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Toxicological information

Developmental toxicity / teratogenicity

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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 we not received the final report from the laboratory. Report will be available in October 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:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
not specified
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
4,4'-Methylenedicyclohexanamine.
Manufactured by Monsanto; no information on purity.

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
Species Hannover Wistar Rat
Supplier Charles River Laboratories, Germany
Breeder Charles River Laboratories, Germany
Sandhofer Weg 7
97633 Sulzfeld
Germany
Number 88 females (the females were nulliparous and non-pregnant)
30 males
Total number of animals ordered 100 females and 30 males (spare animals was used as needed and their exchange and/or sacrifice was documented in the raw data).
Total number of females per group 22
Age (at delivery)
Males: Approximately 10-11 weeks
Females: Approximately 10-11 weeks
Female body-weight range (day 0 post coitum) 191-239 g

Conditions Standard laboratory conditions: air-conditioned with a minimum of 15 20 air changes per hour; the environment monitored continuously with target ranges for temperature of 22 ± 2 °C and for relative humidity between 40 and 70%, 1 2 hours artificial fluorescent light (7:00 am to 7:00 pm)/ 12 hours dark (7:00 pm to 7:00 am). Values outside these ranges occasionally occurred. These transient variations were considered not to have any influence on the study.
Accommodation Cages with Lignocel S8-15 sawdust bedding (J. Rettenmaier & Söhne).
Females:
Acclimatization period (maximum 5 animals/cage) Makrolon cages -IV
Pregnancy (individually) Makrolon cages -III
Males:
Acclimatization and pairing (individually) Makrolon cages –III
Diet Pelleted standard Teklad 2014C rat/mouse maintenance diet ad libitum (supplied by Envigo RMS, S.L., batches no.: 120415MA and 121415MA, expiry dates: 30 August and 09 September 2016, respectively).
The diet was analyzed by the manufacturer for contaminants and to check its composition. The results of analyses will be archived at Envigo CRS, S.A.U.
Water Tap water in bottles ad libitum.


Administration / exposure

Route of administration:
oral: gavage
Vehicle:
propylene glycol
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of dose formulation was determined twice during the study and once (top/middle/bottom) for homogeneity in samples taken from the formulation administered to Groups 2 to 4 by GC. The formulations were quantified following the analytical procedure validated in the present study and using the computerized system EZ Chrom.
The test item was used as analytical standard.
The analytical procedure was successfully validated with respect to specificity of chromatographic analysis, linearity of detector response, method accuracy and precision.
The homogeneity and stability was confirmed for the test item in vehicle formulations at nominal concentrations of 1, 3 and 10 mg/mL when stored refrigerated for 5 days.
The mean concentrations of test item in test formulations analyzed for the study were within 100 ± 10 % of nominal concentrations, confirming accurate formulation.
Details on mating procedure:
After acclimatization, females were housed with sexually mature males in a cage for a period approximately of 16 hours (maximum 2:1). Vaginal smears were taken daily until mating is confirmed. The females were removed and housed individually when:
The vaginal smear was sperm positive, or A copulation plug was observed.
The day of mating was designated day 0 post coitum.
Male rats of the same source and strain were used for mating only. Clinical signs and body weight were recorded weekly but do not report. Data are included in the raw data.
Duration of treatment / exposure:
The females received the test item from days 6 to 19 post coitum.
Frequency of treatment:
Once daily, Administration volume 5 mL/kg
Duration of test:
20 days, The females received the test item from days 6 to 19 post coitum.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control
Dose / conc.:
5 mg/kg bw/day (nominal)
Dose / conc.:
15 mg/kg bw/day (nominal)
Dose / conc.:
50 mg/kg bw/day
No. of animals per sex per dose:
Group 1 2 3 4
Dose Control 5 mg/kg/day 15 mg/kg/day 50 mg/kg/day
Total no. of females 22 22 22 22

Total number of males : 30
Control animals:
yes, concurrent vehicle
Details on study design:
Dose rationale

ECHA’s summary of the PACM OECD 422 study:

Combined repeated dose toxicity study with the reproduction/developmental toxicity screening test (OECD 422; 2010) conducted in rats via the oral route with the registered substance 4,4’-methylenebis(cyclohexanamine), CAS no. 1761-71-3). The following doses were used: 0, 15, 50, 100 mg/kg/day. The following effects were observed (i) Systemic toxicity: at 50 mg/kg/day ‘Treatment related and toxicologically relevant microscopic findings were noted for various organs, including the stomach (vaculolation of the stomach musculature; both sexes), liver (centrilobular vacuolation; both sexes), skeletal muscle (vacuolar myofiber degeneration and myofiber degeneration; both sexes), and the eyes (vacuolation of the cuboidal epithelium of the iris; both sexes).’; and at 100 mg/kg/day a reduced body weight gain, and microscopic findings in the ‘brain (vacuolation of the choroid plexus; both sexes)’ were noted in addition to the findings listed above. (ii) Reproductive/developmental toxicity: ‘Reproduction toxicity was observed at 50 and 100 mg/kg characterized by a reduction in implantation site number. Developmental toxicity included a lower gestation and viability indices and increased postnatal loss for females at 50 mg/kg, and a reduced number of living pups at first litter check at 100 mg/kg.’;


Dose Rationale

In addition to the findings noted by ECHA, the Combined repeated dose toxicity study with the reproduction/developmental toxicity screening test (OECD 422; 2010) conducted in rats via the oral route with the registered substance 4,4’-methylenebis(cyclohexanamine), CAS no. 1761-71-3) (see also IUCLID section 7.5.1 and 7.8.1),resulted in significant clinical signs and mortality. At 150 mg/kg bw/d, a pronounced toxicity was observed in females including clinical signs like hunched posture, rales and piloerection as well as a marked body weight loss. Additionally, on study day 5 one female was euthanized in extremis. Therefore, the dose level was lowered to 100 mg/kg on day 12 of treatment. However, one further female was euthanized after 21 days of treatment at 100 mg/kg. After lowering the dose to 100 mg/kg bw, toxicologically-relevant clinical signs including hunched posture, yellow feces, piloerection, lean appearance, ptosis, lethargy, chromodacryorrhoea, and rales were noted. Lower body weights and body weight gains were observed throughout the treatment period (both sexes) along with reduced food consumption over much of the treatment period (both sexes). Clinical biochemistry parameters were affected including increased aspartate aminotransferase (ASAT) and reduced creatinine levels (both sexes) which are related to histopathological changes seen in skeletal muscle and liver (see below), along with higher kidney to body weight (males) and liver to body weight ratios (both sexes). Treatment related microscopic findings of various organs at 100 mg/kg were noted, including: the stomach (vaculolation of the stomach musculature; both sexes), liver (centrilobular vacuolation; both sexes), brain (vacuolation of the choroid plexus; both sexes), skeletal muscle (vacuolar myofiber degeneration and myofiber degeneration; both sexes), and the eyes (vacuolation of the cuboidal epithelium of the iris; both sexes). At 50 mg/kg bw, treatment related and toxicologically relevant microscopic findings were noted for various organs, including the stomach (vaculolation of the stomach musculature; both sexes), liver (centrilobular vacuolation; both sexes), skeletal muscle (vacuolar myofiber degeneration and myofiber degeneration; both sexes), and the eyes (vacuolation of the cuboidal epithelium of the iris; both sexes). Slight reductions in body weights and food consumption were noted on a few occasions, but at the degree observed were not considered to be biologically significant.
According to the OECD 414 guideline, the highest dose should be chosen with the aim to induce some developmental and/or maternal toxicity (clinical signs or a decrease in body weight) but not death or severe suffering. Since one female had to be euthanized in extremis in the 100 mg/kg bw group and significant toxicological effects were observed in the 50 mg/kg bw group, this half-lethal dosage was chosen as high dose group for the present prenatal developmental toxicity study in Wistar rats. The following doses were set according to the standard interval of 3.
5 mg/kg body weight/day as low-dose level
15 mg/kg body weight/day as mid-dose level
50 mg/kg body weight/day as high-dose level

The oral route was selected since this has proven to be suitable for the detection of a toxicological hazard and this was the route used in the previous OECD 422 study.

Examinations

Maternal examinations:
All females sacrificed during the study were subjected to macroscopic examination. Postmortem examination, including gross macroscopic examination of all internal organs with emphasis on the uterus, uterine contents, position of fetuses in the uterus and the number of corpora lutea was performed and the data recorded. The uteri (and contents) of all females was weighed during necropsy on day 20 post coitum to enable the calculation of the corrected body-weight gain.
Fetal examinations:
Fetuses and placenta were removed from the uterus and weighed individually. Fetuses were sexed, examined for gross external abnormalities (Makris, 2009), sacrificed by intraperitoneal injection of sodium pentobarbital and allocated to one of the following procedures:
1. One-half of the fetuses from each litter were fixed in Bouin's fixative (Wilson, 1965). They were serially sectioned and examined (evaluation of the internal structures of the heads, thoracic and abdominal organs). The tissue was preserved in a solution of 96% ethyl alcohol (one fetus per container). Descriptions of any abnormalities and variations were recorded.
2. The remaining fetuses were eviscerated and fixed in 70% alcohol. Then they were placed in a solution of potassium hydroxide with Alizarin red S (for clearing and staining ossified bone) and a solution of glycerin/alcohol for preservation and storage (Dawson, 1926). The skeletons were examined and all abnormal findings and variations were recorded. The specimens were individually preserved.
When considered appropriate, macroscopic changes in the fetuses were photographed and if necessary samples of tissue fixed in a suitable fixative for possible microscopic examination. The photographs will be archived with the raw data.
The fetuses were shipped (on 09 August 2016) to the PI (Debbie Coulby, Envigo CRS Limited) for evaluation. The necessary necropsy/cesarean data previously recorded in Pristima System was entered manually in the validated VMS System. No Phase number was assigned; CB24RV is used as study identifier.
Statistics:
The following comparisons were performed:
Group 1 vs. Groups 2, 3 and 4 to analyze food consumption, body weights, clinical signs and reproduction data:
Means and standard deviations of various data were calculated and included in the report.
For continuous data, a parametric analysis was performed if Bartlett's test for variance homogeneity is not significant at the 1% level. Treated groups were compared to control using Williams' test, unless there is evidence against a monotonic dose-response when Dunnett's test was performed instead.
A non-parametric analysis was performed if Bartlett's test is still significant at the 1% level following both logarithmic and square-root transformations. Treated groups were compared to control using Shirley's test, unless there is evidence against a monotonic dose-response when Steel's test was performed instead.
Historical control data:
No historical control data were used.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
No treatment-related clinical signs were recorded.
Some findings as coat, hair loss (dorsal or ventral surface, forelimbs and hind limbs) were observed sporadically in all the treatment groups.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Slightly lower mean body weight was recorded at the doses of 50 and 15 mg/kg/day compared to the control group from days 11 and 17 post coitum onwards, respectively. Regarding body-weight gain, statistically significant differences were recorded at the above mentioned doses, from days 11 to 20 of pregnancy at 50 mg/kg/day and from days 18 to 20 of pregnancy at 15 mg/kg/day.
No differences compared to the control group were recorded at the dose of 5 mg/kg/day.
For details of body weight see attached file with tables on pages (38-55, for body weight change pages 55-71

Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
A dose-dependent statistically significant decrease in absolute food consumption (gram per animal per day) compared to the control group was recorded at the doses of 50 and 15 mg/kg/day and on days 9 to 20 and 12 to 18 of pregnancy, respectively.
Food consumption at the dose of 5 mg/kg/day was not affected by the treatment.
No differences compared to the control group were recorded at 5 mg/kg /day or in the females at any of the treated doses
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):
no effects observed
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 test-item-related findings were recorded in the macroscopic examination at hysterectomy in the rats treated with the test item.
Some findings were recorded among all groups, including control: dilated renal pelvis, pale and/or strangulated lobe of liver, thin wall of diaphragm, split cervical thymus.
These findings were not attributed to the test item and were considered to be within the range of normal background lesions that may be seen in rats of this strain.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
Number of pregnant and non-pregnant dams

Group 1 2 3 4
Dose (mg/kg/day) 0 5 15 50
Female numbers
Number of mated females 22 22 22 22
Number of pregnant females 19 20 21 20
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
See attached tables pages 79-83
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
See attached tables pages 79-83
Early or late resorptions:
no effects observed
Description (incidence and severity):
See attached tables pages 79-83
Dead fetuses:
no effects observed
Description (incidence and severity):
See attached tables pages 79-83
Changes in pregnancy duration:
not examined
Description (incidence and severity):
Pregnancy was terminated by hysterectomy on day 20.
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): Pregnancy was terminated by hysterectomy on day 20.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
Number of pregnant and non-pregnant dams

Group 1 2 3 4
Dose (mg/kg/day) 0 5 15 50
Female numbers
Number of mated females 22 22 22 22
Number of pregnant females 19 20 21 20
Details on maternal toxic effects:
There were no relevant test-item related changes.
Differences were recorded in some reproductive parameters. The statistically significant differences observed were devoid of any toxicological relevance. They were considered a consequence of the biological variability and not test-item related.
There were no differences between the left and the right side concerning the distribution of resorptions in the uterine horns nor in the total resorptions nor in the uterus and placenta weight.
For mean foetal/pup body weight see attached tables page 83

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
> 50 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
gross pathology
maternal abnormalities
number of abortions
pre and post implantation loss
total litter losses by resorption
effects on pregnancy duration
early or late resorptions
dead fetuses
changes in pregnancy duration
changes in number of pregnant
necropsy findings

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
Slight higher fetal weights (males) were recorded at 15 and 50 mg/kg/day, approximately 9% and 6%, respectively, when compared with the control group. The differences observed were statistically significant.
No differences compared to the control group were recorded at 5 mg/kg /day or in the females at any of the treated doses
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): See attached tables pages 79-83
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
See attached tables pages 79-83
Changes in sex ratio:
no effects observed
Description (incidence and severity):
See attached tables pages 79-83
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No macroscopic findings which could be attributed to the treatment were observed.
50 mg/kg/day
Litter no. 104: One fetus (no.11) presented left hind paw with restricted movement
15 mg/kg/day
Litter no. 68: One fetus (no. 1) had domed head and short upper jaw
Litter no. 70: One fetus (no. 4) had cleft palate
For numbers and percent of foetuses and litters with malformation please refer to attached tables pages 85-87
Skeletal malformations:
no effects observed
Description (incidence and severity):
At 50 mg/kg/day there was a slightly increased incidence of medially thickened/kinked ribs, misaligned sternebral ossification sites and ossified cervical vertebral centra compared to control group. These findings are considered as minor skeletal abnormalities.
No relevant findings were recorded at 15 or 5 mg/kg/day.For numbers and percent of foetuses and litters with malformation please refer to attached tables pages 85-87
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Some isolated major abnormalities were recorded among the doses 5 and 15 mg/kg/day.
One fetus (no. 4) from litter 50 treated at 5 mg/kg/day presented right sided esophagus; right sided aortic arch; narrow, dorsally displaced retroesophageal pulmonary trunk; diaphragmatic hernia; membranous septal defect ventricular septum; malrotated heart; large posterior caudate liver lobe.
One fetus (no. 1) from litter 75 treated at 15 mg/kg/day presented ascending aorta pulmonary trunk fistula, large right ventricle, large heart accompanied by left umbilical artery, posterior caudate liver lobe fissure.
No major findings were recorded at 50 mg/kg/day. Some minor visceral abnormalities were observed in all the groups, including control (partially undescended lobe of thymus, left umbilical artery).
For numbers and percent of foetuses and litters with malformation please refer to attached tables pages 85-87

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
> 50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
changes in sex ratio
fetal/pup body weight changes
external malformations
skeletal malformations
visceral malformations
Remarks on result:
not determinable
Remarks:
Data will be provided when study report will be available

Fetal abnormalities

open allclose all
Abnormalities:
effects observed, non-treatment-related
Localisation:
external: paw
Description (incidence and severity):
One fetus (no. 11) presented left hind paw with restricted movement (dose 50 mg/kg/day)
Abnormalities:
effects observed, non-treatment-related
Localisation:
other: head + upper jaw
Description (incidence and severity):
Litter no. 68: One fetus (no. 1) had domed head and short upper jaw (dose 15 mg/kg/day)
Abnormalities:
effects observed, non-treatment-related
Localisation:
other: cleft palate
Description (incidence and severity):
Litter no. 70: One fetus (no. 4) had cleft palate (dose 15 mg/kg/day)

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
The test item, 4,4'-methylenebis(cyclohexylamine) was administered once daily by oral gavage to three treatment groups of twenty two pregnant female Wistar rats each from day 6 to day 19 of gestation at dose levels of 5, 15 and 50 mg/kg body weight/day. A control group of twenty two females were administered with vehicle (propyleneglycol) 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. The external and visceral variations observed in foetus were not statistically significant. Therefore these findings were considered as incidental findings and not test item-related. Based on the study 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 50 mg/kg bw/day, under the present experimental conditions. The NOAEL for maternal toxicity was considered as 50 mg/kg bw/day.
Executive summary:

Based on the OECD 414 study findings it is concluded that 4,4'-methylenebis(cyclohexylamine) is non teratogenic in rats and the NOAEL for teratogenicity of the test item can be fixed as 50 mg/kg bw/day, under the present experimental conditions. The NOAEL for maternal toxicity was determined to be 50 mg/kg bw/day.