2,4,6-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

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Data is from peer reviewed publication

Data source

Reference

Reference Type:

publication

Title:

Salmonella Mutagenicity Tests: V. Results from the Testing of 311 Chemicals.

Author:

Errol Zeiger, Beth Anderson, Steve Haworth, Timothy Lawlor, and Kristien Mortelmans

Year:

1992

Bibliographic source:

Environmental and Molecular Mutagenesis Volume 19, Supplement 21: 2-141 (1992)

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: Similar to OECD 471

Principles of method if other than guideline:

Gene mutation toxicity study was performed to evaluate the mutagenic nature of the test chemical

GLP compliance:

not specified

Type of assay:

bacterial gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2,4,6-trichloroaniline

EC Number:

211-219-8

EC Name:

2,4,6-trichloroaniline

Cas Number:

634-93-5

Molecular formula:

C6H4Cl3N

IUPAC Name:

2,4,6-trichloroaniline

Details on test material:

- Name of test material: 2,4,6 trichloroaniline
- Molecular formula: C6H4Cl3N
- Molecular weight: 196.464 g/mol
- Substance type: organic

Method

Target gene:

Histidine

Species / strain

Species / strain / cell type:

S. typhimurium, other: TA 100, TA 1535, TA97 and TA98

Remarks:

Lab 1

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

S9 metabolic activation 10% and 30% hamster liver S9 and 10% and 30% rat liver S9

Test concentrations with justification for top dose:

Lab 1: 0, 1.0, 3.3, 10, 33, 67, 100, 200, 333, 667 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test chemical was soluble in DMSO

Controls

Controls 1

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: sodium azide

Remarks:

For strain TA1535 and TA100

Controls 2

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

For strain TA97

Controls 3

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylenediamine

Remarks:

For strain TA98

Controls 4

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene.

Remarks:

For metabolic activation with all strains

Details on test system and experimental conditions:

METHOD OF APPLICATION: Preincubation

DURATION
- Preincubation period: 20 mins
- Exposure duration: 2 days
- Expression time (cells in growth medium): 2 days

Rationale for test conditions:

No data

Evaluation criteria:

The plates were observed for a dose dependent increase in the number of Histidine- independent (his+) colonies.

Evaluations were made at both the individual trial and chemical levels.

Individual trials were judged mutagenic (+), weakly mutagenic (+ W), questionable (?), or nonmutagenic (-), depending on the magnitude of the increase in his+ revertants, and the shape of the dose response. A trial was considered questionable (?) if the dose-response was judged insufficiently high to support a call of “+ W”, if only a single dose was elevated over the control, or if a weak increase was not dose-related. The distinctions between a questionable response and a nonmutagenic or weakly mutagenic response, and between a weak mutagenic response and mutagenic response are highly subjective. It was not necessary for a response to reach two-fold over background for a trial to be judged mutagenic.

A chemical was judged mutagenic (+) or weakly mutagenic (+W) if it produced a reproducible, dose-related response over the solvent control, under a single metabolic activation condition, in replicate trials. A chemical was judged questionable (?) if the results of individual trials were not reproducible, if increases in his+ revertants did not meet the criteria for a “+W” response, or if only single doses produced increases in his+ revertants in repeat trials. Chemicals were judged nonmutagenic (-) if they did not meet the criteria for a mutagenic or questionable response.

Statistics:

Mean, SEM

Results and discussion

Test results

Species / strain:

S. typhimurium, other: TA 100, TA 1535, TA97 and TA98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: All chemicals were run initially in a toxicity assay to determine the appropriate dose range for the mutagenicity assay. The toxicity assay was performed using TA100. Toxic concentrations were defined as those that produced a decrease in the number of his+ colonies, or a clearing in the density of the background lawn, or both.

COMPARISON WITH HISTORICAL CONTROL DATA: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY: No data

Remarks on result:

other: No mutagenic potential

Any other information on results incl. tables

Table: Results for the test chemical

Dose (µg/plate)	TA100
	NA		10% HLI		30% HLI		10% RLI		30% RLI
	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM
0	147	7.5	102	3.5	150	8.3	121	4.5	176	6.6
1	107	5.2
3.3	120	7.8	101	6.1			115	1.5
10	115	3.4	110	3.2	147	13.7	108	9.6	178	4.9
33	115	1.5	115	3.5	141	9.4	110	3.2	162	7.4
67			119	6.3	148	15.4	115	3.7	166	5.0
100			63s	23.2			26s	22.9
200					129	5.9			6s	4.7
333					T				T
667
Positive control	444	19.3	279	10.1	658	122.3	1197	32.6	1262	30.1

 

Dose (µg/plate)	TA1535
	NA		10% HLI		30% HLI		10% RLI		30% RLI
	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM
41	8.4	23	0.7	15	3.9	25	3.5	17	0.6	6.6
1	42	4.4
3.3	41	1.9	18	1.2	12	0.99	25	2.0	10	0.6
10	42	1.5	22	1.5	14	1.2	25	1.9	14	0.7
33	36	2.1	25	0.7	17	0.9	24	2.9	14	1.5
67	23s	5.0
100			17	3.3	23	2.1	17	2.1	18	0.9
200			6s	1.0			7s	3.5
333					17s	1.7			4s	2.5
667
Positive control	277	14.0	65	6.9	249	1.8	271	7.7	266	7.8

 

Dose (µg/plate)	TA97
	NA		10% HLI		30% HLI		10% RLI		30% RLI
	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM
0	118	4.5	135	6.9	176	4.9	117	5.2	190	3.0
1	115	4.4
3.3	119	4.3	103	4.0	189	5.8	140	7.0	172	10.5
10	117	9.3	120	12.3	187	7.8	135	7.0	155	9.5
33	111	2.8	124	1.8	214	4.7	145	7.3	192	5.0
67	96s	5.5
100			153s	10.0	218	10.6	159s	5.6	199	18.4
200			91s	25.0			86s	1.0
333					146s	15.4			T
667
Positive control	387	72.7	1061	4.3	1341	17.0	1993	3.9	1232	71.6

 

Dose (µg/plate)	TA98
	NA		10% HLI		30% HLI		10% RLI		30% RLI
	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM
0	24	2.0	39	0.3	28	4.3	37	5.5	29	3.3
1	20	1.5
3.3	25	1.7	39	4.8			35	2.6
10	25	2.6	41	3.8	33	4.1	37	4.6	32	3.5
33	19	1.9	37	3.8	28	2.9	36	0.9	27	0.6
67	14s	1.8
100			37	3.7	32	1.5	26	2.8	34	3.8
200			18s	3.2			14s	10.5	6s	4.7
333					19s	9.5			6s	2.8
667					T				T
Positive control	309	19.1	230	7.5	138	12.3	201	11.2	335	9.3

 

T: Toxic

Applicant's summary and conclusion

Conclusions:

The test chemical did not induce gene mutation in Salmonella typhimurium TA98, TA100, TA1538 in the presence and absence of S9 metabolic activation system and hence is not likely to classify as a gene mutant in vitro.

Executive summary:

Gene mutation toxicity study was performed to determine the mutagenic nature of the test chemical. The study was performed using Salmonella typhimurium strains TA98, TA97, TA100 and TA1535 in the presence and absence of S9 metabolic activation system. The chemical was dissolved in DMSO as solvent and used at dose levels 0, 1.0, 3.3, 10, 33, 67, 100, 200, 333, 667 µg/plate by the preincubation method. The doses were selected on the basis of preliminary dose range finding study and concurrent solvent and positive controls were included in the study. The test chemical did not induce gene mutation in Salmonella typhimurium TA98, TA100, TA1538 in the presence and absence of S9 metabolic activation system and hence is not likely to classify as a gene mutant in vitro.