Formaldehyde, polymer with benzenamine, hydrogenated

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

In a repeat dose oral rat study (OECD 407), effects on reproductive organs were not observed at doses up to 300 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:

2012-2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study (GLP)

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

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:

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.

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).

Remarks:

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

Remarks:

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

Remarks:

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

No. of animals per sex per dose:

12

Control animals:

yes

Positive control:

no

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.

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: 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

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.

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

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

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).

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

Reproductive effects observed:

not specified

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.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

280 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Sufficient for assessing endpoint. In addition to the OECD 421 study, no effects were observed on the reproductive organs in an OECD 407 28-day repeat dose oral 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

An oral reproduction and developmenta toxicity screening test (OECD 421) of formaldehyde, polymer with benzenamine, hydrogenated, was conducted in rats. Due to the toxicity of the test substance a dose level higher than 280 mg/kg/day was not possible.

The ability of the male and female rats to achieve pregnancy and give birth to live pups was slightly influenced by test substance treatment but these slight intergroup differences were considered to be of no toxicological significance. The number 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 slightly at the middle and the highest dose level.  These slight changes were probably influenced by parental systemic toxicity rather than a specific effect on reproductive ability.

Administration of the test substance at a dose level of 280 mg/kg/day had no adverse effect on the ability of male and female animals to successfully mate and produce viable offspring. The NOAEL (No Observed Adverse Effect Level) for REPRODUCTION was established as higher than 280 mg/kg body weight/day.

Short description of key information:
An oral reproduction and developmental toxicity screening test (OECD 421) of formaldehyde, polymer with benzenamine, hydrogenated, was conducted in rats. Due to the toxicity of the test substance a dose level higher than 280 mg/kg/day was not possible. Slight effects on mating, fertility and survival of offspring were observed but these effects were not statistically or toxicologically significant. In addition, these slight changes were probably influenced by systemic parental toxicity.
Administration of the test substance at the dose levels 280 mg/kg/day had no adverse effect on the ability of male and female animals to successfully mate and produce viable offspring. The NOAEL (No Observed Adverse Effect Level) for REPRODUCTION was established as higher than 280 mg/kg body weight/day.

Justification for selection of Effect on fertility via oral route:
Guideline OECD 421 GLP study specifically designed to look for effects on fertility.

Effects on developmental toxicity

Description of key information

An oral reproduction and developmental toxicity screening test (OECD 421) of formaldehyde, polymer with benzenamine, hydrogenated, was conducted in rats. Due to the toxicity of the test substance a dose level higher than 280 mg/kg/day was not possible.  An effect on live births was not observed but slight effects on offspring survival were observed.   These effects on survival were not statistically or toxicologically significant.  In addition, these slight effects on offspring survival were probably influenced by systemic parental toxicity.  No significant or treatment-related difference in the development of the surviving pups was observed.
  
Administration of the test substance at the high dose level 280 mg/kg/day did not adversely affect the birth or development of offspring. The NOAEL (No Observed Adverse Effect Level) for developmental toxicity was established as higher than 280 mg/kg body weight/day.  

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: At this moment lead registrant did not receive the final report from the laboratory. Report will be available on beginning of June and endpoint summary will be updated immediately with the data from the report.

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

Copolymer of benzenamine and formaldehyde, hydrogenated, other names : Mixed Polycycloaliphatic Amines; MPCA ; Commercial name Ancamine 2168Batch number 1485489MANUFACTURE DATE 31.1. 2013EXPIRY DATE 31.1. 2018

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

SourceAnimal BreedingRCC Laboratories India Private LimitedGenome Valley, TurkapallyShameerpet (Mandal)Ranga Reddy DistrictHyderabad - 500 078IndiaRegistration Number1010/RO/bc/06/CPCSEABody weight when treatedFemale: 235.6 – 279.6 gWeight variation within groups did not exceeded ± 20% of mean body weightIdentificationBy unique cage number and individual animal numbers marked with an indelible marker pen on the tail. The animals were marked with permanent animal numbers towards the base of tail before the start of test item administration and refreshed weekly thereafter. The animals were marked with the temporary animal numbers on the tip of the tail at start of acclimatization. Foetuses were identified with tag.RandomizationAnimals were selected and grouped based on stratified randomization of body weights using Excel based computerized program.AcclimatizationUnder laboratory conditions for 12 days post veterinary examination. Only animals without any visible signs of illness were used for the study. Standard Laboratory ConditionsThe animal room was air-conditioned with adequate (above 10) air changes per hour (about 10 air changes). The experimental room was continuously monitored for temperature and relative humidity. The ranges for room tem¬pera¬ture and relative humidity were 20.2°C to 22.9°C and 50 to 64%, respectively. The animals were provided with a light cycle of 12 hours light and 12 hours dark.AccommodationDuring acclimatization and randomization period all animals were housed in groups of two in polycarbonate cages (approximate internal dimensions of 365 mm x 202 mm x 180 mm height) with corn cob bedding. After randomization, males and females were housed individually. During the mating phase, animals were housedon one male: one female basis within each dose group. After successful mating, the females were returned to their original cage and housed individually during gestation. Results of analyses for contaminants of corn cob will be archived at RCC Laboratories India Private Limited DietTeklad Certified Global 14% Protein Rodent Maintenance Diet (Lot Number: 2014C-012415MA) from Harlan Laboratories and Teklad Certified Global 14% Protein Rodent Maintenance Diet (Lot Number: 2014C-111215MA) from ENVIGO was provided ad libitum. Results of analyses for contaminants will be archived at RCC Laboratories India Private Limited.WaterAquaguard filtered tap water was provided ad libitum. Results of bacteriological, chemical and contaminant analyses will be archived at RCC Laboratories India Private Limited

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on exposure:

Animals were exposed to following test levels0 mg/kg b.w.70 mg/kg b.w.140 mg/kg b.w.280 mg/kg b.w.Test material was administered at the volume of 10 ml/kg b.w.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of the dose formulations from one set per dose group was taken immediately after preparation once before commencement of dosing (i.e. on day 5 of gestation on day 11 of gestation and on day 19 of gestation (days were considered from first dosing of the group) for homogeneity (mean of homogeneity given as dose concentration). Analyses were performed by Analytical Chemistry Department, RCC Laboratories India Private Limited according to an analytical method provided by the sponsor.

Details on mating procedure:

Animals were paired on a one male: one female basis within each dose group, for a maximum period of fourteen days. Each female was examined for vaginal smear or the presence of a copulation plug in the vagina. The presence of sperm in the vaginal smear and/or vaginal plug was taken as positive evidence of mating (Day 0 of gestation).

Duration of treatment / exposure:

Dosing from Day 5 to day 19 of the gestation

Frequency of treatment:

Once a day

Duration of test:

20 days

Dose / conc.:

70 mg/kg bw/day (nominal)

Dose / conc.:

140 mg/kg bw/day (nominal)

Dose / conc.:

280 mg/kg bw/day (nominal)

No. of animals per sex per dose:

24 females per group

Control animals:

yes, concurrent vehicle

Details on study design:

PRETESTPretest was performed using 3 females. The three females were treated with the highest dose of 280 mg/kg/body weight for seven consecutive days to confirm the doses.MATINGAnimals were paired on a one male: one female basis within each dose group, for a maximum period of fourteen days. Each female was examined for vaginal smear or the presence of a copulation plug in the vagina. The presence of sperm in the vaginal smear and/or vaginal plug was taken as positive evidence of mating (Day 0 of gestation).

Maternal examinations:

OBSERVATIONSClinical sign observations were recorded approximately at the same time(s) each day taking into consideration the peak period of anticipated effects post test item administration. The condition of the animals were recorded including mortality, pertinent behavioural changes, and all signs of overt toxicity.Mortality/ Viability: Twice dailyClinical Signs:Daily cage-side clinical observations-Acclimatization Period:Once daily- Treatment Period:Twice daily on initial 3 days after treatment; once daily thereafterBody Weights-Acclimatization Period:Once weekly- Premating:On first day of pairing and weekly thereafter - Gestation:On gestation day 0, 3, 5, 8, 11, 14, 17 and 20 Feed Consumption- Gestation:On gestation day 3, 5, 8, 11, 14, 17 and 20

Ovaries and uterine content:

On observation of vaginal smear, all female rats were confirmed to have mated and assumed to be pregnant. At necropsy 4 females each of control, low and high and 3 female rats of intermediate dose group were found to be non-pregnant. For consistency 20 pregnant females of each group were taken for evaluation/analysis. Gravid uteri including the cervix were weighed. The uteri of females were examined for the presence and number of implantation sites and the number of corpora lutea in the ovaries were determined for pregnant animals. The uterine content was examined for number of resorption, live and/or dead foetuses. The degree of resorption was described in order to estimate the relative time of death of the conceptus.

Fetal examinations:

EXTERNAL EXAMINATIONThe foetuses were removed, identified, weighed, sexed and evaluated for external malformation/variation. Foetuses were sacrificed by intraperitoneal injection of Thiopental sodium at approximately 200 mg/kg body weight. Each fetus was subject to external examination, which included all visible structures, surfaces and orifices (including the oral cavity). One-half of the fetuses (alternating foetuses within the litter) independent of sex were processed for skeletal alterations and remaining half of each litter were examined for visceral (soft tissue) alterations.VISCERAL (SOFT TISSUE) EXAMINATIONAll the foetuses selected for visceral examination were observed by Staple’s dissection technique for visceral malformations/alterations and discarded. All the organs and structures of the head, neck, thorax and abdomen were observed.SKELETAL EXAMINATIONAll the foetuses selected for skeletal examination were eviscerated, processed, stained with Alizarin red S (single staining) and examined for skeletal malformations/alterations by using stereomicroscope. All the bone structures of the head, spine, rib cage, pelvis and limbs were observed.

Statistics:

The following statistical methods were used to analyze the body weight, body weight change, feed consumption, reproduction and external, visceral and skeletal alterations/variations:Data was summarized in tabular form. Statistical analysis was performed using Statplus program. All the data was checked for Normality with Shapiro-Wilk W test and for Homogeneity with Bartlett Chi-Square test. Each group of animals was subjected to Analysis of Variance (ANOVA) among the groups, and Bonferroni Test for unequal replications. For discontinuous data, nonparametric test (Mann-Whitney U-Test) was used. Values are given as mean ± standard deviation (SD). P ≤ 0.05 (5% level of significance) was considered to represent significance in the respective parameters, P > 0.05 was considered not Significant.

Indices:

INDICES CALCULATIONSThe following formulae were used for calculating the indices: Pre–implantation loss (%)(Number of Corpora Lutea - number of implantation sites)/ Number of Corpora Lutea x100Post–implantation loss (%)(Number of implantation sites - Total number of live foetuses)/ Number of implantation sites x 100Sex Ratio (% males) Number of male foetuses (Day 0) / Total number of foetuses (Day 0) x 100Variation Incidence (%)Number of foetuses with variation/ Total Number of foetuses examined x100

Historical control data:

Will be provided when final report is available

Clinical signs:

no effects observed

Description (incidence and severity):

All the treated animals appeared normal and no systemic or local signs of toxicity were observed from day 0 and continued to be normal till the last day of observation, on day 20th of gestation. No clinical signs were observed in any of the female rats from control (0 mg/kg bw), low (70 mg/kg bw), intermediate (140 mg/kg bw) and high dose (280 mg/kg bw) groups.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No mortality was observed in any of the treated animals. All the female rats survived through out the scheduled treatment period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No significant difference in the body weight and body weight gain (%) was observed in any of the pregnant female rats of low, intermediate and high dose groups when compared with control group animal.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Feed consumption of pregnant female animals of low, intermediate and high dose groups was not significantly different when compared with that of control group except between day 3 - 5 of gestation revealed significantly decreased feed consumption in low and high dose as compare to control group and between day 8 - 11 and 14 - 17 of gestation revealed significantly decreased feed consumption in low dose as compare to control group. These cannot be changes attributed to toxicity to the test item exposure.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

No abnormality was observed in any of the pregnant female animals from control, low, intermediate and high dose groups.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Number of abortions:

no effects observed

Description (incidence and severity):

The number of corpora lutea on the ovaries and implantation sites in uterus, and therefore the extent of pre-implantation loss and post-implantation loss in all treated groups were comparable with control group. There was no significant difference observed in early and late resorption and number of live foetuses in all tretment groups when compared with control group.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

The number of corpora lutea on the ovaries and implantation sites in uterus, and therefore the extent of pre-implantation loss and post-implantation loss in all treated groups were comparable with control group. There was no significant difference observed in early and late resorption and number of live foetuses in all tretment groups when compared with control

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

ParameterGroupG1G2G3G4Dose (mg/kg bw)070140280No. of Dams20202020Gravid Uterine WeightMean55.0849.7053.9355.43SD17.5311.6615.6820.68Total Foetuses Number193189216211Mean4.033.443.583.51SD0.130.120.100.12No. of CLMean14.3813.6713.8315.13SD2.342.782.823.31No. of Implantation Mean9.138.889.759.38SD5.744.835.146.08Early Resorption Mean0.420.420.290.58SD0.650.780.460.83Late Resorption Mean0.040.080.000.00SD0.200.280.000.00Pre implantation loss mean5.254.714.085.75SD5.104.595.017.16%38.335.829.635.7Post implantation lossMean1.041.000.750.6SD1.201.530.901.6%10.710.67.810.3Dam with resorption/sNumber12111212

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not examined

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

GroupG1G2G3G4Dose (mg/kg bw)070140280FEMALE ANIMALS MATED24242424PREGNANT FEMALE ANIMALS 20202120

Other effects:

no effects observed

Dose descriptor:

NOAEL

Effect level:

> 280 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

changes in number of pregnant

changes in pregnancy duration

clinical signs

dead fetuses

early or late resorptions

effects on pregnancy duration

food consumption and compound intake

gross pathology

maternal abnormalities

mortality

number of abortions

organ weights and organ / body weight ratios

pre and post implantation loss

total litter losses by resorption

Abnormalities:

no effects observed

Localisation:

cervix

uterus

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Group →G1G2G3G4Dose (mg/kg bw) 070140280No. of Dams →20202020Total No. of Foetuses 193189216211Mean Litter Size 8.047.889.399.17Live FoetusesNumber193189216211Mean8.047.889.008.79SD5.254.284.905.48
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): GroupFoetus Weight(G)Group 1Mean4.03SD0.13N193Group 2Mean3.44SD0.12N189Group 3Mean3.58SD0.10N216Group 4Mean3.51SD0.12N211

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

Group →G1G2G3G4Dose (mg/kg bw) 070140280No. of Dams →20202020Total No. of Foetuses 193189216211Mean Litter Size 8.047.889.399.17Live FoetusesNumber193189216211Mean8.047.889.008.79SD5.254.284.905.48

Changes in sex ratio:

no effects observed

Description (incidence and severity):

Group/DoseMale%Female%Group 139.9660.04Group 247.0352.97Group 354.0945.91Group 451.5948.41

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

Six small sized foetuses were observed in control, four in low, three in intermediate and one in high dose group.

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

External examination of foetuses revealed dome shaped head in one foetus each of control and low dose group. Short snout was observed in one foetus of low dose group. In intermediate dose group reddish discolouration of body was observed in one foetus. While pale body was observed in one foetus in high dose group.No significant difference was observed in external findings in all treatment groups when compared with control group.

Skeletal malformations:

no effects observed

Description (incidence and severity):

Skeletal examination of high dose group foetuses revealed incompletely ossified interperital, sacral, sternal centers (1 and 2), manubrium and/or Xyphiod; not ossified sternal centers (1 and 2), manubrium and/or Xyphiod; small sized pubis, scapula, humerus, radius, ulna, ilium, ischium, femur, tibia and/or fibula; Dumbbell shaped thoracic and lumbar vertebrae and thin ribs and clavicles. Intermediate dose group revealed incompletely ossified nasal (left and right), premaxilla, frontal, parietal, maxilla, mandible, zygomatic, sphenoid ala, hyoid, sphenoid body, pterygoid process, cervical, thoracic, lumbar vertebrae, sternal centers (1 and 2 manubrium, Xyphiod, clavicle, scapula, humerus, radius, ulna, ilium, femur, tibia and/or fibula; not ossified bascioccipita, interperital, supraoccipital, hyoid, sternal centers (1), manubrium, Xyphiod, ischium and/or pubis; Dumbbell shaped thoracic and lumbar vertebrae; hemivertebrae in lumbar; waviness of ribs; short tail and small sized eye socket, brain case, nasal, premaxilla, frontal, parietal, maxilla, mandible, tympanic ring, lumbar, clavicle, scapula, forelimb, humerus, radius, ulna, ilium, hind limb, femur, tibia and/or fibula.Low dose group revealed incompletely ossified interperital, sternal centers (1 and 2), manubrium, Xyphiod and/or pubis; not ossified sternal centers (1), manubrium, Xyphiod and/or pubis; Dumbbell shaped thoracic and/or sternal centers (1) and small sized scapula.Control group revealed incompletely ossified sternal centers (1 and 2), manubrium, Xyphiod; not ossified sternal centers (1) and/or Xyphiod; Dumbbell shaped thoracic vertebrae; fused sternal centers (1) and manubrium and short ischium.These variations observed in high, intermediate and low dose groups were isolated and when expressed on a fetus/litter basis, it was found to be not statistically significant when compared with incidence in control group. Type and distribution of variations noted during skeletal examination at the dose levels of 70, 140 and 280 mg/kg body weight did not indicate any test item-related effects.

Visceral malformations:

no effects observed

Description (incidence and severity):

Visceral examination of foetuses revealed convoluted ureter in four foetuses of control group and three foetuses each in intermediate and high dose groups. Small sized spleen was observed in one foetus each of control, intermediate and high dose group and two foetuses in low dose group. Dilated ventricles of brain was observed in two foetuses in control and one foetus each of low and high dose groups. Pale spleen was observed in 1 foetus each of control, low and intermediate and 2 foetuses of high dose group.No significant difference was observed in visceral findings in all treatment groups when compared with control group.

Other effects:

not examined

Dose descriptor:

NOAEL

Effect level:

> 280 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

reduction in number of live offspring

changes in sex ratio

fetal/pup body weight changes

changes in litter size and weights

external malformations

skeletal malformations

visceral malformations

Abnormalities:

not specified

Abnormalities:

no effects observed

Description (incidence and severity):

No treatment-related effects were observed in reproduction parameters such as litter weight, foetus weight, corpora lutea count, early and late resorption and number of live and dead foetuses, pre-implantation loss, post-implantation loss as well as sex ratio at any dose level in the female animals treated with 70, 140 and 280 mg/kg body weight/day. The external and visceral variations observed in foetus were randomly distributed across the groups. Therefore these findings were considered as incidental findings and not test item-related.

Developmental effects observed:

no

Conclusions:

Based on the OECD 414 study findings it is concluded that the test item is non teratogenic in rats and the NOAEL for teratogenicity of the test item can be fixed as 280 mg/kg bw/day, under the present experimental conditions. The NOAEL for maternal toxicity was determined to be 280 mg/kg bw/day.

Executive summary:

The test item, copolymer of benzenamine and formaldehyde, hydrogenated refined groundnut oil (Arachis oil) was administered once daily by oral gavage to three treatment groups of twenty four pregnant female Wistar rats each from day 5 to day 19 of gestation at dose levels of 70, 140 and 280 mg/kg body weight/day. A control group of twenty four females were administered with vehicle (refined groundnut oil) alone.The treatment with test item resulted in no mortalities. All the dams survived till the scheduled sacrifice.No test item related clinical signs were observed in any of the pregnant female animals of low, intermediate and high dose groups as well as in control group. The body weight, body weight gain (%) and feed consumption during gestation period in all treatment groups were comparable with control group.No treatment-related effects were observed in reproduction parameters such as litter weight, foetus weight, corpora lutea count, early and late resorption and number of live and dead foetuses, pre-implantation loss, post-implantation loss as well as sex ratio at any dose level in the female animals treated with 70, 140 and 280 mg/kg body weight/day. The external and visceral variations observed in foetus were randomly distributed across the groups. Therefore these findings were considered as incidental findings and not test item-related.Type and distribution of variations noted during skeletal examination at the dose levels of 70, 140 and 280 mg/kg body weight/day did not indicate any test item-related effects. Based on the above findings, it is considered that the test item is non teratogenic in rats and the NOAEL for teratogenicity of the test item can be fixed as 280 mg/kg bw/day, under the present experimental conditions. The NOAEL for maternal toxicity was considered as 280 mg/kg bw/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

280 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Provides preliminary information on pre-natal and post-natal survival and development. Inadequate to fully assess the teratology endpoint

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

An oral reproduction and developmental toxicity screening test (OECD 421) of formaldehyde, polymer with benzenamine, hydrogenated, was conducted in rats. Due to the toxicity of the test substance a dose level higher than 280 mg/kg/day was not possible.

In this screening test, 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 lower in comparison with the control group.

At the highest dose an increasing trend in mortality from birth to Day 4 of lactation may be attributable to a negative influence of the test substance that could be caused by maternal toxicity effects of the substance.

No significant or treatment-related difference in the development of the surviving pups was observed.

No pathological findings were recorded in pups at the lowest and the middle dose levels. One dead 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 hemorrhage).

The NOAEL (No Observed Adverse Effect Level) for developmental effects based on this screening test was established as higher than 280 mg/kg body weight/day.

Justification for selection of Effect on developmental toxicity: via oral route:
Guideline GLP study specifically designed to screen for developmental toxicity effects.

Toxicity to reproduction: other studies

Additional information

In two repeat dose oral rat studies, effects on reproductive organs were not observed at doses up to 300 mg/kg bw/day. At a dose that was toxic to the parental animals (280 mg/kg bw/day) their ability to achieve pregnancy, give birth to live pups, and the survival of the offspring was only slightly influenced by the test substance treatment. These slight treatment-related differences were

considered to be of no statistical or toxicological significance. In addition, these slight changes were probably influenced by systemic parental toxicity rather than a specific effect on reproductive ability. No significant or treatment-related difference in the development of the surviving pups was observed.

The NOAEL (No Observed Adverse Effect Level) for reproductive and developmental effects was established as higher than 280 mg/kg body weight/day.

Justification for classification or non-classification

Based on the findings of OECD 414 teratology studies, it is considered that the test item is non teratogenic in rats and the NOAEL for teratogenicity of the test item can be fixed as 280 mg/kg bw/day, under the present experimental conditions. The NOAEL for maternal toxicity was considered as 280 mg/kg bw/day.

According to the UN Globally Harmonized System of Classification and Labelling of Chemicals (GHS) Part 3 Chapter 3.7 this substance does not require classification for reproductive toxicity.