Reaction products of 3-aminomethyl-3,5,5-trimethylcyclohexylamine with 2,2'-[(1-methylethylidene)bis(4,1-phenyleneoxymethylene)]bisoxirane

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

A reproduction/developmental toxicity screening test was carried out with the test item in the rat according to OECD 421 and draft OECD guidance document 43. CRL:(WI)BR rats were administered repeated oral doses (by gavage) of 25, 80 or 200 mg/kg bw/day compared to control animals (treated with vehicle only). Under the conditions of this study, the no observed adverse effect level (NOAEL) for the test item for parental effects was 80 mg/kg bw/day. For reproduction parameters the NOAEL was 150 mg/kg bw/day. For pup growth rate, the NOAEL was 80 mg/kg bw/day.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2008-08-13 to 2009-06-23

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Draft OECD Guidance Document 43;

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (Europe) Laboratories Inc.; TOXI COOP Ltd. Hungary; 1103 Budapest, Cserkesz u. 90;
- Age at study initiation: 10 weeks;
- Weight at study initiation: Males: 350 - 451 g; Females: 196 - 262 g;
- Fasting period before study: not stated;
- Housing: before mating: 4 animals of same sex/cage; mating: 1 male / 1 female per cage; pregnant females were housed individually; Type II and III polypropylenepolycarbonate cages;
- Diet: SSNIFF SM R/M-Z+H Autoclavable complete feed for rats and mice - breeding and maintenance; ssniff Spezialdiäten GmbH; D-59494 Soest, Germany; ad libitum;
- Water: tap water from municipal supply; ad libitum;
- Acclimation period: 48 days;

ENVIRONMENTAL CONDITIONS
- Temperature: 19.8 - 24.9 °C;
- Humidity: 33 - 70 % R.H.
- Air changes: 8 - 12 air changes per hour;
- Photoperiod: 12 hrs dark / 12 hrs light daily from 06:00 a.m. to 06:00 p.m.;

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Details on exposure:

The test item was administered daily by oral gavage, at similar time. The oral route was selected by the Sponsor as a possible route of human exposure. Control animals were treated and handled in an identical manner to the test groups receiving 5 mL vehicle/kg bw (PEG 400, with no test item). Animals were not treated on the day of gross pathology. Dosing of both sexes began after an appropriate acclimatisation (A) period and two weeks before mating and was continued up to and including the day before necropsy. Mating began soon after the animals attained full sexual maturity. Dosing was continued in both sexes during the mating period.

Details on mating procedure:

Mating began 2 weeks after the initiation of treatment with one female and one male of the same dose group (1:1 mating) in a single cage. Females remained with the same male until copulation occurred (up to 5 days), with the exception of female 4511, as detailed below. Each morning a vaginal smear was prepared and stained with 1 % aqueous methylene blue solution. The smear was 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 OECD 421). Sperm positive females were caged individually.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentration of the test item was determined using reverse phase HPLC with UV detection on a LiChrospher 60 RP select B column.
Apparatus
HPLC system: Merck-Hitachi LaChrom HPLC system:
D-7000 Interface, No.: 1442-122
L-7100 HPLC pump, No.: 1516-030
L-7200 Autosampler, No.: 1406-005
L-7400 UV Detector, No.: 1502-017
L-7360 Column Oven, No.: 00107295
L-7614 Degasser, No.: 14412YA0500
Balances: BP 221S Sartorius, No.: 11809117
Water purification system: MILLIPORE, DIRECT Q3, FOMNO 7334I
Ultrasonic bath: Elmasonic S 300 H, No.: 010890105
Refrigerator: Zanussi, No: ZLKI-262
HPLC Conditions
Detector: UV at 230 nm
Column: LiChrospher 60 RP select B (5 μm), 250x4 μm No.: 737432
Mobil Phase:
A: 0.1 % Trifluoroacetic acid in water
B: 0.1 % Trifluoroacetic acid in Acetonitrile
Gradient:
0 min 80 % A
8 min 10 % A
12 min 10 % A
14 min 80 % A
16 min 80 % A
Flow: 0.8 mL/min
Injection volume: 50 μL
Temperature: 25 °C
Retention time: 7.0 min ± 10 %

Duration of treatment / exposure:

Males were dosed for 28 days, 14 days pre-mating (PM) and 14 days mating/post-mating period (M), then they were euthanised and subjected to necropsy examination. Females were dosed for 14 days pre-mating, for up to 8 days mating period, through gestation (up to 23 days) and day 3 post-partum with necropsy the following day, or shortly thereafter. The day of birth (viz. when parturition was complete) was defined as day 0 post-partum. All F1 offspring were terminated on day 4 post partum or shortly thereafter (up to Day 5 post partum).

Frequency of treatment:

The test item was administered daily by oral gavage, at similar time. The oral route was selected by the Sponsor as a possible route of human exposure. Control animals were treated and handled in an identical manner to the test groups receiving 5 mL vehicle/kg bw (PEG 400, with no test item).

Details on study schedule:

The purpose of this study was to obtain initial information on the possible effects of the test item on reproduction and development when administered orally (by gavage) to CRL:(WI)BR rats at repeated doses of 25 , 80 or 200 mg/kg bw/day compared to control animals (treated with vehicle only).
Due to adverse effects and mortality observed in the high dose group, the dose level was reduced in the remaining female animals from 200 to 150 mg/kg bw/day as of day 36. As a screening test, it was intended to provide initial information on possible effects on male and female reproductive performance such as gonadal function, mating behaviour, conception, pregnancy, parturition and on development of the F1 offspring from conception to day 4 postpartum associated with administration of repeated doses. Stability and homogeneity of the test item in the vehicle, polyethylene glycol 400 (PEG 400), was analytically proven. Assessment of test item stability in this vehicle, in the conditions employed on the study indicated up to 72-hour stability when stored at room temperature, at approximately 0.5 and 50 mg/mL concentration levels. Analytical control of dosing solutions was conducted during the study from all the concentrations employed. The measured concentrations ranged from 96 to 106 % of nominal concentrations. Results were considered suitable for the study purposes. Clinical observations for signs of ill health or reaction to treatment were made once daily. Special attention was paid to evaluation of the mating, pregnancy, parturition and post-partal periods, and relevant parameters and/or indices were measured and/or calculated. Body weight and food consumption were measured at least weekly. Gross necropsy was conducted at the end of the treatment period. The absolute and relative organ weights of a selected list of organs and tissues were determined. A histopathological examination was performed on the selected list of preserved organs and tissues of the animals of groups 1 (control) and 4 (high dose), and on abnormal tissues from low and mid dose groups.

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Dose / conc.:

25 mg/kg bw/day (actual dose received)

Dose / conc.:

80 mg/kg bw/day (actual dose received)

Dose / conc.:

200 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

12 males/12 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

Dose levels were reduced from 200 to 150 mg/kg bw/day (high dose) as of day 36 due to adverse effects and mortality observed. Dose concentration changed accordingly (30 mg/mL at 150 mg/kg bw/day).
A constant dose volume of 5 mL/kg bw/day was administered in all groups. The individual dose volume was based on the most recent individual body weight of the animals (which was determined at least weekly, or more often during the pregnancy period). In the first week of the pre-mating period, animals received the volumes based on the actual body weight on day 0.

Parental animals: Observations and examinations:

Animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each day). The animals that were found dead were processed in the same way as the animals of the terminal necropsy. General clinical observations were made once a day, after treatment at approximately the same time, considering the peak period of anticipated effects after dosing. All signs of toxicity including mortality were recorded including onset, degree and duration of signs. No obvious behavioural changes or signs of difficult or prolonged parturition were noted. Weekly, more detailed examinations were made at the times of weekly weighing, prior to and during the mating until necropsy.

Litter observations:

Females were allowed to litter and rear their offspring. Delivery process was observed as carefully as possible. All observations were recorded. There was no evidence of abnormal deliveries. The duration of gestation was recorded and was calculated from day 0 of pregnancy. Dams were observed for nesting behaviour (whether they made a nest from the bedding material and cover their new-borns or not). The efficiency of the suckling was observed by the presence of milk in the pups' stomach. All observations were recorded. Each litter was examined as soon as possible after delivery to establish the number and sex of pups, stillbirths, live births, runts (pups that were significantly smaller than normal pups), and the presence of gross abnormalities. Live pups were counted, sexed, weighed individually with an accuracy of 0.1 g within 24 hours of parturition (on the first day after parturition was complete), and day 4 post partum with an accuracy of 0.1g. Observations are reported individually for each adult animal.

Postmortem examinations (parental animals):

Gross necropsy was performed on each animal irrespective of the date of its death. Terminally (one day after the last treatment), animals were sacrificed under pentobarbital anaesthesia (details are presented in "Details of Other Materials") by exsanguination. After examination of the external appearance the cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed. Any abnormality was recorded with details of the location, colour, 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. The testes, epididymides (total and caudal), seminal vesicles and prostate; female reproductive organs including ovary, uterus (with and without cervix), and vagina; brain and pituitary of all adult animals were weighed. Paired organs were weighed separately.

Postmortem examinations (offspring):

Pups euthanized at day 4 postpartum, or shortly thereafter (up to day 5 postpartum), were carefully examined at least externally for gross abnormalities. No pups with abnormalities in structure or behaviour were observed. The found-dead pups were subjected to necropsy with macroscopic examination.

Statistics:

The statistical evaluation of appropriate data (marked † below) was performed with the statistical program package SPSS PC+ 4.0. 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. Where significant result was obtained 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 required.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Test item-related clinical signs occurred at 200 mg/kg bw/day, and consisted of noisy respiration, decreased activity, mucoid faeces, and/or red material around the nasal area. The clinical observations at 80 and 25 mg/kg bw/day were not considered to be of toxicological significance.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

Mortality at 200 mg/kg bw/day (4/12 male and 3/12 female animals) was observed. No rats died after the high dose was reduced to 150 mg/kg bw/day. One additional female (1/12) died due to misdosing.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Lower body weights than control animals, and significantly lower body weight gain values were observed in the 200 mg/kg bw/day male animals throughout the study. A small transient statistical difference at 80 mg/kg bw/day noted in male animals on a single occasion was not considered to represent a toxicologically significant event. No significant adverse effects on the body weights and body weight gains were noted in the female animals.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In the male animals, decreased food consumption occurred at 200 mg/kg bw/day during the first two weeks of treatment, attaining statistical significance in week one. There were no such effects observed in the female animals. The difference in males correlated with the lower body weight gains.

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

There were no adverse findings at macroscopic or microscopic examination at up to and including 150 mg/kg bw/day. In the found dead animals at 200 mg/kg bw/day, dilatation of the stomach and/or intestine with the presence of yellow gelatinous material/fluid observed in 7/8 found dead rats were regarded as test item-related and cause of death for these animals.

Other effects:

not specified

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

Description (incidence and severity):

The parental animals displayed no effects related to treatment with regard to the reproductive ability and mating, gestation, parturition or post-partal period.

Key result

Dose descriptor:

NOAEL

Effect level:

80 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

Key result

Critical effects observed:

not specified

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No significant clinical or behavioural changes were noted in the F1 generation following administration of the test item in the parental (P) generation.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Dead pups in the control and low and mid dose treatment were found at first evaluation following burth. No deaths or observations in pups were likely attributed to treatment.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The overall means of individual pup weights and weight gains were significantly below control at 200/150 mg/kg bw/day.

Sexual maturation:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

no effects observed

Description (incidence and severity):

No external abnormalities ascribed to test item administration could be detected at the clinical or macroscopic examinations of the pups.

Histopathological findings:

no effects observed

In the F1 generation, there were no pups with gross abnormalities. No effect on pup survival was noted; gross necropsy of dead pups showed no effects of treatment. The overall means of individual pup weights and weight gains were significantly below control at 200/150 mg/kg bw/day.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

80 mg/kg bw/day

Based on:

test mat.

Sex:

not specified

Basis for effect level:

other: pup growth rate

Key result

Critical effects observed:

not specified

Key result

Reproductive effects observed:

not specified

Conclusions:

In conclusion, under the conditions of this study, the no observed adverse effect level (NOAEL) for the test item for parental effects was 80 mg/kg bw/day. For reproduction parameters, the NOAEL was 150 mg/kg/day. For pup growth rate, the NOAEL was 80 mg/kg bw/day.

Executive summary:

The objective of this study was the Reproduction/Developmental Toxicity Screening Test with the test item in the Rat according to OECD 421 and the draft OECD guidance document 43. The guideline is designed for use with the rat, which is the preferred rodent species for reproduction toxicity testing. The purpose of this study was to obtain initial information on the possible effects of the test item on reproduction and development when administered orally (by gavage) to CRL:(WI)BR rats at repeated doses of 25 , 80 or 200 mg/kg bw/day compared to control animals (treated with vehicle only). Due to adverse effects and mortality observed in the high dose group, the dose level was reduced in the remaining female animals from 200 to 150 mg/kg bw/day as of day 36 (see Dose Administration section). As a screening test, it was intended to provide initial information on possible effects on male and female reproductive performance such as gonadal function, mating behaviour, conception, pregnancy, parturition and on development of the F1 offspring from conception to day 4 postpartum associated with administration of repeated doses. Stability and homogeneity of the test item in the vehicle, polyethylene glycol 400 (PEG 400), was analytically proven. Assessment of test item stability in this vehicle, in the conditions employed on the study (LAB study code 07/523-316AN) indicated up to 72-hour stability when stored at room temperature, at approximately 0.5 and 50 mg/mL concentration levels. Analytical control of dosing solutions was conducted during the study from all the concentrations employed. The measured concentrations ranged from 96 to 106 % of nominal concentrations. Results were considered suitable for the study purposes. Clinical observations for signs of ill health or reaction to treatment were made once daily. Special attention was paid to evaluation of the mating, pregnancy, parturition and post-partal periods, and relevant parameters and/or indices were measured and/or calculated. Body weight and food consumption were measured at least weekly. Gross necropsy was conducted at the end of the treatment period. The absolute and relative organ weights of a selected list of organs and tissues were determined. A histopathological examination was performed on the selected list of preserved organs and tissues of the animals of groups 1 (control) and 4 (high dose), and on abnormal tissues from low and mid dose groups.

The test item administered daily by oral gavage in Wistar rats for 28 days in the male animals, and up to 48 days in the female animals led to adverse effects and mortality at 200 mg/kg bw/day (4/12 male and 3/12 female animals). No remaining female rats died, or displayed systemic adverse effects, after reduction of the high dose to 150 mg/kg bw/day from day 36 of treatment. Test item-related clinical signs occurred at 200 mg/kg bw/day, and consisted of noisy respiration, decreased activity, mucoid faeces, and/or red material around the nasal area. The clinical observations at 80 and 25 mg/kg bw/day were not considered to be of toxicological significance. Slightly lower body weights and significantly lower body weight gain values were observed in the 200 mg/kg bw/day males throughout the study when compared to controls. In the 80 mg/kg bw/day males, a transient statistically significant lower body weight gain was noted during the second week of treatment. However, the animals fully recovered the following week and their body weight gain values remained similar to the control values until the end of the 28-day dose administration. In the female animals, there were no toxicologically significant changes in the mean body weight values during the pre-mating, mating, gestation or postpartal periods at dose levels up to and including 200/150 mg/kg bw/day. In the male animals, decreased food consumption occurred at 200 mg/kg bw/day during the first two weeks of treatment, attaining statistical significance in week one. There were no such effects observed in the female animals. The difference in males correlated with the lower body weight gains. There were no significant differences between the control and test item treated groups with regard to reproductive ability, or in the mating, fertility or gestation indices. Test item administration did not impact the duration of the mating period. Successful coitus (indicated by sperm positive vaginal smears and/or vaginal plugs) generally occurred within 5 days of pairing (cohabitation). One high dose female, although sperm positive, was not pregnant (i.e. did not show evidence of implantation at necropsy examination). This effect was ascribed to a possible pre-coital genital infection. No test item effect was observed in the duration of pregnancy since all females littered in 21 to 23 days. There were no abnormalities in pups that could be ascribed to the treatment. All the parturitions were normal. There were no adverse effects, or biologically significant variations of the parameters related to pregnancy, parturition, or postpartal period noted in the treated groups at dose levels up to and including 150 mg/kg bw/day (previously treated with 200 mg/kg bw/day, as presented in the Experimental Design section) compared to control animals. There were no test item-related alterations in the delivery data of the test item treated dams as compared to the control data.

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

80 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP and guideline study

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

A Reproduction/Developmental Toxicity Screening Test with the test item in the Rat was performed according to OECD 421 and the draft OECD guidance document 43. The guideline is designed for use with the rat, which is the preferred rodent species for reproduction toxicity testing. The purpose of this study was to obtain initial information on the possible effects of the test item on reproduction and development when administered orally (by gavage) to CRL:(WI)BR rats at repeated doses of 25 , 80 or 200 mg/kg bw/day compared to control animals (treated with vehicle only). Due to adverse effects and mortality observed in the high dose group, the dose level was reduced in the remaining female animals from 200 to 150 mg/kg bw/day as of day 36. As a screening test, it was intended to provide initial information on possible effects on male and female reproductive performance such as gonadal function, mating behaviour, conception, pregnancy, parturition and on development of the F1 offspring from conception to day 4 postpartum associated with administration of repeated doses. Stability and homogeneity of the test item in the vehicle, polyethylene glycol 400 (PEG 400), was analytically proven. Assessment of test item stability in this vehicle, in the conditions employed on the study (LAB study code 07/523-316AN) indicated up to 72-hour stability when stored at room temperature, at approximately 0.5 and 50 mg/mL concentration levels. Analytical control of dosing solutions was conducted during the study from all the concentrations employed. The measured concentrations ranged from 96 to 106 % of nominal concentrations. Results were considered suitable for the study purposes. Clinical observations for signs of ill health or reaction to treatment were made once daily. Special attention was paid to evaluation of the mating, pregnancy, parturition and post-partal periods, and relevant parameters and/or indices were measured and/or calculated. Body weight and food consumption were measured at least weekly. Gross necropsy was conducted at the end of the treatment period. The absolute and relative organ weights of a selected list of organs and tissues were determined. A histopathological examination was performed on the selected list of preserved organs and tissues of the animals of groups 1 (control) and 4 (high dose), and on abnormal tissues from low and mid dose groups.

The test item administered daily by oral gavage in Wistar rats for 28 days in the male animals, and up to 48 days in the female animals led to adverse effects and mortality at 200 mg/kg bw/day (4/12 male and 3/12 female animals). No remaining female rats died, or displayed systemic adverse effects, after reduction of the high dose to 150 mg/kg bw/day from day 36 of treatment. Test item-related clinical signs occurred at 200 mg/kg bw/day, and consisted of noisy respiration, decreased activity, mucoid faeces, and/or red material around the nasal area. The clinical observations at 80 and 25 mg/kg bw/day were not considered to be of toxicological significance. Slightly lower body weights and significantly lower body weight gain values were observed in the 200 mg/kg bw/day males throughout the study when compared to controls. In the 80 mg/kg bw/day males, a transient statistically significant lower body weight gain was noted during the second week of treatment. However, the animals fully recovered the following week and their body weight gain values remained similar to the control values until the end of the 28-day dose administration. In the female animals, there were no toxicologically significant changes in the mean body weight values during the pre-mating, mating, gestation or postpartal periods at dose levels up to and including 200/150 mg/kg bw/day. In the male animals, decreased food consumption occurred at 200 mg/kg bw/day during the first two weeks of treatment, attaining statistical significance in week one. There were no such effects observed in the female animals. The difference in males correlated with the lower body weight gains. There were no significant differences between the control and test item treated groups with regard to reproductive ability, or in the mating, fertility or gestation indices. Test item administration did not impact the duration of the mating period. Successful coitus (indicated by sperm positive vaginal smears and/or vaginal plugs) generally occurred within 5 days of pairing (cohabitation). One high dose female, although sperm positive, was not pregnant (i.e. did not show evidence of implantation at necropsy examination). This effect was ascribed to a possible pre-coital genital infection. No test item effect was observed in the duration of pregnancy since all females littered in 21 to 23 days. There were no abnormalities in pups that could be ascribed to the treatment. All the parturitions were normal. There were no adverse effects, or biologically significant variations of the parameters related to pregnancy, parturition, or postpartal period noted in the treated groups at dose levels up to and including 150 mg/kg bw/day (previously treated with 200 mg/kg bw/day) compared to control animals. There were no test item-related alterations in the delivery data of the test item treated dams as compared to the control data.

Effects on developmental toxicity

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data is reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. As a result the substance is not considered to be classified for toxicity to reproduction under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.