4,4'-Methylene-bis-(3-chloro-2,6-diethylaniline)

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

October - December 1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

402-130-7

EC Name:

-

Cas Number:

106246-33-7

Molecular formula:

C21 H28 Cl2 N2

IUPAC Name:

4-[(4-amino-2-chloro-3,5-diethylphenyl)methyl]-3-chloro-2,6-diethylaniline

Test material form:

solid: crystalline

Details on test material:

- Name: LONZACURE® M-CDEA
- Internal substance code: P5367
- Storage: Keep container tightly closed. Keep in a dry, cool and well-ventilated place.
- Aggregate state/appearance: Crystalline solid
- Colour: white to off-white
- Odour: Odourless

Test animals

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK Limited, Margate, Kent, England
- Age at study initiation: approximately 35 days
- Weight at study initiation: between 22 and 24 grams
- Assigned to test groups randomly: yes
- Housing: each group was kept, with the sexes separated, in plastic disposable cages
- Diet (e.g. ad libitum): pelleted Biosure LAD 1 rodent diet
- Water (e.g. ad libitum): tap water
- Acclimation period: yes

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C
- Humidity (%): 55%
- Air changes (per hr): 20 changes/h
- Photoperiod (hrs dark / hrs light): artificial light 12 h/day

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

1% aqueous methylcellulose; suspensions ofthe test item were prepared in CMC (obtained from Courtaulds, batch number T32397) on the morning of the test at the concentrations shown overleaf

Details on exposure:

All animals in all groups were dosed with the standard volume of 20 ml/kg bodyweight. The test substance and negative control were dosed by intraperitoneal injection, and mitomycin C, the positive control compound, orally by intragastric gavage. The animals in the positive control group were deprived of diet overnight prior to and for two hours after dosing.

Duration of treatment / exposure:

24 and 48 hours

Frequency of treatment:

Once

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Remarks:

pretest; 2000 mg/kg = 100 mg/ml

Doses / concentrations 2

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

main study; vehicle control

Doses / concentrations 3

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

main study; 500 mg/kg = 25 mg/ml

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

main study; 1000 mg/kg = 50 mg/ml

Doses / concentrations 5

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Remarks:

main study; 2000 mg/kg = 100 mg/ml

No. of animals per sex per dose:

Males:
- 0 mg/kg; No. of animals: 5; Sacrifice time: 24 hours
- 0 mg/kg; No. of animals: 5; Sacrifice time: 48 hours
- 500 mg/kg; No. of animals: 5; Sacrifice time: 24 hours
- 1000 mg/kg; No. of animals: 5; Sacrifice time: 24 hours
- 2000 mg/kg; No. of animals: 5; Sacrifice time: 24 hours
- 2000 mg/kg; No. of animals: 5; Sacrifice time: 48 hours

Females:
- 0 mg/kg; No. of animals: 5; Sacrifice times: 24 hours
- 0 mg/kg; No. of animals: 5; Sacrifice times: 48 hours
- 500 mg/kg; No. of animals: 5; Sacrifice times: 24 hours
- 1000 mg/kg; No. of animals: 5; Sacrifice times: 24 hours
- 2000 mg/kg; No. of animals: 5; Sacrifice times: 24 hours
- 2000 mg/kg; No. of animals: 5; Sacrifice times: 48 hours

Control animals:

yes

Positive control(s):

A positive control group was dosed orally, by intragastric gavage, with mitomycin C at 12 mg/kg bodyweight.

Examinations

Tissues and cell types examined:

Bone marrow smears were obtained from 5 male and 5 female animals in the negative control, test substance groups and the positive control group 24 hours after dosing. Bone marrow smears were also taken from 5 male and 5 female animals from the negative control and high dose level groups 48 hours after treatment. One smear from each animal was examined for the presence of micronuclei in 1000 polychromatic erythrocytes. The ratio of polychromatic to normochromatic erythrocytes was assessed by examination of at least 1000 erythrocytes from each animal. A record of the incidence of micro nucleated normochromatic erythrocytes was also kept.

Evaluation criteria:

A positive response is normally indicated by a substantial, statistically significant increase (p < 0.01) in the incidence of micronucleated polychromatic erythrocytes compared to the incidence for the concurrent vehicle control group for at least one of the sampling times~ individual and/or group mean values should exceed the laboratory historical control range.

Statistics:

Non-parametric statistical methods, based on rank are chosen for analysis of results because:
- They are suited to analysis of data consisting of discrete/integer values such as the incidence of micronucleated polychromatic erythrocytes.
- 'Outliers' are frequently found in the polychromatic erythrocyte to normochromatic erythrocyte ratios for both control and
- The methods make few assumptions about the underlying distribution of data and therefore the values do not require transformation to fit a theoretical distribution (where data can be approximately fitted to a normal distribution, the results of non-parametric analysis and classical analysis of variance are very similar).

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Toxic signs were only observed in the high dose group after dosing. Thes signs included lethargy, hunched posture, piloerection and altered gait. The ratio of polychromatic to normochromatic erythrocytes was unaltered by treatment.

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Treatment with the test substance did not result in any increase in the number of miconucleated polychromatic erythrocytes.

Any other information on results incl. tables

Only minor clinical signs and no mortalities were obtained in the preliminary toxicity test at a dose level of 2000 mg/kg. Dose levels of 500, 1000 and 2000 mg/kg were therefore selected for use in the micronucleus test. The high dose level of 2000 mg/kg is the standard limit dose for the micronucleus test and we therefore consider it to be an appropriate maximum.

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative
The test substance was found to be non-mutagenic in this test system.

Executive summary:

The study was performed 1996 as GLP-test following OECD test method 474 using bone marrow cells of CD-1 mice. The test item was dissolved in 1% CMC and tested at concentrations of 500 - 2000 ug/ml. Toxic signs were only observed in the high dose group after dosing. Treatment with the test substance did not result in any increase in the number of miconucleated polychromatic erythrocytes. In conclusion, the substance is not mutagenic under the conditions of this test.