4,4'-methylenebis(cyclohexylamine)

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:

guideline study

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)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2,2'-dimethyl-4,4'-methylenebis(cyclohexylamine)

EC Number:

229-962-1

EC Name:

2,2'-dimethyl-4,4'-methylenebis(cyclohexylamine)

Cas Number:

6864-37-5

Molecular formula:

C15H30N2

IUPAC Name:

4,4'-methylenebis(2-methylcyclohexanamine)

impurity 1

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

water

Test material form:

liquid

Test animals

Species:

rabbit

Strain:

New Zealand White

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5% CMC in water

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

GD 6-28 (23 applications)

Frequency of treatment:

once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 mg/kg bw/day

Remarks:

low dose

Doses / concentrations 2

Dose / conc.:

3 mg/kg bw/day

Remarks:

mid dose

Doses / concentrations 3

Dose / conc.:

9 mg/kg bw/day

Remarks:

high dose

No. of animals per sex per dose:

25

Control animals:

yes

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Two females of the high-dose group (Nos. 92, 94 - 9 mg/kg bw/d) had blood in bedding on GD 16-19, respectively. In total, reduced defecation was observed in six control, three low-dose, four mid-dose and eleven high-dose females (0, 1, 3 and 9 mg/kg bw/d). Incidence and distribution of this finding do not indicate a relationship to the test substance. There were no clinical findings in the other does in the study.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One control female (No. 24 - 0 mg/kg bw/d) and one high-dose female (No. 92 - 9 mg/kg bw/d) were sacrificed after abortion ahead of schedule (GD 20 and 18, respectively). Spontaneous abortions in single does are not uncommon findings in the strain of rabbits used for this study. Thus, this was considered to be a spontaneous incident. One control female (No. 17) was sacrificed moribund on GD 14 after an accidental fracture of maxilla and dental root. Furthermore, one female of test group 2 (No. 74 - 3 mg/kg bw/d) died after a gavage error.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The mean body weights (BW) and the average body weight gain (BWC) of the high-dose does (9 mg/kg bw/d) were statistically significantly reduced on GD 16-29 (BW), on GD 9-11 and GD 14-16 (BWC), which is considered to be treatment-related. If calculated for the entire treatment period (GD 6-28) these does gained 82% less body weight (with statistical significance) than the concurrent control rabbits. No statistically significant difference was observed for the BW of the mid-dose does (3 mg/kg bw/d) when compared to the concurrent control group. However, from GD 16 onwards the mid-dose body weights were slightly lower than the control values. Also, the BWC of these rabbits was statistically significantly reduced on GD 9-11. If calculated for the entire treatment period (GD 6-28), the does of the mid-dose group gained 50% less body weight than the control does. The mean body weights and the average body weight gain of the low-dose group (1 mg/kg bw/d) were generally comparable to the concurrent control group throughout the entire study period.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In comparison to the control group the mean food consumption of the does in test group 3 (9 mg/kg bw/d) was reduced during major parts of the treatment period, attaining statistical significance on GD 14-20. If calculated for the entire treatment period (GD 6-28) or the entire study period (GD 0-29), the high-dose does consumed 19% or 14% less food than the concurrent control does (without statistical significance). The food consumption of the low- and mid-dose rabbits (1 and 3 mg/kg bw/d) was comparable to the concurrent control (0 mg/kg bw/d) throughout the entire study period.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Some spontaneous findings were noted in individual females of test groups 0-3 (0, 1, 3 and 9 mg/kg bw/d). These gross findings were:
• Infarct of liver in one control doe (No. 7 - 0 mg/kg bw/d)
• Erosion(s) in stomach in one control doe (No. 5 - 0 mg/kg bw/d) and one low-dose doe (No. 28 - 1 mg/kg bw/d)
• Watery feces in two high-dose does (Nos. 77 and 79 - 9 mg/kg bw/d)
• Absence of uterine horn(s) in one mid-dose doe (No. 65 - 3 mg/kg bw/d)

Two additional findings were noted in test groups 0 and 2. Both were associated with unscheduled
maternal death or sacrifice:
• Fracture of maxilla and dental root in control doe No. 17 (sacrificed moribund on GD 14)
• Findings after gavage error (i.e. thoracic cavity filled with blood) in mid-dose doe No. 74 (died after gavage error on GD 18)

Maternal developmental toxicity

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Female rabbits were placed into the study in four cohorts. The conception rate was 80% in the mid-dose group (3 mg/kg bw/d), 88% in the low-dose group (1 mg/kg bw/d), 92% in the high-dose group (9 mg/kg bw/d) and 96% in the control group (0 mg/kg bw/d). A sufficient number (approximately 20, but not fewer than 16 females with implantation sites) of pregnant females was available for the purpose of the study.
There were no test substance-related and/or biologically relevant differences between the different test groups in conception rate, mean number of corpora lutea or in the values calculated for the pre- and the postimplantation losses and the number of resorptions.
The number of implantation sites and subsequently of live fetuses (live litter size) was statistically significantly lower than the concurrent control in the mid- and high-dose groups (3 and 9 mg/kg bw/d). This apparent decrease in the mid- and high-dose group needs, however, to be considered in view of the unusually high number of implants and litter size of the concurrent control group. These were distinctly above the upper range of the historical control data (implantation sites: 10.9 vs 7.1 – 10.3, litter size: 10.2 vs 6.6 - 9.7). On the other hand the mean implant/litter size values of the mid- and high-dose group were close to the mean of the historical control ranges (8.7 and 8.1, respectively). In addition, no relation to the dose was observed for any of the two apparent effects. Therefore, they were not considered to be treatment-related.
All other differences observed are considered to reflect the normal range of fluctuations for animals of this strain and age.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

1 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Results (fetuses)

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

External malformations occurred in test groups 0 and 2 (0 and 3 mg/kg bw/d) only. One control fetus had multiple external malformations associated with multiple skeletal malformations, while one mid-dose fetus had an open eye and associated soft tissue malformations. The distribution of the findings about the dose groups does not indicate an association to the treatment. No statistically significant differences between the groups were noted.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Skeletal malformations were detected in test groups 0, 1 and 2 (0, 1 and 3 mg/kg bw/d). One control, one low-dose and one mid-dose fetus had associated external and/or soft tissue malformations. No skeletal malformations were observed in the high-dose group (9 mg/kg bw/d). No dose-response relationship was observed and, therefore, all these findings were assessed as not-treatment related.
For the test groups 0-3, skeletal variations of different bone structures were observed, with or without effects on corresponding cartilages. The observed skeletal variations were related to several parts of fetal skeleton and appeared in the majority of cases without a relation to dosing. Although the rate of affected fetuses per litter was statistically significantly increased in the low-, mid- and high-dose groups (1, 3 and 9 mg/kg bw/d), the incidence of test group 3 was well within the historical control range, while the control, low- and mid-dose values were below the historical control range.
Some isolated cartilage findings without impact on the respective bony structures, which were designated as unclassified cartilage observations, occurred in test groups 0-3. The observed unclassified cartilage findings did not show any relation to dosing and were considered to be spontaneous in nature.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Soft tissue malformations occurred in all test groups including control (0, 1, 3 and 9 mg/kg bw/d). The distribution of the findings about the dose groups does not indicate an association to the treatment and no statistically significant differences between the groups were noted. A number of observations affecting the spleen were noted in individual litters. Asplenia was observed in two low-dose litters (fetuses Nos. 36-05 F and 39-02 F) and in one mid-dose litter (fetuses Nos. 51-09 F and 51-10 F). Additionally, small spleens were observed in 12 fetuses of the same mid-dose litter (No. 51) and another small spleen in a different litter. These spleen dysplasia were not observed in the high-dose group and showed, therefore, no relation to dosing. Furthermore, the gestational parameters, in particular the resorption rate, do not indicate any embryofetal mortality in-utero which could have masked the occurence of potentially malformed offspring in the high-dose group. All other malformations can either be found in the historical control data or were single cases (e.g. hydronephrosis in the low dose) without any relation to dosing. Therefore, they were considered as not treatment related.
The examinations of the organs revealed a broad variety of soft tissue variations, i.e. dilated cerebral ventricle, malpositioned carotid branch and absent lung lobe (lobus inferior medialis) in fetuses of all test groups including the control (0, 1, 3 and 9 mg/kg bw/d). The incidences of these variations were neither statistically significantly different from control nor dosedependent and, therefore, not considered biologically relevant. All of them can be found in the historical control data at comparable incidences.
Only one unclassified soft tissue observation was recorded: discolored kidney in one middose fetus (3 mg/kg bw/d). This single incidence was not considered to be biologically relevant.

Details on embryotoxic / teratogenic effects:

There were noted external, soft tissue and skeletal malformations in seven control, four low-dose, eighteen mid-dose and two high-dose litters. The distribution of total malformations about the litters and groups was not related to dose and they were, therefore, considered as not treatment-related.
Nine fetuses were multiple-malformed. For control female fetus No. 10-11 an absent thoracic vertebra and fused ribs were recorded, while the findings in male control fetus No. 11-01 consisted of an absent lumbar vertebra and a branched rib. Female control fetus No. 12-05 had multiple external malformations, i.e. protruding tongue, absent tooth, no bony structure palpable in lower jaw and bent forelimbs, associated with multiple skeletal malformations, i.e. shortened incisive bones, shortened mandible, bent ribs, bent radius and ulna, bent humerus, bent scapula, bent tibia and fibula and bent femur. Furthermore, for male control fetus No. 16-04 a lumbar hemivertebra, a misshapen lumbar vertebra and a branched rib were recorded, while male low-dose fetus No. 28-09 (1 mg/kg bw/d) had a hydronephrosis and a hydroureter. Multiple visceral malformations were seen in female low-dose fetus No. 36-04 (1 mg/kg bw/d), i.e. diaphragmatic hernia, displaced and irregular shaped stomach, absent esophagus, indistinct cartilage rings of trachea, small gallbladder and malpositioned kidney. Female low-dose fetus No. 39-02 (1 mg/kg bw/d) had asplenia combined with malpositioned and bipartite sternebra. For female mid-dose fetus No. 58-09 (3 mg/kg bw/d) an open eye and multiple visceral malformations, i.e. diaphragmatic hernia, cardiomegaly, dilated pulmonary trunk, absent descending aorta and absent subclavian, were recorded, associated with severely fused sternebrae (bony plate) and misshapen caudal vertebra. Finally, female middose fetus No. 64-09 (3 mg/kg bw/d) showed severely fused sternebrae (bony plate) and a misshapen cervical vertebra.
A number of observations affecting the spleen were noted in individual litters. Asplenia was observed in two low-dose litters (fetuses Nos. 36-05 F and 39-02 F) and in one mid-dose litter (fetuses Nos. 51-09 F and 51-10 F). Additionally, small spleens were observed in 12 fetuses of the same mid-dose litter (No. 51) and another small spleen in a different litter. These spleen abnormalities were not observed in the high-dose group and showed, therefore, no relation to dosing. Furthermore, the gestational parameters, in particular the resorption rate, do not indicate any embryofetal mortality in-utero which could have masked
the occurence of potentially malformed offspring in the high-dose group. The distribution of these observations in only very few litters and the absence of any dose response suggest a non-treatment related origin of the spleen findings. Other malformations, such as absent gallbladder, absent subclavian, thoracic hemivertebra and malpositioned kidney were scattered observations in individual fetuses of test groups 0, 2 and 3.
In general, neither the incidence of multiple-malformed offspring nor the individual malformations were dose-related and most of them can be found in the historical control data at comparable or higher frequency. No ontogenetic pattern is recognizable for the individual malformations nor was there any cluster of any of these individual malformations seen in the other offspring of any test group. An association of these findings to the treatment is not assumed.
No external variations were seen in any of the fetuses in this study. A spontaneous origin is assumed for the soft tissue variations and the broad range of skeletal variations which were observed in fetuses of all test groups including the controls. Although the total incidence of skeletal variations was statistically significantly increased in the low-, mid- and high-dose groups (1, 3 and 9 mg/kg bw/d), the incidence in test group 3 was well within the historical control range, while the control, low- and mid-dose values were below. Generally, all skeletal variations are equally distributed about the different test groups including controls, if normal biological variation is taken into account. The changes which were significantly different from the concurrent control were either not related to dose or can be found in the historical control data at a comparable or higher frequency. Although the rates of affected fetuses per litter were statistically significantly increased in test groups 1, 2 and 3 (1, 3 and 9 mg/kg bw/d), the overall incidence of all classified fetal variations in test group 3 was well within the historical control range, while the values in test groups 0, 1 and 2 were below. Thus, an association to the treatment is not assumed.
No unclassified external observations were seen in any of the fetuses in this study. A spontaneous origin is assumed for unclassified soft tissue and unclassified skeletal cartilage observations which were observed in several fetuses of test groups 0, 1, 2 and 3. The distribution and type of these findings do not suggest any relation to treatment. Finally, fetal examinations revealed that there is no adverse effect of the compound on the respective morphological structures up to a dose of 9 mg/kg bw/d.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

9 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: highest dose tested

Overall developmental toxicity

Key result

Developmental effects observed:

no

Applicant's summary and conclusion

Conclusions:

In this prenatal developmental toxicity study, the oral administration of 2,2’-dimethyl-4,4’-methylenebis(cyclohexylamine) to pregnant New Zealand White rabbits from implantation to one day prior to the expected day of parturition (GD 6-28) caused evidence of maternal toxicity at the high- and mid-dose (9 and 3 mg/kg bw/d), such as reduced food consumption and body weights/body weight gain. In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is 1 mg/kg bw/d. Since there was no evidence for toxicologically relevant adverse effects of the test substance on fetal morphology at any dose, the no observed adverse effect level (NOAEL) for prenatal developmental toxicity is the highest dose of 9 mg/kg bw/d.

Executive summary:

2,2’-dimethyl-4,4’-methylenebis(cyclohexylamine) was administered to pregnant New Zealand White rabbits daily by stomach tube from implantation to one day prior to the expected day of parturition (GD 6-28) at dose levels of 1, 3 and 9 mg/kg bw/day. Analyses confirmed the correctness of the prepared concentrations, their homogeneous distribution and the stability of the test substance in the vehicle.

Generally, clinical observations including food consumption and body weight gain revealed no toxicologically relevant effects at the low dose of 1 mg/kg bw/d. At the mid- and the highdose (3 and 9 mg/kg bw/d) signs of maternal toxicity were observed such as a dosedependent reduction of body weights/body weight gain, and reduced food consumption in the high-dose group, only. No differences of toxicological relevance between the control and the treated groups (1, 3 and 9 mg/kg bw/d) were determined for any reproductive parameters, such as conception rate, mean number of corpora lutea, mean number of implantations, as well as pre- and postimplantation loss. Similarly, no adverse effect of the test substance on fetal weight and sex distribution of the fetuses was noted at any dose. Overall, there were no toxicologically relevant adverse effects of the test substance on fetal morphology at any dose.