Formaldehyde, polymer with benzenamine, hydrogenated

Developmental toxicity / teratogenicity

Administrative data

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

not specified

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

Details on test material:

Mixed polycycloaliphatic amines
Best to be used by Jan 31st 2018
Batch 1485489

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

Test animals

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

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

70 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

140 mg/kg bw/day (nominal)

Doses / concentrations 3

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).

Examinations

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

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

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

Maternal developmental toxicity

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

Effect levels (maternal animals)

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

Maternal abnormalities

Abnormalities:

no effects observed

Localisation:

cervix

uterus

Results (fetuses)

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

Effect levels (fetuses)

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

Fetal abnormalities

1

Abnormalities:

not specified

2

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.

Overall developmental toxicity

Developmental effects observed:

no

Applicant's summary and conclusion

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.