Reaction products of m-phenylenebis(methylamine) with 2,2'-[(1-methylethylidene)bis(4,1-phenyleneoxymethylene)]bisoxirane

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2008-07-11 to 2009-06-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Draft OECD Guidance Document 43

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Reaction products of m-phenylenebis(methylamine) with 2,2'-[(1-methylethylidene)bis(4,1-phenyleneoxymethylene)]bisoxirane

EC Number:

641-048-8

Cas Number:

110839-13-9

Molecular formula:

C37H48N4O4

IUPAC Name:

Reaction products of m-phenylenebis(methylamine) with 2,2'-[(1-methylethylidene)bis(4,1-phenyleneoxymethylene)]bisoxirane

Test animals

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: 274 - 426 g; Females: 202 - 274 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: 46 days;

ENVIRONMENTAL CONDITIONS
- Temperature: 21.1 - 24.9 °C
- Humidity: 33 - 69 % 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.

Administration / exposure

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). 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: 6.6 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 220 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 from 220 to 200 mg/kg bw/day and then to 150 mg/kg bw/day. 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 94 to 108 % 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 postpartal 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.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

Doses / concentrations 4

Dose / conc.:

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

No. of animals per sex per dose:

12 males/12 females

Control animals:

yes, concurrent vehicle

Details on study design:

Dose levels were reduced from 220 to 200 mg/kg bw/day (high dose) and then to 150 mg/kg bw/day 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.

Examinations

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 0.1 g. 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.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Other effects:

not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

not specified

Reproductive performance:

not specified

Details on results (P0)

Test item administered daily by oral gavage in Wistar rats, for 28 days in the male animals, and up to 45 days in the female animals led to adverse effects and mortality at 220 and 200 mg/kg bw/day. No toxicologically significant adverse effects of test item administration were observed at dose levels of 25, 80 or 150 mg/kg bw/day. Nasal discharge with noisy, difficult respiration and/or salivation were observed on occasion at 220/200/150 and 80 mg/kg bw/day. These clinical signs were considered to be a consequence of treatment with the test item. However, they were considered to reflect local effects due to oral administration and not systemic toxicity. Slightly lower body weights and significantly lower body weight gain values were observed in the high dose males as compared to the controls. No test item-related changes occurred in the high dose female body weights, or in the other groups at lower dose levels. Food consumption and organ weights measurements did not reveal any treatment-related changes. 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 (sperm positive vaginal smears and/or vaginal plugs) occurred within 5 days of pairing (cohabitation). One control (1/11 surviving sperm positive females) and one high dose female (1/9 surviving sperm positive females) were not pregnant (did not show evidence of implantation at necropsy examination). No test item effect was observed in the duration of pregnancy. All females littered in 21 to 23 days. There were no abnormalities in pups that could be ascribed to the treatment with the test item. 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 220/200 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 test item-treated dams as compared to the control value. The mean numbers of corpora lutea and implantation sites were higher in all the treated groups of females than in the control animals. Pre-implantation, intrauterine mortality, post-natal and total mortality values were lower in the treated animals than in the controls. Litter examination did not reveal any test item-related effects compared to observations noted in the control group. No stillbirths were observed; a few pups were found dead and/or cannibalized, or were runts (pups that were significantly smaller than normal pups). No external abnormalities were detected at the clinical or macroscopic examinations of the pups, as previously presented in the relevant sections. The number of male and female pups, and the sex ratio were similar in the control and treated groups. No significant variations were observed on PN4 compared to PN0. There were no changes that were ascribed to test item administration in the survival index values at PN0 or PN4. There were no adverse findings at macroscopic or microscopic examination at up to and including 150 mg/kg bw/day (animals previously treated with 220/200 mg/kg bw/day). Test item-related dilatation of the stomach and intestine observed at dose levels of 200 and 220 mg/kg bw/day was considered to be the cause of death. Five pups (F1 Generation) died between days 0-3 post partum. A specific cause of death in these pups was not determined.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

150 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

Target system / organ toxicity (P0)

Key result

Critical effects observed:

not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, treatment-related

Mortality / viability:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Sexual maturation:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

effects observed, treatment-related

Histopathological findings:

effects observed, treatment-related

Details on results (F1)

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.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

150 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Target system / organ toxicity (F1)

Key result

Critical effects observed:

not specified

Overall reproductive toxicity

Key result

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

In conclusion, under the conditions of this study, the no observed adverse effect level (NOAEL) for the test item for parental effects was 150 mg/kg bw/day. For reproduction and litter effects the NOAEL was 150 mg/kg/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 220 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 from 220 to 200 mg/kg bw/day,

and then to 150 mg/kg bw/day, as detailed in the 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 as well as 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 94 to 108 % 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 the control and high dose groups, and on gross lesions from low and mid dose groups.

Results and Conclusion

Test item administered daily by oral gavage in Wistar rats, for 28 days in the male animals, and up to 45 days in the female animals led to adverse effects and mortality at 220 and 200 mg/kg bw/day. No toxicologically significant adverse effects of test item administration were observed at dose levels of 25, 80 or 150 mg/kg bw/day.

Nasal discharge with noisy, difficult respiration and/or salivation were observed on occasion at 220/200/150 and 80 mg/kg bw/day. These clinical signs were considered to be a consequence of treatment with the test item. However, they were considered to reflect local effects due to oral administration and not systemic toxicity. Slightly lower body weights and significantly lower body weight gain values were observed in the high dose males as compared to the controls. No test item-related changes occurred in the high dose female body weights, or in the other groups at lower dose

levels. Food consumption and organ weights measurements did not reveal any treatment-related changes. 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 (sperm positive vaginal smears

and/or vaginal plugs) occurred within 5 days of pairing (cohabitation). One control (1/11 surviving sperm positive females) and one high dose female (1/9 surviving sperm positive females) were not pregnant (did not show evidence of implantation at necropsy examination). No test item effect was observed in the duration of pregnancy. All females littered in 21 to 23 days. There were no abnormalities in pups that could be ascribed to the treatment with the test item. All the parturitions were normal. There were no adverse effects, or biologically significant variations of the parameters related to pregnancy, parturition, or post-partal period noted in the treated groups at dose levels up to and including 150 mg/kg bw/day (previously treated with 220/200 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 test item-treated dams as compared to the control value. The mean numbers of corpora lutea and implantation sites were higher in all the treated groups of females than in the control animals. Pre-implantation, intrauterine mortality, post-natal and total mortality values were lower in the treated animals than in the controls. Litter examination did not reveal any test item-related effects compared to observations noted in the control group. No stillbirths were observed; a few pups were found dead and/or cannibalized, or were runts (pups that were significantly smaller than normal pups). No external abnormalities were detected at the clinical or macroscopic examinations of the pups, as previously presented in the relevant sections. The number of male and female pups, and the sex ratio were similar in the control and treated groups. No significant variations were observed on PN4 compared to PN0. There were no changes that were ascribed to test item administration in the survival index values at PN0 or PN4. There were no adverse findings at macroscopic or microscopic examination at up to and including 150 mg/kg bw/day (animals previously treated with 220/200 mg/kg bw/day). Test item-related dilatation of the stomach and intestine observed at dose levels of 200 and 220 mg/kg bw/day was considered to be the cause of death. Five pups (F1 Generation) died between days 0-3 post partum. A specific cause of death in these pups was not determined. In conclusion, under the conditions of this study, the no observed adverse effect level (NOAEL) for the test item for parental effects was 150 mg/kg bw/day. For reproduction and litter effects the no observed effect level (NOAEL) was 150 mg/kg bw/day.