Reaction products of m-phenylenebis(methylamine) 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:

from 2008-01-17 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

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 m-phenylenebis(methylamine) with 2,2'-[(1-methylethylidene)bis(4,1-phenyleneoxymethylene)]bisoxirane

EC Number:

641-048-8

Cas Number:

110839-13-9

Molecular formula:

C37H48N4O4

IUPAC Name:

Reaction products of m-phenylenebis(methylamine) 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, 53 – 60 days old
- Weight at study initiation: Male: 276 – 324 g, Female: 214 – 248 g
- Fasting period before study: None
- 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: 18 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 - 25
- Humidity (%): 30 - 47
- Air changes: 8 - 12 air exchanges/hour by central air-condition system
- Photoperiod: 12 hours daily, from 6.00 a.m. to 6.00 p.m.

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.

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 International Hungary 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. The suitability of the chosen vehicle for the test item at the intended concentrations was analytically verified up front. 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.: 07/524-100PE). 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 3. The test item caused reduction in the body weight development at 250/200 mg/kg bw/day. There were no test item related changes at 25 mg/kg bw/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

Description (incidence):

No mortality occurred during the 28-day treatment period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

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

80 mg/kg bw/day group
In the male animals, the body weight gain was slightly higher than in the control group between days 0 and 7.
For female animals, no difference was noted in the body weight or body weight gain when compared to the control.

200 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.
In summary: There was no test item related alterations in the body weight development. The slight difference at 80 mg/kg bw/day was not considered to be relevant, as there was no similar finding at the high dose.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no differences in the mean daily food intake in between the control and any test item treated groups (25 mg/kg bw/day, 80 mg/kg bw/day and 200 mg/kg bw/day, male and female).

Food efficiency:

no effects observed

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

no effects observed

Ophthalmological findings:

not specified

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related alterations were found at any dose level in the haematological parameters examined.
The only statistically significant difference from the control value for male animals was noted at 80 mg/kg bw/day. A slightly lower mean basophil cell count (BA) was noted for this group.
In the female animals of the 80 mg/kg bw/day dose group, the mean platelet volume (MPV) was slightly lower than the control value.
However, these slight differences between the control and treated animals were not correlated with administered dose and levels and were well within the historical background range.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

A slightly lower concentration of total bilirubine (T-BIL) was found in all test item treated groups (25 mg/kg bw/day, 80 mg/kg bw/day and 200 mg/kg bw/day, male; 80 mg/kg bw/day and 200 mg/kg bw/day, female), when compared to the control. In female of the high dose group (200 mg/kg bw/day), the mean potassium (K+) concentration was above the control value.

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

25 mg/kg bw/day group
The mean testes weights relative to the body and brain weights were slightly less than the control value.
There were no differences in the examined organs between the female group and the female control group.

80 mg/kg bw/day group and 200 mg/kg bw/day group
There were no differences in the examined organs between the male test item treated groups and the male control group.
In the female animals, the liver weights relative to the body weights were slightly higher than the control value.

In summary:
There was no clear evidence of test item effect on the examined organ weights. The differences in the liver weight relative to the body weights between the control and the female 80 mg/kg bw/day and 200 mg/kg bw/day groups were independent from doses. In the absence of any clinical pathology or histological findings these weight differences were not considered to be treatment related. The slight, but significant differences testes weights were not relevant toxicologically, since these were small and within historical control ranges. There were no related pathological findings or dose-response relationship.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Necropsy
25 mg/kg bw/day group
The mean testes weights relative to the body and brain weights were slightly less than the control value.
There were no differences in the examined organs between the female group and the female control group.

80 mg/kg bw/day group and 200 mg/kg bw/day group
There were no differences in the examined organs between the male test item treated groups and the male control group.
In the female animals, the liver weights relative to the body weights were slightly higher than the control value.

In summary:
There was no clear evidence of test item effect on the examined organ weights. The differences in the liver weight relative to the body weights between the control and the female 80 mg/kg bw/day and 200 mg/kg bw/day groups were independent from doses. In the absence of any clinical pathology or histological findings these weight differences were not considered to be treatment related. The slight, but significant differences testes weights were not relevant toxicologically, since these were small and within historical control ranges. There were no related pathological findings or dose-response relationship.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Control group
Focal alveolar emphysema (2/5 male, 2/5 female), focal haemorrhages (1/5 male, 1/5 female) and alveolar histiocytosis (1/5 male) were observed in the lungs. In the kidneys, bilateral pyelectasis along with unilateral focal lympho-histiocytic infiltration and unilateral focal fibroblast proliferation (male animal No.: 8962, each) and cyst (one side, female animal No.: 8981) were noted.

80 mg/kg bw/day group
In the kidney of male animal subjected to histology examination, (No.: 8955), one side pyelectasis was observed.

200 mg/kg bw/day group:
In the lungs, focal alveolar emphysema (3/5 male, 2/5 female) was observed. In one female animal (No.: 8990; 1/5), uterus dilation was found.

There was no morphological evidence of acute or subacute injury of the alimentary tract (particular attention was paid to gastro-intestinal tracts based on the results of the preceding acute study, such as distended, liquid filled intestines and stomach at 2000 mg/kg bw or 300 mg/kg bw LAB study code: 07/524-001P), 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 were similar 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 (pyelectasis, focal lympho-histiocytic infiltration, fibroblast proliferation and the cyst in the cortical region of the kidney) and in the lungs (alveolar histiocytosis) were considered to be individual disorders. These are commonly seen in experimental rats of this strain and age and were present mainly in single control animals and pyelectasis occurred also in a single male animal at 80 mg/kg bw/day. The focal alveolar emphysema and focal haemorrhages in the lungs were due to the hypoxia, dyspnoea and circulatory disturbance developed during exsanguination. Uterus dilatation is indicative of the sexual function in female animals.

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.
Clinical Observations:
Daily and Detailed Weekly Observations
Soft stool was noted in the bedding material for each cage from day 1 to day 6. There was no difference between the control and test item treated groups in severity and duration of this finding, therefore, it was not considered to be related to the test item.
No clinical symptoms were noted for any groups in the course of the daily observations and 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, increased transfer arousal (1/5), lack of finger approach (1/5), and lack of finger withdrawal (1/5) were observed.
In the female control animals, positional struggle to a marked degree (2/5) and vocalisation (2/5) were noted.

Group 2 – 25 mg/kg bw/day
In the male animals, all examined parameters were considered to be normal (5).
Vocalisation (1/5) was noted for one female animal of this group.

Group 3 – 80 mg/kg bw/day
In the male animals, lack of finger approach (1/5) and marked finger withdrawal (1/5), positional struggle to a marked degree (1/5) and vocalisation (1/5) were found.
In the female animals, positional struggle to a marked degree (1/5) and vocalisation (1/5) were observed.

Group 4 – 200 mg/kg bw/day
In the male group, increased positional struggle (1/5) and vocalisation (1/5) were noted.
In the female animals, positional struggle to a marked degree (2/5) and vocalisation (2/5) were noted.

The results of the functional battery observation and motor activity determinations 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 transfer arousal, finger approach and finger withdrawal, 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.
In summary:
There were no test item related changes in the clinical chemistry parameters examined. The slightly lower concentrations of bilirubine were within the normal ranges and not related to doses. The mean potassium concentration of female animals at 200 mg/kg bw/day represents a marginal value but within historical control range. There were no related findings in clinical pathology or organ pathology for potassium and bilirubine therefore the slight changes were not considered to be of toxicological significance.

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. The no observed effect level (NOEL) was 200 mg/kg bw/day.

Effect levels

Key result

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: highest dose tested

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 and 200 mg/kg bw/day. The no observed effect level (NOEL) 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. 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 (98 % and 93 %, respectively). Analytical control of dosing solutions was conducted on days 1 and 23. The measured concentrations ranged from 96 % to 101 % 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 week of the study. 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 was observed 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 test item related body weight and body weight gain changes. There were no differences in the mean daily food consumption between the control and test item treated groups. Haematology and clinical chemistry examinations did not reveal test item-related pathologic changes in the evaluated haematological or clinical chemistry parameters. 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 and 200 mg/kg bw/day. The no observed effect level (NOEL) was 200 mg/kg bw/day.