2,4,5-trichloroaniline

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

Study period:

1988-11-23 to 1989-02-28

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study, 1 strain missing

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1989

Report date:

1989

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Not mentioned; but the study complies with OECD guideline 471 (adopted 26 May 1983).

Deviations:

yes

Remarks:

One bacterial strain is missing. According to the current OECD guideline 471 (adopted 21 July 1997) at least 5 tester strains are required, whereas before 4 tester strains were sufficient.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2,4,5-trichloroaniline

EC Number:

211-254-9

EC Name:

2,4,5-trichloroaniline

Cas Number:

636-30-6

Molecular formula:

C6H4Cl3N

IUPAC Name:

2,4,5-trichloroaniline

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Metabolic activation system:

S9 mix prepared from Aroclor 1254 induced livers of male Sprague Dawlwy rats

Test concentrations with justification for top dose:

Experiment I (plate incorporation): 0, 20, 100, 500, 2500, 12500 µg/plate both with and without metabolic activation
Experiment II (plate incorporation): 0, 40, 80, 160, 320, 640 µg/plate both with and without metabolic activation
Experiment III (plate incorporation): 0, 20, 40, 80, 160, 320 µg/plate both with and without metabolic activation

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Details on test system and experimental conditions:

DURATION
- Exposure duration: 48 h

Evaluation criteria:

A reproducible and dose-related increase in mutant counts for at least one strain is considered positive. For TA 1535, TA 100 and TA 98 in principle a twofold increase compared to the corresponding negative controls should be reached, whereas for TA 1537 at least a threefold increase should be reached. Otherwise, the result is evaluated as negative.

Statistics:

not mandatory

Results and discussion

Test results

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

occurred > 40µg/plate, at 12500µg/plate; the substance precipitated

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: at 12500µg/plate

RANGE-FINDING STUDIES: Experiment I. Only two doses could be used for assessment

COMPARISON WITH HISTORICAL CONTROL DATA: Yes

ADDITIONAL INFORMATION ON CYTOTOXICITY: see below

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

The assay was performed in three independent experiments all with and without liver microsomal activation. Each concentration, including the controls, was tested in quadruplicate. The plates incubated with the test item showed normal background growth up to 40 µg /plate. At higher doses the substance had a strong strain specific bacteriotoxic effect, so that this range could only be used to limited extend up to 500µg/plate for evaluation purposes.

Substance precipitation occurred at the dose 12500µg/plate.12500 µg/plate is clearly exceeding the current recommendations for the maximum test dose of 5000 µg/plate. However, evidence of mutagenic activity of 2,4,5 -trichloroaniline was not found. No biologically relevant increase in the mutant count, in comparison with the negative controls, was observed. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

In the first trial only two doses could be used for assessment. Therefore, this test was used only as range finding test.

An increase in mutant counts of strain TA98 in the third trial revealed to double those of the negative controls (see table below). This increase did however not correlate with dose, and is therefore to be regarded as a random result due to the low spontaneous rate of the strain.It must also be considered that the negative control with S9 mix was higher than the range of values for treatment goups without S9 mix, and therefore the increase without S9 mix can be seen as biologically insignificant.

Table: Summary of mean number of revertants (mean of 4 plates) in Salmonella typhimurium strains with and without metabolic activation

Experiment I (range finding test)
Concentration	TA1535		TA100		TA1537		TA98
[µg/plate]	- S9 mix	+ S9 mix^1	- S9 mix	+ S9 mix^1	- S9 mix	+ S9 mix^1	- S9 mix	+ S9 mix^1
Control	11	13	84	113	6	9	17	56
20	8	19	84	121	8	11	21	63
100	9	20	81	109	5	12	25	52
500	b	7	b	b	5	8	10	37
2500	0	0	0	0	0	0	0	0
12500	p	p	p	p	p	p	p	p
Experiment II
	TA1535		TA100		TA1537		TA98
	- S9 mix	+ S9 mix^1	- S9 mix	+ S9 mix^1	- S9 mix	+ S9 mix^1	- S9 mix	+ S9 mix^1
Control	16	23	91	180	8	9	20	38
40	16	21	81	153	9	9	14	40
80	18	18	82	153	8	11	16	41
160	18	17	77	144	7	13	14	45
320	16	11	42	121	9	19	8	31
640	b	b	b	b	b	b	b	b
Experiment III
	TA1535		TA100		TA1537		TA98
	- S9 mix	+ S9 mix^1	- S9 mix	+ S9 mix^1	- S9 mix	+ S9 mix^1	- S9 mix	+ S9 mix^1
Control	13	17	81	139	9	9	7	35
20	14	11	88	152	7	12	16	33
40	10	14	85	140	6	9	17	34
80	15	8	82	156	5	9	15	36
160	11	20	73	137	8	7	11	35
320	10	14	37	115	3	6	9	30

¹ S9 mix content of 30%, b= background; p= precipitation

Applicant's summary and conclusion

Conclusions:

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations in the genomes of the strains used.

Executive summary:

This study was performed to investigate the potential of 2,4,5-Trichloroaniline to induce gene mutations in the plate incorporation test (experiment I, II and III, respectively) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100. The assay was performed in three independent experiments all with and without liver microsomal activation. Each concentration, including the controls, was tested in quadruplicate. The test item was tested at the following concentrations: 20, 100, 500, 2500 and 12500 µg/plate in experiment I, 40, 80, 160, 320 and 640 µg/plate in experiment II and 20, 40, 80, 160 and 320µg/plate in experiment III. Substance precipitation occurred at the dose 12500µg/plate. No biologically relevant increase in the mutant count, in comparison with the negative controls, was observed. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations in the genomes of the strains used.