bw/day.

Occasional statistically significant differences in the body weight gain were regarded as incidental and without toxicological significance.

Food consumption
There were no test substance related differences in the mean daily food consumption in any test substance treated group when compared to the controls.

Occasional statistically significant differences were regarded as incidental and of no toxicological significance.

Clinical pathology

Haematology
When compared to the controls, there were no differences that could be considered toxicologically significant in the treated animals.

There was no effect of treatment on coagulation parameters investigated, i.e. Activated Partial Thromboplastin Time (APTT) and Prothrombin Time (PT).

Variations were noted in a few parameters, on occasion attaining statistical significance, including higher Activated Partial Thromboplastin Time (APTT) in low dose females (p<0.05), or lower Platelet (thrombocyte) count (Plt) in mid dose females (p<0.05).

Evaluation of the mean and individual results in comparison with the control data did not reveal any test substance related cause of the changes noted, and/or no consistent dose or gender-related response was observed. Therefore, these differences observed between the control and treated groups were considered to be incidental or individual findings, which were not related to treatment, were generally comparable with the expected physiological range and of no toxicological significance.

Clinical chemistry
In the animals evaluated at the completion at termination (on Day 28 in males and on PPD5 in females), there were no toxicologically significant changes or adverse effects on the animal serum chemistry that could be ascribed to test substance administration in the conditions of this study.

On Day 28, Cholesterol concentration was slightly higher than control in the high dose males. The differences attained statistical significance (p<0.05); however, there was no dose or gender response or the values were within the physiological ranges. For this reason, these variations were not considered toxicologically significant or related to treatment.

Urinalysis
There was no effect of treatment noted during urinalysis.

Slight differences which attained statistical significance were record in urine gravity in mid and high dose males (p<0.05 and p<0.01, respectively). However, these findings were regarded as minor variations and to be of no toxicological importance.

Reproductive ability assessment and indices
There were no differences between the control and test substance treated groups with regard to reproductive ability, mating or gestation indices, and no effects considered adverse or toxicologically significant in correlation with test substance administration. The mating indices were 100% in all groups and the fertility index was 92, 92, 100 and 100% in control, low, mid and high dose groups, respectively. The gestation index was 100% in all groups.

Test substance administration was considered to have no impact on the duration of the mating period. Successful coitus (sperm positive vaginal smears and/or vaginal plugs) generally occurred within up to 5 d of pairing (cohabitation). In one low dose female, the duration of the mating period was 14 d, with almost continuous dioestrus observed during the oestrus cycle, however this female successfully paired on Day 14. This variation was regarded as individual, normal biological variability and without toxicological significance.

Mean pre-coital interval was 3.2, 3.2, 2.6 and 3.0 d in control, low, mid and high dose groups, respectively.

Evaluation of the gestation, parturition and post-partal period

There was no effect of treatment noted during gestation, parturition or the post-partal period.

The mean duration of pregnancy was comparable in the control and test substance treated groups.

The number of corpora lutea and number of implantation sites was comparable to the control mean at all dose groups.

There were no significant differences or effects that could be ascribed to treatment on the pre-implantation, post-natal or total mortality values (litter mean and %) at up to and including 100 mg/kg bw/day.

Compared to the control group, higher intrauterine mortality (abs and %) was observed in low dose females (by 21%) with attaining statistically significance. In the absence of the similar effect in the mid and High dose group, these changes were considered incidental and unrelated to the treatment.

PATHOLOGY EVALUATION AND ORGAN WEIGHTS

Pathology evaluation

TERMINAL / Parental Generation (Males, Day 28, Females, PND5)

Macroscopic Findings

No treatment related macroscopic findings were noted at necropsy.

The changes such as enlarged spleen, or adrenal, multifocal, dark red discoloration of the glandular mucosa of the stomach, based on the low incidence, and distribution in control and dosed animals, were considered as incidental or background.

Microscopic Findings

Test substance-related vacuolation of the epithelial cells in the body of the epididymides was observed in the high dose males (100 mg/kg bw/day). Bilateral minimal to mild microvesicular vacuolation affected 12/12 males. The small, clear cytoplasmic vacuoles, without degenerative/necrotic/inflammatory signs or increased cell debris in the lumina, were present in the epithelial cells of the body segment, correlated with organ weight changes. No microscopic changes were detected in the epididymis of the mid dose male rats.

The histopathological picture of the male gonads as well as testicular interstitial cell structure, the spermatogenic cells representing different phases of the development and differentiation of the spermatozoa were similar in control and high dose males. The follicular, luteal and interstitial compartments of the ovary as well as epithelial capsule and stroma were similar histological structure in both control and high dose females.

Other changes including dilatation in the kidney (1/5 control male), congestion/haemorrhage in the thymus (1/5 control male) and in the gastric mucosa (1/1 low dose female), minimal/mild extramedullary hematopoiesis of the spleen both in the control (5/6) and high dose (3/5) and in one low dose female (2506) and one mid dose female (3509) as well, based on the low incidence and/or severity and/or distribution cross control and dosed animals were incidental or a common background.

Organ weights
Test substance related differences were found in epididymides weights at high dose males.

The absolute and brain weight relative epididymides weight was higher than the control in the high dose males attaining statistical significance (by 10%, p<0.01). These findings were associated with the microscopic changes.

Compared to controls, slightly higher weights of liver were recorded for high dose males. The difference was approximately 11-12% and attained statistical significance (p<0.01) for absolute and relative values. The changes were not associated with any findings in clinical pathology or microscopic changes and were regarded as physiological variation.

There were no other toxicologically significant differences among groups in the weights of other organs measured when compared to controls.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Effect level:
ca. 100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: No test substance related adverse effects up to the highest tested dose.
Dose descriptor:
NOAEL
Effect level:
ca. 30 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Adverse effect on epididymides weights and histopathological changes of microvacuolation of the epithelial cells in the body segment of the epididymis at 100 mg/kg bw/day in males.

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Gross pathological findings:
no effects observed

Details on results (F1)

OFFSPRING (F1) GENERATION

Mortality and clinical observations
The number of viable pups on PND4 as well as pups survival indices on PND0 and PND4 were comparable to control values at up to and including 100 mg/kg bw/day. Overall, there were not treatment-related effects on pup mortality. There were no treatment related effect on the viability of pups on PND 0 and PND 4.

Body weight and body weight gain

There were no effects considered adverse on the offspring weight or weight gain following administration of test substance at 10, 30 or 100 mg/kg bw/day to parental generation under the conditions of this study.

When evaluated per litter basis, the mean litter body weights and/or body weight gain on PND 0 and 4 showed no statistically or toxicologically significant differences compared to controls in the F1 generation.

There were no effects of treatment on pup weights or weight gains.

PATHOLOGY EVALUATION AND ORGAN WEIGHTS

TERMINAL / F1 Generation

Macroscopic Findings
No macroscopic changes were seen in F1 offspring generation euthanized and examined externally at scheduled termination on PND 4.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
ca. 100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: There were no adverse effects on the F1 offspring viability, clinical signs, development or at observations following euthanasia

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The NOAEL for the test substance is considered to be 100 mg/kg bw/day for females and 30 mg/kg bw/day for males parental/adult generation and 100 mg/kg bw/day for the F1/pups generation.
Executive summary:

A study was conducted to assess the toxicity of the test substance following repeated administration according to OECD Guideline 422, in compliance with GLP. Male and female Wistar rats were treated for 2 weeks pre-mating and then during the mating/post-mating periods. This was 28 d in total for males. Females were treated throughout gestation and up to and including postpartum/lactation Day PPD4. Parameters measured during the study included signs of morbidity and mortality twice daily, daily or detailed weekly observation of clinical signs, neurological assessment, weekly body weight and food consumption, and clinical pathology evaluation, including haematology, coagulation, clinical chemistry and urinalysis. Neurological assessment including functional observation battery (FOB) and measurements of the landing foot splay, grip strength and motor activity were performed during the last week of the treatment. At termination, necropsy with macroscopic examination was performed. Weights of selected organs were recorded and representative tissues/organs were sampled and preserved in appropriate fixatives from the adult animals. For the adult animals, a detailed histological examination was performed on the selected list of retained organs in the control and high dose groups. Due to treatment related changes in the epididymis in high dose males, the histological examination of this tissue was extended to the mid dose males. Daily administration of test substance by oral gavage to Wistar rats at dose levels of 10, 30, or 100 mg/kg bw/day during the treatment period under the conditions of this study did not result in test substance related mortality, clinical adverse effects, or changes in neurological assessment, body weight, food consumption, haematology, coagulation, clinical chemistry or urinalysis parameters. No test substance related changes were noted in the reproductive parameters during mating and gestation, delivery and post-partum/lactation period until PPD5. There were no adverse effects on the F1 offspring viability, clinical signs, development or at observations following euthanasia.Test substance related effects were found in epididymides weights at 100 mg/kg bw/day in males, in correlation with the histopathological changes. There were no test substance related organ weight changes in females. Test substance-related microvacuolation of the epithelial cells in the body segment of the epididymis in the high dose males (100 mg/kg bw/day) was noted, in correlation with the organ weight changes. No test substance-related microscopic changes were noted in the female reproductive organs. In conclusion, under the conditions of this study, the NOAEL for the test substance is considered to be 100 mg/kg bw/day for females and 30 mg/kg bw/day for males parental/adult generation and 100 mg/kg bw/day for the F1/pups generation (Török-Bathó M, 2015).

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