Registration Dossier

Administrative data

Description of key information

Data from a combined repeated dose and reproductive/developmental toxicity screening test of 4,4'-methylenebis (2-chlorobenzenamine) by oral administration in rats performed according to GLP principles and OECD guidelines were available to cover this endpoint.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 Augustus 2003 - 20 January 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: No deviations reported. The study fully meets the current guidelines.
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Japan
- Age at study initiation: 8 weeks
- Weight at study initiation: males; 335-435 g; females; 198-267 g
- Fasting period before study: not mentioned
- Housing: individually in wire-mesh steel cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 13 day

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25
- Humidity (%): 45 -65
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
olive oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: once weekly

VEHICLE
- Justification for use and choice of vehicle (if other than water): test substance is soluble in olive oil, not in water
- Amount of vehicle (if gavage): 5 ml/kg
- Lot/batch no. (if required): KH 21
- Purity: not mentioned
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The first lot of each dosing solution was analyzed by HPLC. The concentrations measured were as expected.
Duration of treatment / exposure:
males 42 days; females 42-55 days
Frequency of treatment:
once daily
Remarks:
Doses / Concentrations:
0, 0.4, 2, 10, 50 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on dose-range finding study of 14 days
- Rationale for animal assignment (if not random): at random
- Rationale for selecting satellite groups: not mentioned
- Post-exposure recovery period in satellite groups: not mentioned
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: first hour after dosing

BODY WEIGHT: Yes
- Time schedule for examinations: Males: day 1, 7, 14, 21, 28, 35, 42 during dosing period; day 7, 14 during recovery period
Females: day 1, 7, 14 during dosing; day 0, 7, 14, 20 on gestation days; day 0, 4 lactation days

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

HAEMATOLOGY: Yes
- Time schedule for collection of blood: day of last dosing and end of recovery period
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes
- How many animals: all
- Parameters checked: erythrocytes (+ Heinz body containing), hemoglobin, hematocrit, reticulocytes, methemoglobin, platelets, leucocytes

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day of last dosing and end of recovery period
- Animals fasted: Yes
- How many animals: all
- Parameters checked: LDH, GPT, gamma-GTP, choline-esterase, total protein, albumin, A/G ratio, total cholesterol, triglyceride, Ca, inorganic phosphorus, Cl, K.

URINALYSIS: Yes
- Time schedule for collection of urine: males on day 40 and recovery day 8
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No data
- Parameters checked: gross appearance, qualitative pH, occult blood, protein, glucose, ketone body, urobilinogen, bilirubin, sedimentation, volume, specific gravity, concentration sodium and potassium.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: last day of dosing and end of recovery period
- Battery of functions tested: sensory activity and reflex functions
Sacrifice and pathology:
GROSS PATHOLOGY: Yes - organ weight
HISTOPATHOLOGY: Yes
Other examinations:
Observation of newborn pups: number of littermates, sex, malformations of pups, clinical signs, body weight, pathological examinations.
Statistics:
Where there was a significant difference (<5% level of significance) of mean values or frequencies between the control and treated groups were analyzed with the following statistical tests. (As for the data of pups born, the mean value per littermate was treated as one sample): If there was more than three treated groups, the following parametric data was first examined for uniformness of variance by the Bartlett's test: body weight, body weight gain, food consumption, landing foot splay width, grip strength, locomotor activity, quantitative data of urinalysis, hematological data, blood biochemical data, organ weight, number of corpora lutea, number of implantation sites, paring days until copulation, gestation length, number of pups born, number of live pups, number of dead pups . The parametric data with uniform variance were then examined by a one-way analysis of variance (ANOVA). Both parametric data with non-uniform variance and non-parametric data such as differential leukocyte count, qualitative data of urinalysis, implantation index, live birth index, delivery index, estrous cycle, viability index were analyzed with the Kruskal-Wallis rank test. Results with significant difference found in these analyses were examined using a multiple comparison procedure with the Dunnett or Dunnett-type test. When comparing data between two test groups, parametric data was firstly examined using the F test. The data with uniform variance was next examined by Student t-test and the data with non-uniform variance was examined by Aspin-Welch's t-test. Non-parametric data was examined by Mann-Whitney U test. The categorical data including clinical signs, detailed observations, sensory and reflex function test, incidence of abnormality in necropsy and histopathological test, copulation index, fertility index, delivery index, and sex ratio of pups was examined by Fisher's exact probability test.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
salviation in 50 mg/kg group 8 days after dosing
Mortality:
mortality observed, treatment-related
Description (incidence):
salviation in 50 mg/kg group 8 days after dosing
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
females 50 mg/kg group showed decreased body weight
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
males 50 mg/kg group decreased at day 1
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
both sexes at 50 mg/kg group
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
in females of 10 mg/kg group and in both sexes of 50 mg/kg group
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
males 50 mg/kg group decreased at day 1
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
both sexes at 50 mg/kg group
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
both sexes at 50 mg/kg group
Details on results:
CLINICAL SIGNS AND MORTALITY
During the administration period, salviation was observed in 6 male and 3 female rats out of 12 in the 50 mg/kg group 8 days after dosing. Salviation was also observed in 3 of 5 rats in the 50 mg/kg female satellite group. Salviation was generally observed from 10 to 30 minutes after dosing or immediately after dosing in some cases. During the recovery period, there were no rats showing changes in clinical signs. No deaths occurred during administration and recovery periods.

BODY WEIGHT AND WEIGHT GAIN
During the administration period, the body weight of female rats of the 50 mg/kg group was significantly decreased on gestation days 14 and 20, and significant decreased body weight gain was also observed during the gestation period. Body weights of females of the same group on the lactation day 0 and 4 had a tendency to show values lower than those of the control group, but there was no difference observed in the body weight gain during the period. In the female satellite group, the body weight tended to be slightly lower than that in the control group, but the difference was not significant. In male rats, there were no changes in either body weight or body weight gain. During the recovery period, there were no significant changes in either body weight or body weight gain in both sexes.

FOOD EFFICIENCY
During the administration period, food consumption of male rats on dosing day 1 in the 50 mg/kg group was significantly decreased.

HAEMATOLOGY
In the hematological test performed at the end of the administration period, significant changes were observed in the 50 mg/kg group: decrease in erythrocyte count and methemoglobin in both sexes, decrease in hemoglobin concentration and hematocrit value and increase in reticulocyte count and platelet count in the males, and increase in Heinz bodies-containing erythrocytes in the females. Also observed in the 50 mg/kg group, there was a tendency for hemoglobin concentration and hematocrit value to be lower and for reticulocyte count to be higher. At the end of the recovery period, significant differences in the hemoglobin concentration and hematocrit value of male rats was found, but the differences became smaller. Except for these points, no changes were found. In female rats at the end of the recovery, methemoglobin was noted as having slightly increased. Despite this significant difference, the value was within the normal range.

CLINICAL CHEMISTRY
In the blood biochemical test performed at the end of the administration period, a significant decrease in total protein and albumin of female rats in the 10 mg/kg group was found. In the 50 mg/kg group, there was a significant decrease in total protein and albumin of both sexes, and A/G ratio showed significant decrease in female rats and showed the tendency of having a lower value in male rats. In the same group, there were significant increases in total cholesterol, triglyceride and inorganic phosphorus in male rats, and in LDH and gamma-GTP in female rats; LDH and gamma-GTP in male rats and potassium of both sexes had also tendency to show a higher value. At the end of the recovery period, no significant changes in any test items found.

URINALYSIS
During the administration and recovery period, there were no significant changes found in any items of urinalysis.

NEUROBEHAVIOUR
There were no significant changes at the measurement time points of dosing week 6 and recovery week 2.

ORGAN WEIGHTS
In animals sacrificed after the administration period, a significant increase in relative weight of the kidney was found in female rats in the 10 mg/kg group. In the 50 mg/kg group, a significant increase in relative weight of the kidney was found in female rats. Significant increases were also found in absolute and relative weight of the liver and relative weight of the spleen in male rats; and in relative weight of both the liver and thyroid, and absolute and relative weight of the spleen in female rats. In animals sacrificed after the recovery period, there were significant differences in relative weights of male spleen, female liver and female kidney but the differences tended to decrease. There weren't any changes in male liver, female spleen and female thyroid. There was a significant increase in relative weight of testis in rats sacrificed after recovery period.

HISTOPATHOLOGY: NON-NEOPLASTIC
Changes attributed to the test article were found in the liver, spleen and kidneys.
Liver:
In the 50 mg/kg group, those changes characterized by swelling (centrilobular zone) and fatty degeneration (in intermediate zone) of hepatocytes were found in all of 6 male rats and 6 out of 8 female rats, and 3 of these male rats showed single cell necrosis of hepatocytes in the centrilobular zone. In female rats in the same group, there was another rat showing prominent fatty degeneration only. There was significant difference in the incidences of swelling and fatty degeneration of hepatocytes in both sexes compared with those in the control group. These changes were not found in the recovery group.

Kidney:
Among 5 male rats in each group, mildly basophilic change of renal tubules was found in 1 control groups whereas there was an increase tendency of the incidence with 3 in the 10 mg/kg group and 4 in the 50 mg/kg group, and the degree of this change tended to be more severe. In female rats, there wasn't any increase tendency of basophilic change of renal tubules found. In the male recovery group, there was no difference in the incidence of basophilic change of renal tubules compared to that in the control group.

Spleen:
Mild hemosiderin deposits in splenic red pulp were found in all rats of both sexes in the control group and moderate in 4 male rats from the 10 mg/kg group and in 5 male rats and 4 female rats from the 50 mg/kg group, and there was a significant difference in the incidence of moderate hemosiderin deposits. As for extramedullary hematopoiesis in splenic red pulp in female rats, only mild cases were found except for one rat in the control group with a moderate degree, but 4 rats showed moderate extramedullar hematopoiesis in the 50 mg/kg group and tendency for the enhancement. In the recovery group, increased tendency for hemosiderin deposits remained in female rats, which was not significantly different, and there were no differences in both hemosiderin deposits and extramedullary hematopoiesis in male rats compared with those in the control group.
Dose descriptor:
NOEL
Effect level:
2 mg/kg bw/day (actual dose received)
Sex:
male/female
Critical effects observed:
not specified

none

Conclusions:
Sprague-Dawley rats were treated with 0, 0.4, 2, 10, and 50 mg/kg 4,4'-methylene bis (2-chloro-aniline) once daily during 42 days (males) or up to 4 days after delivery (females). 4,4'-methylene bis (2-chloro-aniline) had toxic effects on the spleen, liver and kidneys following repeated administration in rats. Overall, a NOEL of 2 mg/kg was reported for 4,4'-methylene bis (2-chloro-aniline).
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
2 mg/kg bw/day
Study duration:
subacute
Species:
rat

Additional information

Sprague-Dawley rats were treated with 0, 0.4, 2, 10, and 50 mg/kg 4,4'-methylene bis (2-chloro-aniline) once daily during 42 days (males) or up to 4 days after delivery (females). 4,4'-methylene bis (2-chloro-aniline) had toxic effects on the spleen, liver and kidneys following repeated administration in rats. Overall, a NOEL of 2 mg/kg was reported for 4,4'-methylene bis (2-chloro-aniline).


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Carcinogenicity is used to derive the definitive DMEL.

Justification for classification or non-classification

Based on the above mentioned results, the substance does not need to be classified according to the Dangerous Substance Directive 67/548/EC andCLP Regulation (EC) 1272/2008.