Reaction products of 3-aminomethyl-3,5,5-trimethylcyclohexylamine with 2,2'-[(1-methylethylidene)bis(4,1-phenyleneoxymethylene)]bisoxirane

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2008-01-16 to 2008-06-06

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

1995

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Version / remarks:

96/54/EC

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Reaction products of 3-aminomethyl-3,5,5-trimethylcyclohexylamine with 2,2'-[(1-methylethylidene)bis(4,1-phenyleneoxymethylene)]bisoxirane

EC Number:

614-657-1

Cas Number:

68609-08-5

Molecular formula:

C41H68N4O4

IUPAC Name:

Reaction products of 3-aminomethyl-3,5,5-trimethylcyclohexylamine with 2,2'-[(1-methylethylidene)bis(4,1-phenyleneoxymethylene)]bisoxirane

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: CHARLES RIVER (EUROPE) LABORATORIES INC. TOXI COOP Ltd. 1103 Budapest, Cserkesz u. 90.
- Age at study initiation: Young adult rats, 7 – 8 weeks old
- Weight at study initiation: Male: 265 – 299 g Female: 204 – 233 g
- Fasting period before study: No
- Housing: Group caging (5 animals/cage) Type III. polypropylene/polycarbonate
- Diet: Animals received ssniff® SM R/M-Z+H “Autoclavable complete feed for rats and mice – breeding and maintenance” produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany ad libitum
- Water: tap water, as for human consumption, ad libitum
- Acclimation period: 15 days

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Details on oral exposure:

The test item was administered by oral gavage on a 7 days/week basis. Control animals were treated concurrently with the vehicle only. A constant treatment volume of 5 mL/kg body weight was applied in all groups. The actual treatment volume was calculated according to the most recent body weight. Animals were not treated on the day of gross pathology.
Duration of treatment: 28 days.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test item was formulated in PEG 400 in concentrations of 5 mg/mL, 16 mg/mL and 40 mg/mL. Formulations were prepared daily except weekends in the Central Dispensary of LAB Research Ltd. Analytical control (concentration, homogeneity) of dosing solutions was performed in the Analytical Laboratory of LAB Research Ltd. on the first and last treatment weeks. Results of analysis are attached to the Draft Report (Appendix 2.9). The suitability of the chosen vehicle for the test item at the intended concentrations was analytically verified up front (LAB study code 07/523-316AN). A sufficient stability (up to 3 days at room temperature) was verified in the chosen vehicle over the range of relevant concentrations.

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

Doses / concentrations 4

Dose / conc.:

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

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

The doses were chosen on the basis of the results of a 14-day oral dose range finding toxicity study in CRL:(WI)BR rats (LAB study no.: 07/523-100PE). Doses assessed in the dose range finding study were 25 mg/kg bw/day, 80 mg/kg bw/day, 250 mg/kg bw/day and an additional dose level of 2000 mg/kg bw/day. The 250 mg/kg bw/day dose was reduced to 200 mg/kg bw/day from day 5.

Reaction Product of Bisphenol A (BADGE) with IPDA caused mortality at 250 mg/kg bw/day (1/5 female) and clinical signs, as well as changes in body weight and daily mean food consumption, in enzyme activity of alanine aminotransferase and aspartate aminotransferase at 200 mg/kg bw/day.
There were no test item related changes at 25 mg/kg bw/day. The maximum acceptable daily dose was considered to be 200 mg/kg/day.

Examinations

Observations and examinations performed and frequency:

General clinical observations were made once daily. A detailed clinical examination was made before the first treatment, then once a week outside the home cage. A functional observation battery was conducted on the last day of the treatment period. Body weight and food consumption were measured weekly.

Sacrifice and pathology:

Clinical pathology examinations and gross necropsy were conducted at the end of the treatment period. The absolute and relative organ weights of adrenals, brain, epididymides, heart, liver, kidney, spleen, testes, and thymus were determined. A full histopathological examination was performed on the preserved organs and tissues of the animals of Groups 1 and 4.

Other examinations:

Haematology:
The following haematology parameters were assessed in all animals:
RBC Red Blood Cell (erythrocyte) count, (10E12/L) M/µL
Hgb Haemoglobin concentration, (g/dL)
Hct Haematocrit (relative volume of erythrocytes) (%)
MCV Mean Corpuscular (erythrocyte) Volume (fL)
MCH Mean Corpuscular (erythrocyte) Haemoglobin, (pg)
MCHC Mean Corpuscular (erythrocyte) Haemoglobin Concentration, (g/dL)
RDW Red Cell (erythrocyte) volume (%)
Plt Platelet (thrombocyte) count, (10E9/L) K/µL
MPV Mean Platelet Thrombocyte volume (fl)
RETIC % Reticulocyte count (%)
WBC White Blood Cell (leukocyte) count, (10E9/L) K/µL
NE % Neutrophil (%)
LY % Lymphocyte (%)
MO % Monocyte (%)
EO % Eosinophil (%)
BA % Basophil (%)
LUC % Large Unstained Cells (%)
APTT Partial Thromboplastin Time (sec)
PT Prothrombin Time (sec)

Statistics:

Statistical analysis was done with SPSS PC+ software for the following data:
- body weight
- haematology
- clinical chemistry
- organ weight

The heterogeneity of variance between groups was checked by Bartlett's homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance was carried out. If the obtained result was positive, Duncan's Multiple Range test was used to assess the significance of inter-group differences.
Where significant heterogeneity was found, the normal distribution of data was assessed by a Kolmogorov-Smirnov test. In case of a not normal distribution, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was applied. If there was a positive result, the inter-group comparisons were performed using a Mann-Whitney U-test.
Frequency of clinical observations, mean daily food consumption by groups and sex, frequency of pathological and histopathological findings by sex and dose were calculated.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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

no effects observed

Ophthalmological findings:

not specified

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not specified

Details on results:

Results and Discussion

A 28-day oral (by gavage) toxicity study was performed in CRL:(WI) BR rats to obtain information on the toxic potential of the test item.

Mortality
No mortality occurred during the 28-day treatment period.

Clinical Observations

Daily and Detailed Weekly Observations
Soft stool was noted in the bedding material for each group including control from day 1 to 9. Taking into account that there was no difference between the control and test item treated groups in the degree and duration of this finding, it was interpreted as a vehicle induced slight change, which disappeared after adaptation.

No individual clinical symptoms were noted for any groups in the course of the daily observations and the detailed weekly observations. The general physical condition and behaviour of animals were considered to be normal throughout the entire observation period.

Functional Observation Battery
Group 1 – Control
In the male control animals, lack of finger approach (1/5) and lack of finger withdrawal (1/5) positional struggle to a marked degree (2/5) and vocalisation (3/5) were observed.
In the female control animals, marked positional struggle (3/5) and vocalisation (3/5) were noted.

Group 2 – 25 mg/kg bw/day
In the male animals, reduced transfer arousal (1/5), of finger approach (1/5), an elongated finger approach (1/5), reduced finger withdrawal (1/5), positional struggle to a marked degree (3/5) and vocalisation (3/5) were found.
For female animals, increased touch escape response (1/5) and positional struggle (1/5) and vocalisation (1/5) were noted.

Group 3 – 80 mg/kg bw/day
In the male animals, increased touch escape response (1/5) and positional struggle (1/5) and vocalisation (1/5) were found.
In the female animals, an increased startle reaction (1/5) and positional struggle to a marked degree (1/5) and vocalisation (4/5) were observed.

Group 4 – 200 mg/kg bw/day
In the male group, lack of finger approach (1/5), increased finger withdrawal (1/5) and touch escape response (1/5), positional struggle to a marked degree (1/5) and vocalisation (2/5) were noted.
In the female animals, increased touch escape response (1/5) and positional struggle (2/5) and vocalisation (2/5) were noted.
The results of the functional battery observation demonstrated no treatment-related differences to the control in the behaviour or in reactions to different type of stimuli at the end of the treatment period. Variations in startle reaction, transfer arousal, finger approach and finger withdrawal, touch escape response, positional struggle, as well as vocalisation were within the normal biological variation with respect to behaviour, reactions to different type of stimuli or manipulations. Incidences and extent of reactions observed in groups of treated animals were comparable to those observed in control animals and are without any toxicological significance.

Body Weight
25 mg/kg bw/day group
In the male animals, the body weight gain was slightly lower than in the control group between days 7 and 14.
For female animals, no difference was noted in the body weight or body weight gain when compared to the control.

80 mg/kg bw/day group
No differences were noted for the body weight and body weight gain between the control and test item treated group (male and female) during the entire observation period.

200 mg/kg bw/day group
In the male animals, no difference was found in the mean body weight and body weight gain when compared to the control.
In the female animals, the body weight gain was significantly lower than in the controls on week 3.

In summary: There was no test item related alterations in the body weight development. The slight differences noted in the body weight gain at 25 mg/kg bw/day and at 200 mg/kg bw/day were transient and not considered of toxicological relevance. All observations were well within the historical control ranges.

Food Consumption
There were no test item related differences in the mean daily food intake in any test item treated groups (25 mg/kg bw/day, 80 mg/kg bw/day or 200 mg/kg bw/day, male or female) when compared to the control.

Haematology
25 mg/kg bw/day group
All the examined haematological parameters were similar to the control values in male and female rats at the termination of the treatment period.

80 mg/kg bw/day group
For male animals of this dose group, the mean platelet cell volume (MPV) was slightly lower than the control value
For female animals, no statistically significant differences were found in the examined haematological parameters. One animal (No.: 8941) showed a high ratio of neutrophil cells (NE) and low ratio of lymphocytes (LY) (marginal to the normal range) but the white blood cell (WBC) count was normal.

200 mg/kg bw/day group
In male rats, the mean platelet cell volume (PLT) was statistically significantly less than in the control group. The ratio of neutrophil cells (NE) was above and ratio of lymphocytes (LY) was below the control value.
In the female animals, the mean prothrombin time (PT) was less than the control value. The ratio of neutrophil cells (NE) was above and ratio of lymphocytes (LY) was below the control value, but without statistical significance.

In summary, the only statistically significant test item related change was noted in the differential white blood cell picture of male animals at 200 mg/kg bw/day. Changes in the female high dose group were comparable, but not statistically significant. Similar changes were also observed in one single female animal at 80 mg/kg bw/day.
At 200 mg/kg bw/day, for both male and female animals, the mean ratio of neutrophil cells was higher and the ratio of lymphocytes was lower when compared to the control value. The white blood cell count was normal and changes in ratio of neutrophil cells and lymphocytes were marginal and values remained within the historical control ranges, therefore findings in these haematological parameters were not considered to be indicative of a toxicologically adverse effect. The same findings, only noted for one single female animal at 80 mg/kg bw/day, were similarly considered to be of no toxicological significance.
The slight statistical differences in the volume of platelet cells and prothrombin time between the control and treated groups were considered to be individual findings, which were not related to treatment and remained within the historical control ranges.
(Appendices 1.4, 2.4, Historical control data are presented in Appendix 2.13)

Clinical Chemistry
25 mg/kg bw/day group
There were no statistical differences between the control and test item treated groups (male and female animals) in the examined haematological parameters.

80 mg/kg bw/day group
In the male animals, all clinical chemistry parameter values were similar to the control value.
In the female animals, lower creatinine (Creat.) concentration was noted for this group.

200 mg/kg bw/day group
In the male animals, the activity of alanine aminotransferase (ALT) was higher than the control.
In the female animals, the concentration of creatinine (Creat.), total protein (Tot. Prot.), albumin (Alb.) and total bilirubin (T-BIL.) was lower, the enzyme activity of alanine aminotransferase (ALT) exceeded the control value.

In summary:
The slightly higher activity of alanine aminotransferase at 200 mg/kg bw/day was within the normal values for all animals. Even though these could indicate a potential test item influence on liver function, there were no related findings in organ weight or pathology and therefore the findings were considered to be without toxicological relevance.
The mean total protein level at 200 mg/kg bw/day female group was marginal to historical control range and did not represent toxicologically meaningful change.
The statistically significant differences in creatinine, albumin and total bilirubin concentrations represent biological variations within the historical control data range and were not correlated with administered dose.

Necropsy
Control group
In the male animals, in the lungs pinprick-sized haemorrhages (1/5) and in the small intestines red oedemic mucous membrane (1/5) were found.
In the female animals, there were no macroscopic findings.

25 mg/kg bw/day group
In the male animals, pinprick-sized haemorrhages were observed in the lungs (1/5).
In the female group, pinprick-sized haemorrhages (2/5) and pale raised areas (1/5) were found in the lungs. Hydrometra was noted in one female rat (1/5).

80 mg/kg bw/day group
In the male animals, pinprick-sized haemorrhages were observed in the lungs (1/5).
In the female group, one side pyelectasis (1/5) and hydrometra (1/5) were observed in single animals.

200 mg/kg bw/day group
In the male animals, pinprick-sized haemorrhages were found in the lungs (1/5) and one side pyelectasia (1/5) in the kidney.
Hydrometra was observed in one female animal (1/5).

In summary:
Gross necropsy did not reveal any macroscopic changes related to the test item. Pyelectasis is a common finding in untreated rats and it occurred in single animals, only. Pulmonary findings (haemorrhages and pale raised areas in the lungs) were due to the exsanguination procedure occurring in the control and treated groups without any toxicological relevance.
Hydrometra, related to the female sexual cycle, is a frequent observation in experimental rats, which is of no toxicological significance.
Red oedemic mucous membrane occurred in a control animal and was considered to be individual change.

Organ Weight
There were no test item related differences in the examined organ weight between the control and test item treated groups.
Thymus weights (absolute and relative to the body and brain weights) were below the control value in male animals at 200 mg/kg bw/day group. As these were of low magnitude and without any related pathological (necropsy or histology) findings were not considered to be toxicologically significant. All other examined organ weights (absolute and relative to the body and brain weights) were similar in the control and test item treated groups.
(Appendix 1.7, 2.7)

Histopathology
Control group:
In the male group, focal alveolar emphysema (1/5) and focal haemorrhages (1/5) were observed in the lungs. Hyperaemia was observed in the mucous membrane of the small intestines, without inflammatory or degenerative lesions in animal No.: 8896 (1/5).
In the female group, focal alveolar emphysema (2/5) was observed in the lungs.

80 mg/kg bw/day group:
One side pyelectasia was detected in the kidneys processed histologically in female animal No.: 8941.

200 mg/kg bw/day group:
In the male animals, focal alveolar emphysema (1/5) and focal haemorrhages (2/5) were observed in the lungs. One rat showed one side pyelectasia in the kidney (1/5).
In the female group, focal alveolar emphysema in the lungs (1/5) and uterus dilation (1/5) were found.

There were no morphological evidence of acute or subacute injury of the alimentary tract (particular attention was be paid to gastro-intestinal tracts based on the results of the preliminary study, such as distended and liquid filled intestines; LAB study code 07/523-100PE), the liver, the pancreas, the cardiovascular system, the haematopoietic system, the immune system, the skeleton, the muscular system, the male and female reproductive system, or the central or peripheral nervous system. The structure and the cell morphology of the endocrine glands was the same at the control and treated animals.

In summary:
The histopathological examination of organs and tissues revealed no lesions related to treatment with the test item. The histopathological findings in the kidneys (one side pyelectasia) were considered to be individual disorders commonly seen in experimental rats of this strain and age. The findings occurred in single animals without any relationship of incidences and/or severity to administered dose. The focal alveolar emphysema and focal haemorrhages in the lungs were due to the hypoxia, dyspnoea and circulatory disturbance developed during exsanguination. The incidence and severity of these lesions was the same in the control and treated groups. Uterus dilatation is indicative of the sexual function in female animals. The hyperaemia in the mucous membrane of small intestine in a control rat is a physiological phenomenon.

Conclusion
The test item caused no adverse toxic effects in male or female CRL:(WI) BR rats after 28-day subsequent oral (by gavage) administration at 25 mg/kg bw/day, 80 mg/kg bw/day and 200 mg/kg bw/day.
However, a slight statistically significant test item influence was found on the white blood cell picture in male animals at 200 mg/kg bw/day. Changes in the female 200 mg/kg bw/day dose group were comparable, but not statistically significant. Similar changes were also observed in one single female animal at 80 mg/kg bw/day.
At 200 mg/kg bw/day, test item related changes in enzyme activity of alanine aminotransferase were observed.
At 25 mg/kg bw/day, there were no test item related effects.
The no observed effect level (NOEL) was 25 mg/kg bw/day.
The no observed adverse effect level (NOAEL) was 200 mg/kg bw/day.

Effect levels

1

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

haematology

2

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

Target system / organ toxicity

Key result

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The test item caused no adverse toxic effects in male or female CRL:(WI) BR rats after 28-day subsequent oral (by gavage) administration at 25 mg/kg bw/day, 80 mg/kg bw/day or 200 mg/kg bw/day. The no observed effect level (NOEL) was 25 mg/kg bw/day. The no observed adverse effect level (NOAEL) was 200 mg/kg bw/day.

Executive summary:

A 28-day oral toxicity study was performed with the test item in male and female CRL (WI) Wistar rats. Dose levels administered in this study were selected from a preceding 14-day dose-range-finding toxicity study performed in the same strain of rats (LAB study code: 07/523-100PE).

A control and three dose groups (n= 5 animals per group and sex) were involved in the study. The test item was administered in concentrations of 0 mg/mL, 5 mg/mL, 16 mg/mL and 40 mg/ml prepared in Polyethylene glycol 400 corresponding to 0 mg/kg bw/day 25 mg/kg bw/day, 80 mg/kg bw/day and 200 mg/kg bw/day doses at a 5 mL/kg bw treatment volume.

Stability and homogeneity of test item in this vehicle was analytically proven. The test item was stable at concentrations of 0.5 mg/mL and 50 mg/mL in this vehicle at room temperature for 72 hours (92 % and 101 %, respectively; LAB study code 07/523-316AN). Analytical control of dosing solutions was conducted on days 3 and 25. The measured concentrations ranged from 91 % to 105 % of nominal concentrations.

General clinical observations were made once daily. A detailed clinical examination was made before the first treatment, then once a week outside the home cage. A functional observation battery was conducted on the last day of the treatment period. Body weight and food consumption were measured weekly. Clinical pathology examinations and gross necropsy were conducted at the end of the treatment period. The absolute and relative organ weights of adrenals, brain, epididymides, heart, liver, kidney, spleen, testes, and thymus were determined. A full histopathological examination was performed on the preserved organs and tissues of the animals of Groups 1 and 4.

No mortality occurred during the study. There were no test item related clinical signs during the treatment period. The behaviour and the general condition of the test animals were normal during the study. There was no treatment-related effect on motor activity or in the functional observation battery tests across groups of treated male and female animals and no findings indicative for neurotoxicity were observed. There were no significant differences in the body weight and body weight gain and in the mean daily food consumption between the control and test item treated groups. Haematology revealed marginal changes in the mean ratio of neutrophil cells and in the ratio of lymphocytes at 200 mg/kg bw/day male and female animals and in a single female animal at 80 mg/kg bw/day. The mean values of changes were within historical control ranges and thus not considered adverse toxicological effects. At the clinical chemistry examinations, a slightly elevated activity of alanine aminotransferase was observed in male and female animals at 200 mg/kg bw/day, but within the historical control ranges and with no correlations in organ weight or histopathology. Therefore, this variation was not considered toxicologically relevant. Specific alterations related to treatment with the test item were not observed at the necropsy, organ weight or histopathology examinations.

The test item caused no adverse toxic effects in male or female CRL:(WI) BR rats after 28-day subsequent oral (by gavage) administration at 25 mg/kg bw/day, 80 mg/kg bw/day or 200 mg/kg bw/day. However, a slight statistically significant test item influence was found on the white blood cell picture in male animals at 200 mg/kg bw/day. Changes in the female 200 mg/kg bw/day dose group were comparable, but not statistically significant. Similar changes were also observed in one single female animal at 80 mg/kg bw/day. At 200 mg/kg bw/day, test item related changes in enzyme activity of alanine aminotransferase were observed. At 25 mg/kg bw/day, there were no test item related effects. The no observed effect level (NOEL) was 25 mg/kg bw/day. The no observed adverse effect level (NOAEL) was 200 mg/kg bw/day.