3(or 4)-methylbenzene-1,2-diamine

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: fully reported guideline GLP study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

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:

Diaminotoluene

EC Number:

246-910-3

EC Name:

Diaminotoluene

Cas Number:

25376-45-8

Molecular formula:

C7H10N2

IUPAC Name:

3-methylbenzene-1,2-diamine

Details on test material:

33.2 % 2,3-TDA
66.4% 3,4-TDA

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male

Details on test animals or test system and environmental conditions:

Initially 6 to 12 weeks old
Acclimatised for at least 5 days
Singly caged on soft wood granules

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

The negative control was the vehicle deionized water(20 ml/kg).

Details on exposure:

Groups of 5 male mice were administered o-TDA 75, 150 and 300 mg/kg twice via intraperitoneal injection (20 ml/kg), separated by 24 hours.

Post exposure period:

Animals were sacrificed 24 hours after the last treatment,

Doses / concentrations

Remarks:

Doses / Concentrations:
75, 150 and 300 mg/kg
Basis:

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

The positive control: Animals, received a single intraperitoneal treatment with 20 mg/kg, cyclophosamide. Results indicated a clear clastogenic
effect, as is shown by the biologically relevant increase in polychromatic erythrocytes with micronuclei. The ratio of polychromatic to normochrom
atic erythrocytes was not altered.

Examinations

Tissues and cell types examined:

The femoral marrow of all groups was prepared 24 hours after the last administration.
Normally 2000 polychromatic erythrocytes were counted per animal. The number of normochromatic erythrocytes per 2000 polychromatic ones was noted. In addition to the number of normochromatic erythrocytes showing micronuclei was also established.

Details of tissue and slide preparation:

Schmids method was used to prepare the smears.

Evaluation criteria:

The rate of normochromatic erythrocytes containing micronuclei was examined if the micronuclear rate for polychromatic erythrocytes was already relevantly increased. A one sided chi2-test was performed when the group with the highest mean was compared iwth the negative control.

Statistics:

Wilcoxon's non-parametric rank sum test was performed with respect to the number of polychromatic erythrocytes having micro-nuclei and the number of normochromatic erythrocytes. A variation was considered statistically signifcant 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. A one sided chi2-test was performed when the group with the highest mean was compared iwth the negative control.
Standard deviations were (1s ranges) were calculated for all means.

Results and discussion

Test results

Sex:

male

Genotoxicity:

positive

Toxicity:

yes

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Males treated twice with o-TDA in doses up to 300 mg/kg showed symptoms of toxicity after administration starting at 75 mg/kg. These symptoms demonstrate relevant systemic exposure of males to o-TDA. However, all males survived until the end of the test.

Treated males showed the following compound related symptoms until sacrifice: apathy, roughened fur, loss of weight, sternal recumbency, spasm, difficulty in breathing, slited eyes and closed eyes. No symptoms were reported in the control group. No animals died in these groups.

The ratio of polychromatic to normochromatic erythrocytes in males was not altered by treatment with o-TDA, being 2000:4317 (1s=1396) in the
negative control. 2000:3750 (1s=1575) in the 75 mg/kg group, 2000:5481 (1s=3157) in the 150 mg/kg group and 2000:3948 (1s=988) in the 300 mg/kg group. No variations were thus noted for males.

Indications of clastogenic effect of o-TDA was detected at 300 mg/kg. At 300 mg/kg biologically important and statistically signficant variation
existed for males with the respect to the incidence of micronucleated polychromatic erythroytes. No biologically relevant differences were observed for 75 and 150 mg/kg doses. The incidence of these micronucleated cells was 4.2/2000 (1s=1.3) in the negative control, and 3.2/2000 (1s=3.3), 5.6/2000 (1s=1.9) and 13.2/2000 (1s=4.1) in the o-TDA groups.

There was a clear indication of a clastogenic effect of o-TDA administered i,p. in a somatic cell test system in vivo.

Any other information on results incl. tables

Results of Micronucleus Test with o-TDA

 Negative Control

 

Random number and sex	Body weight in g	Number of evaluated PCE	Number of NCE per 2000 PCE	MNNCE per 2000 NCE	MNPCE per 2000 PCE
5 ♂	39	2000	3525	1.1	3
11 ♂	42	2000	3564	1.7	5
17 ♂	38	2000	4253	0.9	6
25 ♂	39	2000	3492	0	4
28 ♂	37	2000	6749	1.5	3
Mean	39	2000	4317	1.0	4.2
1s	2		1396	0.7	1.3

 

 

Results of Micronucleus Test with o-TDA

 2 x 75 mg/kg i.p.

 

Random number and sex	Body weight in g	Number of evaluated PCE	Number of NCE per 2000 PCE	MNNCE per 2000 NCE	MNPCE per 2000 PCE
1 ♂	41	2000	3467	3.5	9
8 ♂	38	2000	3327	1.8	2
12 ♂	41	2000	2013	1.0	1
13 ♂	40	2000	6324	1.3	2
21 ♂	40	2000	3617	2.2	2
Mean	40	2000	3750	1.9	3.2
1s	1		1575	1.0	3.3

 

 

Results of Micronucleus Test with o-TDA

2 x 150 mg/kg i.p.

 

Random number and sex	Body weight in g	Number of evaluated PCE	Number of NCE per 2000 PCE	MNNCE per 2000 NCE	MNPCE per 2000 PCE
2 ♂	39	2000	5369	2.2	5
14 ♂	40	2000	4094	1.0	3
20 ♂	43	2000	5055	1.6	7
22 ♂	43	2000	10679	1.7	8
27 ♂	40	2000	2206	2.7	5
Mean	41	2000	5481	1.8	5.6
1s	2		3157	0.7	1.9

 

 

 

Results of Micronucleus Test with o-TDA

 2 x 300 mg/kg i.p.

 

Random number and sex	Body weight in g	Number of evaluated PCE	Number of NCE per 2000 PCE	MNNCE per 2000 NCE	MNPCE per 2000 PCE
16 ♂	40	2000	4916	4.1	10
23 ♂	41	2000	3162	0.6	12
26 ♂	39	2000	4646	1.3	17
29 ♂	40	2000	2649	1.5	18
30 ♂	41	2000	4365	1.8	9
Mean	40	2000	3948	1.9	13.2
1s	1		988	1.3	4.1

 

 

 

Results of Micronucleus Test with o-TDA

Positive Control Cyclophosphamide

 1 x 20 mg/kg i.p.

 

Random number and sex	Body weight in g	Number of evaluated PCE	Number of NCE per 2000 PCE	MNNCE per 2000 NCE	MNPCE per 2000 PCE
3 ♂	41	2000	2243	2.7	26
4 ♂	39	2000	3188	1.3	49
9 ♂	42	2000	3313	0.6	40
10 ♂	43	2000	2334	2.6	49
19 ♂	40	2000	2527	2.4	48
Mean	41	2000	2721	1.9	42.4
1s	2		496	0.9	9.9

 

 

 

Summary of Results of the Micronucleus Test with o-TDA

  

Experimental Groups	Number of evaluated PCE	Number of NCE per 2000 PCE	MNNCE per 2000 NCE	MNPCE per 2000 PCE
Negative control	10,000	4317 ±      1396	1.0 ±         0.7	4.2 ±         1.3
o-TDA 2 x 75 mg/kg	10,000	3750 ±      1575	1.9 ±         1.0	3.2 ±         3.3
o-TDA 2 x 150 mg/kg	10,000	5481 ±      3157	1.8 ±         0.7	5.6 ±         1.9
o-TDA 2 x 300 mg/kg	10,000	3948 ±        988	1.9 ±         1.3	13.2* ±         4.1
Positive control Cyclophosphamide 20 mg/kg	10,000	2721 ±        496	1.9 ±         0.9	42.4* ±         9.9

*P <0.01 in non-parametric Wilcoxon ranking test

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): positive
There was a clear indication of a clastogenic effect of o-TDA administered i,p. in a somatic cell test system in vivo.