Registration Dossier

Administrative data

Endpoint:
toxicity to reproduction
Remarks:
other: reproduction and first generation pups
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 September - 1 November 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: No deviations reported. The study fully meets the current guidelines.
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2005

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Name of test material (as cited in study report): 4, 4'-methylene bis (2-chlorobenzen amine)
- Substance type: white, needlelike crystalline powder
- Physical state: solid
- Analytical purity: 99.76%
- Impurities (identity and concentrations): not mentioned
- Purity test date: 2002-12-12
- Lot/batch No.: FG-3002
- Expiration date of the lot/batch: not mentioned
- Stability under test conditions: stable during study period; purity was 98.98% at 2004-01-01
- Storage condition of test material: protected from air and moisture at 3-5 °C

Test animals

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

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
olive oil
Details on 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
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: two weeks
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
Doses / concentrations
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

Examinations

Parental animals: Observations and examinations:
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
Oestrous cyclicity (parental animals):
estrous cyclus observed during 10 days acclimation and during mating period
Sperm parameters (parental animals):
Parameters examined in P male parental generations: spermatogenesis cycle (stage II-III, V, VII and XII)
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, all pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in [F1] offspring: number and sex of pups, live births, postnatal mortality, presence of gross abnomalities, weight gain

GROSS EXAMINATION OF DEAD PUPS: yes, for external and internal abnormalities
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals - as soon as possible after the last litters in each generation were produced
- Maternal animals: All surviving animals - after the last litter of each generation was weaned

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera

The reader is referred to paragraph 7.5.1 for detailed description.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals were sacrificed at 4 days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows: major thoracic and abdominal organs of pups were observed with naked eye

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.
Reproductive indices:
Number of live pups on day 0/number of pups born X 100
Offspring viability indices:
Number of live pups on lactation day 4/number of live pups on day 0 X 100

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
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):
effects observed, treatment-related
Description (incidence and severity):
females 50 mg/kg group showed decreased body weight
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
Other effects:
no effects observed
Description (incidence and severity):
Test substance intake: oral gavage

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
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 FOOD CONSUMPTION (PARENTAL ANIMALS)
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 any significant changes in either body weight or body weight gain in both sexes.
During the administration period, food consumption of male rats on dosing day 1 in the 50 mg/kg group was significantly decreased.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
Not applicable: oral gavage.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
From the day after group assignment to before mating, all rats repeated estrous cycle at 4 to 5 -day intervals, and there were no significant changes in estrous cycle.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
No changes were found related to the test article. Among male rats in the 50 mg/kg group and the control groups which succeeded to cause pregnancy, lymphocyte infiltration into prostate interstitium was found in 1 out of 5 rats in each group, and unilateral spermatic granuloma in epididymis was found in one rat from the 50 mg/kg group. Dose-independent atrophy of seminiferous tubules of testis was found in 2 out of 5 rats in the 0.4 mg/kg group, 1 out of 4 rats in each the 2 and 10 mg/kg group, and 2 out of 5 rats in the 50 mg/kg group. No changes were found in seminal vesicle. Also, there were no changes found in the spermatogenic cycle test in both control and 50 mg/kg groups.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Unsuccessful copulation was found in one pair from the 2 mg/kg group and in two pairs from the 10 mg/kg group, and the copulation indexes were 91.7% and 83.3%, respectively, in which no significant difference and dose-dependency were found. In other groups, copulation was confirmed in all pairs. The pairing days until copulation was significantly longer in the 50 mg/kg group, especially in three pairs that needed 7, 13 and 14 days. As the result of vaginal smear test during mating period, anestrus started at mating day 2 in two rats (animal No. 558 and 563) in the 50 mg/kg group. Signs of the recurrence of estrus were found at mating day 12 and 13 respectively, and copulations were confirmed the next day. Another rat (No. 564) showed a normal estrous cycle, but copulation was not successful during the first optimal mating period and it took 7 days until successful copulation during the next optimal period. Except for these three rats, no significant differences in the number of days until copulation was
confirmed in the dosing group compared with that of the control group. In one rat from the 2 mg/kg group (No. 533) and two rats from the 10 mg/kg group (No. 544 and 550), anestrus started at mating day 2 and continued to the end of the mating period with unsuccessful copulation. These female rats, of which the estrous cycle was normal in the estrus test before mating, were in metestrus-I (III) at the mating day 1 on vaginal smear examination except for one rat. Pregnancy was observed in all rats with successful copulation except for one in the 50 mg/kg group. Thus, the fertility index of the 50 mg/kg group was 91.7%, no significant difference was found.

ORGAN WEIGHTS (PARENTAL ANIMALS)
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 (PARENTAL ANIMALS)
The reader is referred to paragraph 7.5.1 for a detailed description of the changes attributed to the test article found in the liver, spleen and kidneys.

Effect levels (P0)

Dose descriptor:
NOEL
Effect level:
50 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: no effects

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

VIABILITY (OFFSPRING)
There were no significant changes observed in the number of littermates, delivery index, the number of pups alive on day 0 and day 4 of lactation, live birth index, sex ratio, or viability index on day 4 of lactation, and no abnormalities were found in the clinical signs of pups.

CLINICAL SIGNS (OFFSPRING)
There were no significant changes observed in the number of littermates, delivery index, the number of pups alive on day 0 and day 4 of lactation, live birth index, sex ratio, or viability index on day 4 of lactation, and no abnormalities were found in the clinical signs of pups.

BODY WEIGHT (OFFSPRING)
The body weights at the lactation day 0 and 4 showed slightly decreasing tendencies in the 50 mg/kg group, but they were within the normal range shown in historical baseline data (lactation day 0: 6.2 - 7.4 g in males, 5.8 - 7.0 g in females, lactation day 4: 9.8 - 12.0 g in males, 9.3 - 11.4 g in females). Thus, there were no significant differences in these values compared with those in control group.

GROSS PATHOLOGY (OFFSPRING)
There were no significant differences in the incidence of these visceral abnormalities between control and dosing groups, nor any increased tendency of dose-dependency was found.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

none

Applicant's summary and conclusion

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). No effects on the estrous cycle, paring days until copulation, copulation index, fertility index, gestation length, the number of corpora lutea, implantation index, live birth index delivery index, parturition and parenting state were observed in dams. In addition, effects after the administration of test article on total number of pups, number of live pups, sex ratio, live birth index, body weight, body formation and viability index of live pups on day 4 were observed. In conclusion, the NOEL in the reproductive potential of parents in both sexes and the growth and development in pups was 50 mg/kg/day.