4-methyl-m-phenylenediamine

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1979

Report date:

1979

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

Not to GLP

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

4-methyl-m-phenylenediamine

EC Number:

202-453-1

EC Name:

4-methyl-m-phenylenediamine

Cas Number:

95-80-7

Molecular formula:

C7H10N2

IUPAC Name:

4-methylbenzene-1,3-diamine

Constituent 2

Reference substance name:

2-methyl-m-phenylenediamine

EC Number:

212-513-9

EC Name:

2-methyl-m-phenylenediamine

Cas Number:

823-40-5

IUPAC Name:

2-methylbenzene-1,3-diamine

Details on test material:

2,4-TDA CAS 95-80-7
2,6-TDA CAS 823-40-5

Method

Species / strain

Species / strain / cell type:

other: Salmonella typhimurium strains, TA100, TA98, TA1535, TA1537 and TA1538

Metabolic activation:

with

Metabolic activation system:

Aroclor 1254 induced rat liver S-9

Vehicle / solvent:

dimethylsulphoxide (DMSO)

Results and discussion

Additional information on results:

2,4-Toluenediamine (2,4-TDA) was positive in the presence of S-9 mix in Salmonella typhimurium strains TA 98, TA 100, TA 1537 and TA 1538 with respect to induction of bacterial gene mutations.

2,4-Toluenediamine (2,4-TDA) was negative in the absence of S-9 mix in all Salmonella typhimurium strains tested, with respect to induction of bacterial gene mutations.

2,6-Toluenediamine (2,6-TDA) was positive in the presence of S-9 mix in Salmonella typhimurium strains TA 98, TA 100, TA 1537 and TA 1538 with respect to induction of bacterial gene mutations.

2,6-Toluenediamine (2,6-TDA) was negative in the absence of S-9 mix in all Salmonella typhimurium strains tested, with respect to induction of bacterial gene mutations.

See Tables 1 and 2. Positive controls were used as detailed in Tables 1 and 2 (see Remarks on Results below). Toxicity data are available in Table 3 (see Remarks on Results below).

Any other information on results incl. tables

Table 1. Bacterial mutagenicity of 2,4-TDA (see Additional information on results, above)

Chemical	Amount per plate (ug)	S-9 (b)	Revertants per plate (a)
			TA98	TA100	TA1535	TA1537	TA1538
Negative Control	-	-	13	54	54	6	8
Negative Control	-	+	17	55	52	7	11
Positive Control (c)	-	-	558	237	78	122	1209
Positive Control (d)	-	+	1178	242	0	379	1156
2,4-TDA	500	-	14	67	61	9	10
	1077	-	12	76	60	8	6
	2320	-	13	80	63	8	12
	5000	-	10	72	58	8	11
	500	+	213	98	46	23	154
	1077	+	376	140	56	34	362
	2320	+	738	167	66	82	750
	5000	+	1144	218	60	101	1138

(a) Average of two plates <br>(b) Aroclor 1254 induced Fisher 344 rat liver.<br>(c) The following positive controls were used without activating medium: dinitrotoluene (500 ug, TA1535 and TA100), quinacrine mustard (10 ug, TA1537) and nitrofluorene (100 ug, TA1538 and TA98).<br>(d) The following positive controls were used with activating medium: anthramine (100 ug, TA1535 and TA 100), aminoquinoline (100 ug, TA1537) and acetamidofluorene (500 ug, TA1538 and TA98).

Table 2. Bacterial mutagenicity of 2,6-TDA (see Additional information on results, above)

Chemical	Amount per plate (ug)	S-9 (b)	Revertants per plate (a)
			TA98	TA100	TA1535	TA1537	TA1538
Negative Control	-	-	13	54	54	6	8
Negative Control	-	+	17	55	52	7	11
Positive Control (c)	-	-	558	237	78	122	1209
Positive Control (d)	-	+	1178	242	0	379	1156
2,6-TDA	500	-	12	72	64	10	7
	1077	-	14	64	63	6	8
	2320	-	14	80	62	8	14
	5000	-	12	74	66	6	10
	500	+	151	418	66	20	176
	1077	+	205	645	61	20	261
	2320	+	348	1086	66	30	380
	5000	+	395	1000	64	34	528

(a) Average of two plates. <br>(b) Aroclor 1254 induced Fisher 344 rat liver.<br>(c) The following positive controls were used without activating medium: dinitrotoluene (500 ug, TA1535 and TA100), quinacrine mustard (10 ug, TA1537) and nitrofluorene (100 ug, TA1538 and TA98).<br>(d) The following positive controls were used with activating medium: anthramine (100 ug, TA1535 and TA 100), aminoquinoline (100 ug, TA1537) and acetamidofluorene (500 ug, TA1538 and TA98).

Table 3. Toxicity of TDA isomers for S. Typhimurium (see Additional information on results, above)

Chemical	Amount per plate (ug)	S-9 (b)	Relative survival (a)
			TA98	TA100	TA1535	TA1537	TA1538
2,4-TDA	500	-	0.150	0.776	0.776	1.032	0.453
2,4-TDA	1077	-	0.183	0.680	0.629	0.886	0.432
2,4-TDA	2320	-	0.187	0.482	0.755	0.889	0.456
2,4-TDA	5000	-	0.162	-	0.598	0.912	0.345
2,4-TDA	500	+	0.406	0.541	1.083	1.054	0.506
2,4-TDA	1077	+	0.240	0.646	0.882	0.931	0.424
2,4-TDA	2320	+	0.233	0.584	0.802	0.915	0.367
2,4-TDA	5000	+	0.229	0.619	0.917	0.905	0.357
2,5-TDA	500	-	0.000	0.000	0.595	0.076	0.042
2,5-TDA	1077	-	0.000	0.000	0.269	0.0024	0.000
2,5-TDA	2320	-	0.000	0.000	0.000	0.000	0.000
2,5-TDA	5000	-	0.000	0.000	0.000	0.000	0.000
2,5-TDA	500	+	0.222	0.578	1.051	0.860	0.287
2,5-TDA	1077	+	0.257	0.438	0.962	0.860	0.284
2,5-TDA	2320	+	0.017	0.000	0.683	0.231	0.209
2,5-TDA	5000	+	0.000	0.000	0.000	0.000	0.000
2,6-TDA	500	-	0.150	0.488	0.898	0.927	0.356
2,6-TDA	1077	-	0.158	0.540	1.033	0.931	0.355
2,6-TDA	2320	-	0.166	0.475	0.771	0.907	0.335
2,6-TDA	5000	-	0.168	0.409	0.733	0.912	0.342
2,6-TDA	500	+	0.208	0.511	0.993	1.015	0.295
2,6-TDA	1077	+	0.205	0.374	0.948	0.856	0.302
2,6-TDA	2320	+	0.200	0.473	1.078	0.808	0.334
2,6-TDA	5000	+	0.259	0.464	0.990	0.927	0.332
3,4-TDA	1000	-	0.107	0.463	0.803	0.794	0.341
3,4-TDA	1710	-	0.094	0.473	0.767	0.811	0.302
3,4-TDA	2924	-	0.068	0.471	0.598	0.712	0.185
3,4-TDA	5000	-	0.018	0.411	0.407	0.326	0.042
3,4-TDA	1000	+	0.224	0.471	0.901	0.872	0.320
3,4-TDA	1710	+	0.247	0.501	0.904	0.868	0.313
3,4-TDA	2924	+	0.240	0.422	0.816	0.861	0.309
3,4-TDA	5000	+	0.194	0.385	0.773	0.875	0.309

(a) The amount of chemical indicated, + or - rat S-9, was added to a tube of top agar containing 0.1 ml of a 10^-6 dilution of the relevant tester strain. The mixture then was poured onto a nutrient agar plate, was allowed to harden, and the plates were incubated at 37°C for 24 hr. <br>Relative survival = <br>Number of colonies on plate + chemical <br>Number of colonies on control plate <br>(b) Aroclor 1254 induced Fisher 344 rat liver

There are various other in vivo bacterial mutation assays reported on TDA isomers. The EU Risk Assessment for 2,4-TDA discusses these reports thus:

"Bacterial gene mutations

Bacterial genotoxicity tests were clearly positive with S-9 mix in several tests. 2,4-Toluenediamine (2,4-TDA) was positive with S-9 mix in Salmonella typhimurium strains TA 98, TA 100, TA 1537 and TA 1538 with respect to induction of bacterial gene mutations for doses from 20 µg/plate upward (JETOC, 1996; George and Westmoreland, 1991; Shahin et al., 1985; Haworth et al., 1983; Shahin et al., 1980; Green et al, 1979). All investigations showed strong and dose-dependent effects; no toxic effects were observed. These Salmonella typhimurium strains were negative without S-9 mix up to dose of 10000 µg/plate. 2,4-TDA was also positive in TA98 with S-9 mix in a microsuspension bioassay (George et al., 2001).

Cunningham and Matthews (1990) described the role of bacterial acetyltransferase on mutagenic effect of 2,4-TDA in bacterial mutation test with metabolic activation as shown by experiments with strain Salmonella typhimurium strain TA98/1,8-DNP6 (deficient in acetyltransferase), TA 98 (normal acetyltransferase) and the overproducer of acetyltransferase TA 98(pYG219). Compared with TA 98 the effect of strain TA98/1,8-DNP6 resulted in approximately 90% decrease in the mutagenic potency whereas the strain TA 98(pYG219) greatly enhanced the mutagenic effect. The authors concluded that after N-hydroxylation of 2,4-TDA by S-9 mix cytochrome P450 the resulting hydroxylamino intermediate is further activated by bacterial acetyltransferase to form the ultimate reactive intermediate, which is postulated to be 4-acetoxyamino-2-aminotoluene.

Negative results with and without S-9 mix were obtained in Salmonella typhimurium strain TA 1535 up to 10000 µg/plate (JETOC, 1996; Haworth et al., 1983) and in E. coli WP2uvrA up to 5000 µg/plate (JETOC, 1996)."

Table 1.   In vitro tests: bacterial genotoxicity

Test system	Concentration range		Result	Toxicity	Remarks	Reference
	with S-9 mix	without S-9 mix
Gene mutation, Salm. typh. TA 98, TA 100, TA 1535, TA 1537, E.coli WP2uvrA	0.07 63- 5000 µg/plate	0.07 63- 5000 µg/plate	positive	no toxic effects	positive only with S-9 mix	JETOC, 1996
Gene mutation, Salm. typh. TA 98	100 - 3333 µg/plate	not done	positive	no toxic effects	.	George and Westmoreland, 1991
Gene mutation, Salm. typh. TA 97, TA 1537, TA 1538	50 - 500 µg/plate	not done	positive	no toxic effects	.	Shahin et al., 1985
Gene mutation, Salm. typh. TA 98, TA 100, TA 1535, TA 1537	10 - 10'000 µg/plate	10 - 10'000 µg/plate	positive	no toxic effects	positive only with S-9 mix	Haworth et al., 1983
Gene mutation, Salm. typh. TA 98, TA 100, TA 1538	5.0 -1000 µg/plate	5.0 - 1000 µg/plate	positive	no toxic effects	positive only with S-9 mix	Shahin et al., 1980
Gene mutations Salm. typh. TA 98; TA 100, TA 1535, TA 1537, TA 1538	500 - 5000 µg/plate	500 - 5000 µg/plate	positive	no toxic effects	positive only with S-9 mix	Green et al., 1979
Gene mutation, Salm. typh. TA 98	100 - 1666 µg/plate	not done	positive	no data	.	Cunningham and Matthews, 1990
Gene mutation, Salm. typh. TA 98, TA 100 (microsuspension bioassay)	10-500 µg/plate	10-500 µg/plate	positive	no data	positive only with S-9 mix	George et al., 2001

These studies identified in the EU Risk Assessment are consistent with the results of the industry Key endpoint study.

Applicant's summary and conclusion

Conclusions:

Both 2,4-TDA and 2,6-TDA were capable of producing frameshift and basepair substitution mutations in S. typhimurium, after metabolic activation by induced rat liver S-9 fraction.