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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1993
Report date:
1993

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Constituent 1
Chemical structure
Reference substance name:
4,4',4''-methylidynetrianiline
EC Number:
208-952-0
EC Name:
4,4',4''-methylidynetrianiline
Cas Number:
548-61-8
Molecular formula:
C19H19N3
IUPAC Name:
4,4',4''-methylidynetrianiline

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: F. Winkelmann, Borchen, Germany
- Weight at study initiation: mean weight of about 28-42 g
- Assigned to test groups randomly: yes
- Diet (e.g. ad libitum): standard pellet feed ad libitum
- Water (e.g. ad libitum): drinking water from bottles; ad libitum

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil (test substance); water (positive control)
- Amount of vehicle: 5 mL/kg bw for test substance and 10 mL/kg bw for positive control
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance was suspended in corn oil. The stability in the vehicle was analytically verified for at least 24 hours.
The selection of the test substance dose was based on a pilot test, in which groups of five animals, including both males and females, were intraperitoneally administered 500 mg/kg and 1000 mg/kg in cremophor emulsion and 750 mg/kg, 1000 mg/kg (two groups), 1500 mg/kg and
2500 mg/kg test substance in corn oil. The following symptoms were recorded for up to 48 hours, starting at 500 mg/kg: apathy, increased motility, uncoordinated movements, roughened fur, staggering gait, spasm, extension spasm, rolling over and orange to red discolored urine. A cremophor suspension could not be used as vehicle due to instability of the test substance suspension.
Based on these results, 750 mg/kg bw test substance was chosen as MTD for this test.
Duration of treatment / exposure:
single intraperitoneal administration
Frequency of treatment:
single treatment
Post exposure period:
16, 24 and 48 h sampling time for treatment group; negative and positive control groups only 24 h
Doses / concentrations
Remarks:
Doses / Concentrations:
750 mg/kg bw
Basis:
nominal conc.
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Route of administration: i. p.
- Doses / concentrations: 20 mg/kg bw

Examinations

Tissues and cell types examined:
At least one intact femur was prepared from each sacrificed animal. The bone marrow was prepared and centrifuged in calf serum. The numbers of polychromatic erythrocytes (PCE) and normochromatic erythrocytes (NCE) with and without micronuclei (MN) were counted.
Details of tissue and slide preparation:
DETAILS OF SLIDE PREPARATION: One drop of the cell suspension resulting from centrifugation was used to prepare smears. Dried slides were automatically stained with an Ames Hema-Tek Slide Stainer from the Miles Company. The slides were then 'destained' with methanol and rinsed with deionized water.

METHOD OF ANALYSIS: A total of 1000 PCE (with and without micronuclei) from each animal were evaluated. The number of NCEs per 1000 PCEs was noted. The number of NCE showing micronuclei was also counted.
Evaluation criteria:
A test was considered positive if, at any of the intervals, there was a relevant and significant increase in the number of polychromatic erythrocytes showing micronuclei in comparison to the negative control.
A test was considered negative if there was no relevant or significant increase in the rate of micronucleated polychromatic erythrocytes at any time. A test was also considered negative if there was a significant increase in that rate which, according to the laboratory's experience was within the range of negative controls.
In addition, a test was considered equivocal if there was an increase of micronucleated polychromatic erythrocytes above the range of attached historical negative controls, provided the increase was not significant and the result of the negative control was not closely related to the data of the respective treatment group. In this case, a second test had to be performed at the most sensitive interval.
Statistics:
The test substance group(s) with the highest mean (provided this superseded the negative control mean) and the positive control were checked by Wilcoxon's non-parametric rank sum test with respect to the number of polychromatic erythrocytes having micronuclei and the number of normochromatic erythrocytes. A variation was considered statistically significant if its error probability was below 5 % and the treatment group figure was higher than that of the negative control.
The rate of normochromatic erythrocytes containing micronuclei was examined if the micronuclear rate for polychromatic erythrocytes was already relevantly increased. In this case, the group with the highest mean was compared with the negative control using the one-sided chiz-test. A variation was considered statistically significant if the error probability was below 5 % and the treatment group figure was higher than that of the negative control. In addition, standard deviations (1s ranges) were calculated for all the means.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Toleration by the animals:
After single intraperitoneal administration of 750 mg/kg test substance, treated animals showed the following compound-related symptoms until sacrifice: apathy, roughened fur, spasm and orange to red discoloured urine. Their feeding behaviour was normal. There were no substance-induced mortalities. No symptoms were recorded for the control groups. No animals died in these groups.

Any other information on results incl. tables

Treatment group

Dose

[mg/kg]

Dissection interval

[h]

Mean MN-PCE

[‰ ± SE]

Mean MN-NCE

[‰ ± SE]

PCE/NCE

Corn oil

0

24

1.9 ± 1.7

1.5 ± 0.8

1000/1173

test substance

750.0

16

2.1 ± 1.0

1.8 ± 1.1

1000/1073

750.0

24

2.7 ± 1.4

1.4 ± 1.5

1000/1380

750.0

48

2.2 ± 1.1

2.0 ± 1.0

1000/1537

Cyclophosphamide

25.0

24

16.2* ± 5.2

1.8 ± 1.6

1000/899

* P < 0.01 in non-parametric Wilcoxon ranking test

The test substance does not show any chromosome-damaging (clastogenic) effect in the bone marrow cells of mice.

The administration of the test substance led to clinical signs such as apathy, roughened fur, spasms and orange to red discoloured urine. The PCE/NCE ratio in the 48 hour dissection interval group was considered to be an indication for a biologically relevant weak toxic effect in the bone marrow, although not statistically significant.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Executive summary:

A micronucleus test on male and female NMRI mice was performed according to OECD TG 474 to investigate potential clastogenic effects of the test item. Groups of 5 mice/sex received a single intraperitoneal injection of either the test substance (750 mg/kg bw), the vehicle (corn oil), or the positive control substance cyclophosphamide (20 mg/kg bw). The femoral marrow of the groups treated with the test item was prepared 16, 24 and 48 hours after administration.

All animals treated with the test item showed symptoms of toxicity after treatment, as apathy and roughened fur. A weak altered ratio between poly- and normochromatic erythrocytes in the 48 h treated group pointed to exposure of the bone marrow. No indications of clastogenic effects of the test item were found. Thus, the test item was not clastogenic under the conditions of this test.