Formaldehyde, polymer with benzenamine, hydrogenated

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study (GLP)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2,4-bis[(4-aminocyclohexyl)methyl]aniline; 2,4-bis[(4-aminocyclohexyl)methyl]cyclohexan-1-amine; 2-[(1-aminocyclohexyl)methyl]aniline; 4-[(4-aminocyclohexyl)methyl]cyclohexan-1-amine; 4-{[4-({4-[(4-aminocyclohexyl)methyl]cyclohexyl}amino)cyclohexyl]methyl}cyclohexan-1-amine

EC Number:

603-894-6

Cas Number:

135108-88-2

Molecular formula:

Exact identification is not feasible

IUPAC Name:

2,4-bis[(4-aminocyclohexyl)methyl]aniline; 2,4-bis[(4-aminocyclohexyl)methyl]cyclohexan-1-amine; 2-[(1-aminocyclohexyl)methyl]aniline; 4-[(4-aminocyclohexyl)methyl]cyclohexan-1-amine; 4-{[4-({4-[(4-aminocyclohexyl)methyl]cyclohexyl}amino)cyclohexyl]methyl}cyclohexan-1-amine

Test material form:

liquid: viscous

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

All the study proceeded in individually ventilated cages (IVC) in special animal house of CETA, 2 rats of the same sex in one polysulfone cage (floor area – 900 cm2) containing sterilised clean shavings of soft wood. During mating period – one male and one female in one cage, pregnant females – individually, offspring – with mother.
Animals were housed in controlled environment, in which optimal conditions were considered to be approximately 15 air changes per hour, a temperature of 22+3°C, a relative humidity of 30-70% and 12-hour light/12 hour dark cycle.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: dimethylsulphoxide

Details on exposure:

The application form was administered to the stomach by gavage. The treated groups were administered daily for the period: males and females – 2 weeks prior to the mating period and 2 weeks during the mating period. Pregnant females were administered during pregnancy and till 4th day of lactation. Males were administered till 28th day of study, females showing no-evidence of copulation till 54thday of study and non-pregnant females till the 25th day after confirmed mating. The animals were treated 7 days per week at the same time (8.00 – 10.00 am).

Details on mating procedure:

Animals were mated from the 15th day of study. Mating 1 : 1 (one male to one female) was used in this study. Each morning the females were examined for presence of spermatozoa in vaginal smears. Day 0 of pregnancy was defined as the day the sperms were found.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analyses were performed by high-performance liquid chromatography based on a method developed at the test facility.
The determination of homogeneity and stability of application form was performed on two concentration levels: 10 mg/mL and 300 mg/mL. The application form was prepared by dissolving the test substance in dimethylsulfoxide.
This phase of study was delegated to principal analytical chemist: Michaela Nováková, Ph.D., CETA, GLP Test Facility, Analytical Group I. Details of analytical work was specified in study phase plan which was attached to study plan as Annex 1.

Homogeneity of the application form was checked by determination of peak area sum of the test substance in three places of solution (at the bottom, in the middle and at the surface).
Stability of the application form was checked by analyses of the application form within 120 min (at the time 0, 30, 60, 90 and 120 min). Time interval 0 min represents for both concentration the time after 30 minutes of mixing by magnetic stirrer (850 rpm).
The results of analytical work were a subject of an Annex 1 to final report of the study.

Duration of treatment / exposure:

Males were administered till 28th day of study, females showing no-evidence of copulation till 54thday of study and non-pregnant females till the 25th day after confirmed mating.

Frequency of treatment:

The animals were treated 7 days per week at the same time (8.00 – 10.00 am).

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
70 mg/kg bw
Basis:
actual ingested

Doses / concentrations 2

Remarks:

Doses / Concentrations:
140 mg/kg bw
Basis:
actual ingested

Doses / concentrations 3

Remarks:

Doses / Concentrations:
280 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

12

Control animals:

yes

Positive control:

no

Examinations

Parental animals: Observations and examinations:

Methods of Investigation
- Body Weight monitoring
- Food Consumption monitoring
- Mortality Control
- Health conditions control
- Clinical Observations of Males and Females
- Clinical Observations of Pups
- Observation of Sperm
- Pathological observations
- Biometry of Reproductive Organs
- Histological Technique

Sperm parameters (parental animals):

In all males of all groups surviving to scheduled necropsy the sperm parameters were examined: sperm motility and sperm morphology. Sperm specimens were prepared and examined according to internal SOP No. M/45.

Sperm motility
Sperm samples were taken from one epididymis and sperm motility was assessed from these samples. The motility of sperms was determined by microscopic examination of the prepared sperm suspension. The result of observation was evaluated subjectively according to following grades: 1 – fast progressive motility, 2 - slow progressive motility, 3 – no progressive motility, 4 – non-motile sperm.

Sperm morphology
Sperm samples were taken from one epididymis and sperm morphology was assessed from these samples. A smear from the sperm suspension was prepared and stained (Giemsa staining). The morphology of sperm was determined by microscopic examination.
All deviations – e.g. broken tail, abnormal form of tail, double head, amorphous head, no head, abnormal form of neck ¬– were recorded.

Postmortem examinations (parental animals):

- Pathological Examination
Parental males were killed at the end of the administration period – after 28 days of administration. Parental females were killed on the 4th day of lactation. Mated females without delivery were killed 26th day after confirmed mating.
Then they were macroscopically examined for any pathological changes with special attention to the organs of the reproductive systems. All macroscopic abnormalities were recorded.
Dead pups were sexed and externally examined; the stomach was examined for the presence of milk. Pups killed on the 4th day of lactation were sexed and subjected to external examination of the cranium, and to macroscopic examination of the thoracic and abdominal tissues and organs. All macroscopic changes were recorded.
- Biometry of Reproductive Organs
The absolute weights of testes, epididymis, prostate gland and pituitary gland were recorded in males and absolute weight of ovaries, uterus (incl. uterine tube and cervix) and pituitary gland were recorded in females. Afterwards the somatic indexes - SI (= relative weight of organ) were computed according to the following formula: SI = weight of organ x 100/ body weight.
- Histological Technique
The following tissues and organs were collected from all killed males and females at necropsy and fixed in neutral 4% formaldehyde suspension (v/v) for further histopathological evaluation: relevant gross lesions, pituitary gland, coagulation gland, prostate gland, seminal vesicles, epididymis and testes (fixed in Davidson´s suspension), cervix of uterus, ovaries, uterus and vagina.
For histopathological processing the routine histopathological paraffin technique with haematoxylin-eosin staining was used.
The full histopathology of the preserved organs and tissues was performed for all high dose and control animals. Organs demonstrating treatment-related changes: thymus, brain, cerebellum, kidneys, spleen, heart, thyroid gland, thoracic spinal cord in males and females and trachea (larynx) in females and organs with macroscopical changes were examined at the lowest and middle dose level groups.
Detailed histological examination was performed on testes of males from Reproduction toxicity part of study (with special emphasis on stages of spermatogenesis and histopathology of interstitial testicular cell structure). Spermatogenesis and spermatogenic cycle were evaluated according to the following publications:
1. Hess, R.A.; Quantitative and qualitative Characteristics of the Stages and Transitions in the Cycle of the Rat Seminiferous Epithelium: Light Microscopic Observation of Perfusion-Fixed and Plastic-Embedded Testes (Biology of Reproduction 43, 525-542, 1990);
2. Creasy, D.M.; Evaluation of Testicular Toxicity in Safety Evaluation Studies: The Appropriate Use of Spermatogenic Staging (Toxicologic Pathology 25, 119-131, 1997)
3. Guidance Document for Histologic Evaluation of Endocrine and Reproductive Tests in Rodents, ENV/JM/MONO(2009)11.

Postmortem examinations (offspring):

Dead pups were sexed and externally examined; the stomach was examined for the presence of milk. Pups killed on the 4th day of lactation were sexed and subjected to external examination of the cranium, and to macroscopic examination of the thoracic and abdominal tissues and organs. All macroscopic changes were recorded.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

piloerection and hunched posture, were observed in treated males of the highest dose level during the 4th week of application.

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:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Slightly increased presence of sperms with changed motility .

Reproductive performance:

no effects observed

Details on results (P0)

Sporadic histopathological changes of the male reproductive system were detected at the control group and at the treated groups.
Slight focal chronic inflammation was described in the prostate gland of 1-3-3-0 males (dose levels 0-70-140-280 mg/kg/day/day). In the epididymis of one control male, unilateral spermatogranuloma was observed.

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

body weight and weight gain

food consumption and compound intake

organ weights and organ / body weight ratios

gross pathology

reproductive function (sperm measures)

reproductive performance

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

no effects observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Details on results (F1)

Body Weight
The mean body weight of the pups at the highest dose level was slightly lower compared to control. At the lowest and middle dose levels the mean body weights of pups was well-balanced with the control group. No statistically significant differences were detected.
Development of Pups
At the first check of litters, dead pups were found in control group and at the middle and the highest dose levels.
Control: One male pup from litter of female No.101 and one male pup from the litter of female No.104 were found dead at first check of litter. Pups died after delivery (aerial lung).
Cannibalism of one male pup from litter of female No. 105 was reported at the check of litter on 4th day of lactation.
140 mg/kg/day: Two female pups from litter of female No.152 were found dead at first check of litter. Pups died after delivery (aerial lung).
280 mg/kg/day: One female pup from litter of female No.162 was found dead at first check of litter. This pup was delivered dead (anaerial lung).
In female No. 165, four dead pups (anaerial lung) were reported at the first check of litter; and on the second day, cannibalism of one female pup was recorded.
In female No.166, two dead pups (1male and 1 female with anaerial lung) were found at the first check of litter. On the second day, cannibalism of two pups (1male and 1 female) was reported.
Cannibalism of one male pup on the second day of lactation was also reported in female No. 170.
One male pup was found dead at the check of litter on 4th day of lactation in female No.172.
No serious differences in development of pups were observed at the control and the treated groups.
Pathological Examination
The macroscopic examination was performed in all pups. No pathological findings were recorded in pups at the lowest and the middle dose levels. One death pup from the control group showed empty stomach – without milk. One pup from the highest dose level showed focal changes on kidneys (histopathological examination: marked focal interstitial hemmorhage).

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

> 280 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

mortality

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Due to the toxicity of the formaldehyde, polymer with benzenamine, hydrogenated, a dose level higher than 280 mg/kg/day was not possible. Administration of the test substance at the dose levels 280 mg/kg/day had no adverse effect to ability of male and female animals to successfully mate and produce viable offspring. Also development of pups was not significantly changed in treatment groups. The NOAEL (No Observed Adverse Effect Level) for the REPRODUCTION was established as higher than 280 mg/kg body weight/day.

Executive summary:

Formaldehyde, polymer with benzenamine, hydrogenated was orally administered in graduated doses (70, 140 and 280 mg/kg/day) to three groups of males and females. Before mating the males and females were dosed for two weeks. All animals were treated during the whole time of mating period. Application of females continued during pregnancy and lactation period. Males were administered for 28 days (in total). 

 

The application of the test substance caused the death of two females at the highest dose level (280 mg/kg/day). The last week of application slight symptoms of toxicity were observed in males and females at the highest dose level.

The test substance slightly affected growth of parental animals of the highest dose level (280 mg/kg/day). Mean body weight of males was lower after the first week of application. The decreased food consumption in males of the highest dose level was also recorded. Body weight of females was lower during the whole study. The decreased food consumption in females of the highest dose level was recorded only during the lactation period.

The biometry of organs, pathological and histopathological examination revealed no significant changes of reproductive organs in males and females at the control and treated groups.

 

Observation of sperm motility and sperm morphology detected slightly impaired quality of sperms in treated males versus control but without toxicological significance.

 

Observation of pups– the statistical evaluation of the number of live pups at birth did not detect significant differences between control and treated groups. But total number of live pups in mothers at the middle and highest dose levels was decreased in comparison with the control group. On the contrary at the lowest dose level the total number of pups per litter and mean weight of litter was higher than in control group.

 

Mortality occurred in pups in control and groups treated by the test substance at the middle and highest dose levels. The increasing trend at the highest dose level may be attributable to maternal toxicity effects of the substance.

 

No mortality of pups until 4^thday was observed at lowest dose and sporadic mortality was recorded at the middle dose level and at the control group. At the highest dose the increasing trend in mortality may be attributable to a negative influence of the test substance that could be caused by maternal toxicity effects of the substance.

Reproduction parameters– ability of males and females to achieve pregnancy and give birth to live pups was slightly influenced by the test substance treatment but these slight intergroup differences were considered to be of no toxicological significance.

Numbers of females achieving pregnancy was lower at the middle and highest dose levels. Duration of mating and duration of pregnancy were not changed. Mating and fertility indexes were decreased at the middle and the highest dose level. The survival index was decreased at the highest dose level. These changes were probably influenced by maternal toxicity.