2,4,6-trichloroaniline

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In Vitro: Ames: Negative, OECD 471, Sokolowski 2014 Ames: Negative, OECD 471, Zeiger 1992 Chromosome Aberration: Negative, OECD 473, JETOC 1997

Link to relevant study records

Reference

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:

2013-12-13 to 2014-06-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: the study was conducted under GLP and according to OECD 471 (1997), EPA OPPTS 870.5100 (1998) and EC 440/2008 B.13/14 (2008) without deviations.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

The S. typhimurium histidine (his) and the E. coli tryptophan (trp) reversion system measures his-/his+ and trp-/trp+ reversions, respectively. The S. typhimurium and Escherichia coli strains are constructed to differentiate between base pair (TA1535, TA100, WP2 uvrA pKM101, and WP2 pKM101) and frameshift (TA1537, TA98) mutations.

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Additional strain / cell type characteristics:

other: tryptophan biosynthesis, DNA repair process

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/ß-naphthoflavone induced rat liver S9 was used as the metabolic activation system. The S9 was prepared from 8 – 12 w old male Wistar rats (Hsd Cpb: WU; weight approx. 220 – 320 g, Harlan Laboratories B. V., 5960 AD Horst, The Netherlands).

Test concentrations with justification for top dose:

In the pre-experiment the concentration range of the test substance was 3 - 5000 µg/plate. The pre-experiment is reported as experiment I as at least five concentrations were analysable. Since toxic effects were solely observed at high concentrations, 5000 µg/plate was also chosen as maximal concentration of the second experiment.
The concentration range included two logarithmic decades. The following concentrations were tested in experiment II:
3, 10, 33, 100, 333, 1000, 2500, and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO (purity 99 %)
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubilisation properties and its relative non-toxicity to the bacteria

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine (4-NOPD), 2-aminoanthracene (2-AA)

Details on test system and experimental conditions:

METHOD OF APPLICATION: direct plate incorporation and pre-incubation method

DURATION
- Preincubation period: 60 minutes
For the pre-incubation method 100 µL test solution (solvent or reference mutagen solution (positive control)), 500 µL S9 mix / S9 mix substitution buffer (7 parts of the 100 mM sodium-ortho-phosphate-buffer pH 7.4 with 3 parts of KCl solution 0.15 M) and 100 µL bacteria suspension were mixed in a test tube and incubated at 37 °C for 60 minutes. After pre-incubation 2.0 mL overlay agar (45 °C) was added to each tube. The mixture was poured on selective agar plates. After solidification the plates were incubated upside down for 72 hours at 37°C in the dark, plates were then stored at 4°C until counted.
- Exposure duration: 60 minutes (pre-exposure) + 72 hours (exposure)
- Expression time (cells in growth medium): in the pre-culture 10^8 to 10^9 cells/mL
- Selection time (if incubation with a selection agent): not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): not applicable

SELECTION AGENT (mutation assays): not applicable
SPINDLE INHIBITOR (cytogenetic assays): not applicable
STAIN (for cytogenetic assays): not applicable

NUMBER OF REPLICATIONS: 3

NUMBER OF CELLS EVALUATED: number of revertant colonies

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

OTHER EXAMINATIONS:
- Determination of polyploidy: not applicable
- Determination of endoreplication: not applicable

Evaluation criteria:

Acceptability of the assay
The Salmonella typhimurium and Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
- regular background growth in the negative and solvent control
- the spontaneous reversion rates in the negative and solvent control are in the range of the historical data
- the positive control substances should produce a significant increase in mutant colony frequencies
- a minimum of five analysable concentrations should be present with at least four showing no signs of toxic effects, evident as a reduction in the number of revertants below an induction factor of 0.5.

Evaluation of results
A test substance is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice the colony count of the corresponding solvent control is observed.
A concentration-dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A concentration-dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no
- Effects of osmolality: no
- Evaporation from medium: no
- Water solubility: DMSO was used as solvent.
- Precipitation: The test substance precipitated in the overlay agar in the test tubes from 1000 to 5000 µg/plate with and without S9 mix in both experiments. Precipitation of the test substance in the overlay agar was also observed on the incubated agar plates from 1000 to 5000 µg/plate in experiment I and from 333 to 5000 µg/plate in experiment II. The undissolved particles had no influence on the data recording.
- Other confounding effects: no

RANGE-FINDING/SCREENING STUDIES:
To evaluate the cytotoxicity of the test substance a pre-experiment was performed with all strains. Eight concentrations were tested for cytotoxicity and mutation induction each with three replicate plates. The experimental conditions in this pre-experiment were the same as described below for experiment I (plate incorporation test).
Cytotoxicity of the test substance results in a reduction in the number of spontaneous revertants (below a factor of 0.5) or a clearing of the bacterial background lawn.
The pre-experiment is reported as the main experiment I since the criteria mentioned under Acceptability of the assay were met.

COMPARISON WITH HISTORICAL CONTROL DATA:
The laboratory historical control range was not quite reached in the untreated control of strain WP2 uvrA pKM101 with S9 mix in experiment I. Since this deviation is rather small, this effect is judged to be based upon statistical fluctuations and has no detrimental impact on the outcome of the study.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Reduced background growth was observed at the following concentrations (µg/plate):
TA 1535: Experiment I (without S9 mix): 5000 --- Experiment I (with S9 mix): 2500, 5000 --- Experiment II (without S9 mix): 333 - 5000 --- Experiment II (with S9 mix): 333 - 5000
TA 1537: Experiment I (without S9 mix): 1000 - 5000 --- Experiment I (with S9 mix): 1000 - 5000 --- Experiment II (without S9 mix): 333 - 5000 --- Experiment II (with S9 mix): 333 - 5000
TA 98: Experiment I (without S9 mix): 1000 - 5000 --- Experiment I (with S9 mix): 2500, 5000 --- Experiment II (without S9 mix): 333 - 5000 --- Experiment II (with S9 mix): 333 - 5000
TA 100: : Experiment I (without S9 mix): 1000 - 5000 --- Experiment I (with S9 mix): 1000 - 5000 --- Experiment II (without S9 mix): 333 - 5000 --- Experiment II (with S9 mix): 333 - 5000
WP2 pKM101: : Experiment I (without S9 mix): 1000 - 5000 --- Experiment I (with S9 mix): 2500, 5000 --- Experiment II (without S9 mix): 333 - 5000 --- Experiment II (with S9 mix): 333 - 5000
WP2 uvrA pKM101: : Experiment I (without S9 mix): 2500, 5000 --- Experiment I (with S9 mix): 2500, 5000 --- Experiment II (without S9 mix): 333 - 5000 --- Experiment II (with S9 mix): 333 - 5000

Toxic effects, evident as a reduction in the number of revertants (below an induction factor of 0.5), were observed at the following concentrations (µg/plate):
TA 1535: Experiment I (without S9 mix): / --- Experiment I (with S9 mix): 2500, 5000 --- Experiment II (without S9 mix): 5000 --- Experiment I (with S9 mix): 2500, 5000
TA 1537: Experiment I (without S9 mix): 5000 --- Experiment I (with S9 mix): 1000 - 5000 --- Experiment II (without S9 mix): 1000 - 5000 --- Experiment I (with S9 mix): 333 - 5000
TA 98: Experiment I (without S9 mix): 5000 --- Experiment I (with S9 mix): 2500, 5000 --- Experiment II (without S9 mix): 1000 - 5000 --- Experiment I (with S9 mix): 333 - 5000
TA 100: Experiment I (without S9 mix): 1000 - 5000 --- Experiment I (with S9 mix): 1000 - 5000 --- Experiment II (without S9 mix): 1000 - 5000 --- Experiment I (with S9 mix): 333 - 5000
WP2 pKM101: Experiment I (without S9 mix): 1000 - 5000 --- Experiment I (with S9 mix): 2500, 5000 --- Experiment II (without S9 mix): 333 - 5000 --- Experiment I (with S9 mix): 333 - 5000
WP2 uvrA pKM101: Experiment I (without S9 mix): 2500, 5000 --- Experiment I (with S9 mix): 2500, 5000 --- Experiment II (without S9 mix): 1000 - 5000 --- Experiment I (with S9 mix): 333 - 5000
/ = no toxic effects observed

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 1 Summary of Results Pre-Experiment/Experiment I

Metabolic Activation	Test group	Concentration (per plate)	Revertant Colony Counts (Mean ±SD)
			TA 1535	TA 1537	TA 98	TA 100	WP2 pKM101	WP2 uvrA pKM101
Without Activation	DMSO	---	16 ± 1	14 ± 6	22 ± 4	91 ± 3	228 ± 24	327 ± 30
	Untreated	---	11 ± 2	13 ± 2	26 ± 6	100 ± 15	249 ± 12	373 ± 1
	test item	3 µg	13 ± 4	9 ± 1	20 ± 3	91 ± 9	231 ± 23	331 ± 29
		10 µg	16 ± 3	12 ± 2	23 ± 7	85 ± 10	209 ± 16	334 ± 46
		33 µg	18 ± 1	10 ± 2	19 ± 7	91 ± 5	190 ± 18	294 ± 37
		100 µg	19 ± 5	14 ± 1	25 ± 1	72 ± 5	224 ± 24	312 ± 42
		333 µg	14 ± 3	12 ± 6	23 ± 3	54 ± 17	174 ± 26	311 ± 19
		1000 µg	20 ± 3^P	12 ± 4^P R	16 ± 1^P R	31 ± 6^P M R	61 ± 4^P R	205 ± 41^P
		2500 µg	14 ± 3^P M	6 ± 3^P R M	11 ± 3^P M R	24 ± 1^P M R	33 ± 4^P M R	93 ± 9^P M R
		5000 µg	9 ± 2^P M R	5 ± 2^P R M	4 ± 1^P M R	16 ± 4^P M R	26 ± 3^P M R	58 ± 12^P M R
	NaN3	10 µg	2919 ± 285	---	---	2303 ± 99	---	---
	4-NOPD	10 µg	---	---	388 ± 12	---	---	---
	4-NOPD	50 µg	---	58 ± 5	---	---	---	---
	MMS	2.0 µL	---	---	---	---	3663 ± 93	4303 ± 35
With Activation	DMSO	---	15 ± 3	22 ± 6	32 ± 3	100 ± 8	217 ± 2	366 ± 5
	Untreated	---	16 ± 2	20 ± 6	35 ± 2	116 ± 16	269 ± 35	373 ± 43
	test item	3 µg	16 ± 4	25 ± 4	33 ± 2	101 ± 14	243 ± 19	349 ± 14
		10 µg	13 ± 1	17 ± 2	34 ± 7	105 ± 6	192 ± 11	316 ± 28
		33 µg	15 ± 6	17 ± 3	35 ± 8	114 ± 12	201 ± 15	295 ± 21
		100 µg	17 ± 1	14 ± 7	34 ± 4	103 ± 8	203 ± 58	304 ± 8
		333 µg	14 ± 1	20 ± 5	30 ± 7	83 ± 16	192 ± 19	310 ± 58
		1000 µg	11 ± 1^P	9 ± 2^P M R	23 ± 6^P	42 ± 6^P M R	104 ± 17^P	212 ± 21^P
		2500 µg	3 ± 1^P M R	5 ± 2^P M R	8 ± 2^P M R	16 ± 3^P M R	24 ± 3^P M R	98 ± 16^P M R
		5000 µg	2 ± 1^P M R	1 ± 1^P M R	1 ± 1^P M R	0 ± 1^P M R	4 ± 2^P M R	12 ± 2^P M R
	2-AA	2.5 µg	498 ± 40	349 ± 36	1852 ± 77	2408 ± 268	---	---
	2-AA	10.0 µg	---	---	---	---	3552 ± 129	1822 ± 155

NaN3. sodium azide

2-AA: 2-aminoanthracene

4-NOPD: 4-nitro-o-phenylene-diamine

MMS: methyl methane sulfonate

P: Precipitate

M: Manual count

R: Reduced background growth

Table 2 Summary of Results Experiment II

Metabolic Activation	Test group	Concentration (per plate)	Revertant Colony Counts (Mean ±SD)
			TA 1535	TA 1537	TA 98	TA 100	WP2 pKM101	WP2 uvrA pKM101
Without Activation	DMSO	---	16 ± 5	9 ± 2	27 ± 4	96 ± 17	243 ± 2	407 ± 11
	Untreated	---	17 ± 4	10 ± 5	32 ± 1	105 ± 7	276 ± 27	419 ± 6
	test item	3 µg	15 ± 1	9 ± 3	19 ± 5	93 ± 16	247 ± 12	412 ± 23
		10 µg	13 ± 6	8 ± 1	17 ± 2	83 ± 11	232 ± 17	413 ± 10
		33 µg	14 ± 2	11 ± 1	16 ± 3	91 ± 10	235 ± 11	460 ± 10
		100 µg	19 ± 5	6 ± 2	21 ± 2	72 ± 14	112 ± 31	259 ± 15
		333 µg	16 ± 3^P R	6 ± 1^P R	17 ± 4^P R	59 ± 10^P R	68 ± 7^P R	207 ± 16^P R
		1000 µg	14 ± 5^P M R	3 ± 1^P M R	9 ± 2^P M R	13 ± 5^P M R	32 ± 7^P M R	112 ± 6^P M R
		2500 µg	8 ± 3^P M R	3 ± 2^P M R	2 ± 1^P M R	21 ± 1^P M R	15 ± 4^P M R	47 ± 5^P M R
		5000 µg	3 ± 2^P M R	0 ± 0^P M R	0 ± 0^P M R	0 ± 0^P M R	8 ± 4^P M R	17 ± 4^P M R
	NaN3	10 µg	2706 ± 139	---	---	2241 ± 83	---	---
	4-NOPD	10 µg	---	---	341 ± 25	---	---	---
	4-NOPD	50 µg	---	75 ± 16	---	---	---	---
	MMS	2.0 µL	---	---	---	---	3107 ± 175	2908 ± 14
With Activation	DMSO	---	11 ± 4	15 ± 1	36 ± 2	104 ± 12	266 ± 23	453 ± 3
	Untreated	---	10 ± 3	17 ± 4	31 ± 9	131 ± 5	314 ± 22	449 ± 1
	test item	3 µg	7 ± 4	19 ± 7	33 ± 4	106 ± 13	262 ± 5	431 ± 49
		10 µg	12 ± 2	11 ± 3	29 ± 5	115 ± 1	252 ± 5	444 ± 7
		33 µg	14 ± 2	10 ± 4	26 ± 3	95 ± 18	267 ± 21	486 ± 6
		100 µg	15 ± 6	13 ± 5	37 ± 8	105 ± 3	256 ± 19	424 ± 28
		333 µg	8 ± 5^P M R	3 ± 5^P M R	16 ± 5^P M R	23 ± 6^P M R	32 ± 9^P M R	78 ± 9^P M R
		1000 µg	8 ± 4^P M R	0 ± 1^P M R	4 ± 1^P M R	3 ± 2^P M R	25 ± 6^P M R	50 ± 11^P M R
		2500 µg	2 ± 1^P M R	4 ± 1^P M R	0 ± 0^P M R	0 ± 0^P M R	2 ± 1^P M R	21 ± 2^P M R
		5000 µg	1 ± 1^P M R	0 ± 0^P M R	0 ± 0^P M R	0 ± 0^P M R	0 ± 1^P M R	0 ± 0^P M R
	2-AA	2.5 µg	408 ± 5	327 ± 12	2406 ± 113	2974 ± 707	---	---
	2-AA	10.0 µg	---	---	---	---	2072 ± 44	2205 ± 126

NaN3. sodium azide

2-AA: 2-aminoanthracene

4-NOPD: 4-nitro-o-phenylene-diamine

MMS: methyl methane sulfonate

P: Precipitate

R: reduced background growth

M: Manual count

Conclusions:

Interpretation of results (migrated information):
negative

During the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, the substance is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

This study was performed to investigate the potential of the test item to induce gene mutations in the plate incorporation test and the pre-incubation test using the Salmonella typhimurium strains and the Escherichia coli strains. The plates incubated with the test substance showed reduced background growth at higher concentrations with all strains used. Cytotoxic effects were observed in all strains at higher concentrations with the exception of strain TA 1535 in the first experiment without metabolic activation. No increase in revertant colony numbers of any of the six tester strains was observed. There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies. The test item is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

In vivo data:

Kugler (1989) carried out a somatic mutation and recombination test in Drosophila melanogaster: Larvae (72 h-old) transheterozygous for the mutations multiple wing hair (mwh, 3-0.0) and flare (flr, 3-38.8) were fed the test substance at a concentration of 5 nM for 48 h. Spontaneous mutation frequency was determined from solvent controls. Induced spots on the wings were scored and subject to statistical analysis. The results indicated in the somatic mutation and recombination test (SMART) in drosophila is not considered relevant for the purpose of risk assessment as it is a non-guideline assay in a non-mammalian species. The study was diregarded.

In vitro data:

From the available data, three studies were not conducted following recent guidelines.

Shimizu (1984) conducted an Ames test to assess the mutagenicity of nineteen aniline derivatives were tested and the co-mutagenic effect of norharman. The study produced the only positive result for mutagenicity in vitro from the available dataset. Given that the method description is lacking details such as the concentrations used and the strains tested, the result is deemed unreliable.

Ono (1992) performed an Umu Chromotest and Yoshimi (1988) carried out an in vitro UDS. Both studies produced a negative result for mutagenicity which is in good agreement with OECD guideline studies.

JETOC (1997) carried out a Chromosome Aberration test with Chinese hamster lung fibroblasts. The exposure period was of 24 - 48 hours. The study included two sets of cells, one set was cultivated without metabolic activation and the other set with S9 mix. Negative control (solvent) and positive control were reported valid. The type of aberrations were classified into six groups, none of which came back positive. Under the experimental conditions investigated, the test item did not induce gene mutations.

Zeiger (1992) performed a study according to OECD guidelines 471 where S. typhimurium strains TA100, TA1535, TA97, TA98 were exposed to the test item in agar for two days. The tests were conducted silmutaneously in two different laboratories at nine to eleven concentrations ranging from 1.000 to 3333.000 µg/Plate, determined by a preliminary range-finding study. Experiments were run in triplicates. A negative and positive controls were reported. The test item was found to be non-mutagenic in the Ames test.

Sokolowski (2014) conducted an Ames test under GLP using the S. typhimurium strains and the E. coli strains. Cytotoxic effects were observed in all strains at higher concentrations with the exception of strain TA 1535 in the first experiment without metabolic activation. No increase in revertant colony numbers of any of the six tester strains was observed. There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies. The test item is considered to be non-mutagenic in this S. typhimurium and E. coli reverse mutation assay. The result is in good agreement with the rest of the database.

The study is considered complete, reliable and adequate. The result is carried forward for the purposes of risk assessment and classification.

Justification for selection of genetic toxicity endpoint
Study carried out according to OECD guideline and under GLP.

Justification for classification or non-classification

The results indicated in the somatic mutation and recombination test (SMART) conducted by Kugler (1989) in drosophila is not considered relevant for classification for the following reasons: The classification of mammalian germ cell mutagenicity is based on in vivo mammalian somatic  cell data, or other in vivo data supported by in vitro mutagenicity assays. In this instance there is one result in a non-guideline assay in a non-mammalian species. This finding is not supported by other in vivo mammalian somatic cell data or in vitro data in for example mammalian cell lines. Hence, the SMART assay is not considered sufficient or relevant for classification of the test item.

A bacterial reverse mutation test was conducted following OECD guideline 471 and in compliance with GLP Directive 2004/10/EC. The result is valid, reliable and adequate for the purpose of risk assessment, classification and labelling.

The substance does not meet the criteria for classification as a germ cell mutagen according to Regulation (EC) 1272/2008, 3.5.2.