α,α,α,4-tetrachlorotoluene

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1986-11-5 to 1986-11-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1986

Report date:

1986

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

α,α,α,4-tetrachlorotoluene

EC Number:

226-009-1

EC Name:

α,α,α,4-tetrachlorotoluene

Cas Number:

5216-25-1

Molecular formula:

C7H4Cl4

IUPAC Name:

1-chloro-4-(trichloromethyl)benzene

Method

Target gene:

Histidine: Salmonella typhimurium strains
One of the genes for tryptophan biosynthesis: Escherichia coli strain

No more data

Species / strain

Species / strain 1

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: deficient in the complete structure of the lipopolysaccharide layer and in DNA excision repair system

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1538

Additional strain / cell type characteristics:

other: deficient in the complete structure of the lipopolysaccharide layer and in DNA excision repair system

Species / strain 3

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

other: deficient in the uvrA system of DNA repair

Metabolic activation:

with and without

Metabolic activation system:

rat liver homogenate (S-9 mix)

Test concentrations with justification for top dose:

Dose range: 0.16 to 500 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol

No further data available

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

executed for strain S. typhimurium TA 100 without metabolic activation

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA 100, TA 1535; without metabolic activation

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

TA 1537; without metabolic activation

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

TA 98, TA 1538; without metabolic activation

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)

Remarks:

WP2uvrA; without metabolic activation

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

executed for strain S. typhimurium TA 100 with metabolic activation

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

TA 98, TA 100, TA 1535, TA 1537, TA 1538, WP2uvrA; with metabolic activation

Controls 6

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

executed for strain S. typhimurium TA 100 with metabolic activation

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

TA 98, TA 100, TA 1535, TA 1537, TA 1538, WP2uvrA; with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 to 72h

NUMBER OF REPLICATIONS: 3

No further data available

Evaluation criteria:

Amino-acid requiring strains of bacteria are used to detect reverse gene mutations. Based on the number of revertants one can assess the mutagenicity of the test compound.

Statistics:

No data

Results and discussion

Test results

Test results 1

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

see table 1 and 2 in overall remarks sections

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

thinning of bacterial lawn and reduction in number of colonies at 20 or 100 µg/plate depending on strain

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

see table 1 and 2 in overall remarks sections

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

thinning of bacterial lawn and reduction in number of colonies at 100 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

see table 1 and 2 in overall remarks sections

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

thinning of bacterial lawn and reduction in number of colonies at 100 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: visible precipitation of the test compound on the plates at 2500 and 10000 µg/plate for all strains with and without metabolic activiation


RANGE-FINDING/SCREENING STUDIES:
An experiment was performed with all tester strains (3 replicates per dose) to obtain information on mutagenicity and toxicity for calculation of an appropriate dose range. As indicators for toxicity a reduced rate of spontaneously occuring colonies as well as visible thinning of the bacterial lawn (controlled microscopically) were used.
Based on this test 500 µg/plate was chosen as highest dose for mutagenicity testing.


No further data available

Remarks on result:

other: all strains/cell types tested

Any other information on results incl. tables

Table 1: Result for the mutagenicity experiment with 4 -chloro-benzotrichloride without metabolic activation for all tested strains, including controls. The values presented are the mean value obtained from the three replicates. (ibl: incomplete bacterial lawn; nbl: no bacterial lawn)

		S. typhimurium	S. typhimurium	S. typhimurium	S. typhimurium	S. typhimurium	E. coli
		TA-100	TA-1535	TA-1537	TA-1538	TA-98	WP2uvrA
Positive control*
Test compound	0 µg/plate	168	19	8	17	26	46
	0.16 µg/plate			7		28
	0.8 µg/plate	175	16	10	15	29	50
	4 µg/plate	216	22	10	17	37	58
	20 µg/plate	318 (ibl)	20	17	16	67	63
	60 µg/plate	113 (ibl)	15 (ibl)	22 (ibl)	15 (ibl)	36 (ibl)	67
	100 µg/plate	85 (nbl)	2 (ibl)	10 (ibl)	7 (ibl)	20 (ibl)	22 (ibl)
	500 µg/plate	nbl	nbl	nbl	nbl	nbl	ibl

*	TA-100	Sodium-azide	1 µg/plate	602
	TA-1535	Sodium-azide	1 µg/plate	510
	TA-1537	9-aminoacridine	50 µg/plate	242
	TA-1538	2-nitrofluorene	2.5 µg/plate	603
	TA-98	2-nitrofluorene	2.5 µg/plate	366
	WP2uvrA	MNNG	2.5 µg/plate	330
		p-chlorbenzotrichlorid TTR	0.8 µg/plate	0

Table 2: Result for the mutagenicity experiment with 4 -chloro-benzotrichloride with metabolic activation for all tested strains, including controls. The values presented are the mean value obtained from the three replicates. (ibl: incomplete bacterial lawn; nbl: no bacterial lawn)

Metabolic activation (rat liver)		S. typhimurium	S. typhimurium	S. typhimurium	S. typhimurium	S. typhimurium	E. coli
		TA-100	TA-1535	TA-1537	TA-1538	TA-98	WP2uvrA
Positive control**
Test compound	0 µg/plate	182	23	10	20	34	54
	0.16 µg/plate			6		43
	0.8 µg/plate	216	23	9	18	39	54
	4 µg/plate	274	26	13	19	43	55
	20 µg/plate	392 (ibl)	23	24	23	75	65
	60 µg/plate	173 (ibl)	13 (ibl)	14 (ibl)	18 (ibl)	66 (ibl)	71
	100 µg/plate	97 (ibl)	13 (ibl)	9 (ibl)	11 (ibl)	25 (ibl)	34 (ibl)
	500 µg/plate	nbl	nbl	nbl	nbl	20 (nbl)	nbl

**	TA-100	2-aminoanthracen	0.5 µg/plate	718
	TA-100	Benzo(a)pyrene	10 µg/plate	619
	TA-1535	2-aminoanthracen	1 µg/plate	160
	TA-1535	Benzo(a)pyrene	10 µg/plate	25
	TA-1537	2-aminoanthracen	1 µg/plate	74
	TA-1537	Benzo(a)pyrene	10 µg/plate	112
	TA-1538	2-aminoanthracen	0.5 µg/plate	470
	TA-1538	Benzo(a)pyrene	10 µg/plate	162
	TA-98	2-aminoanthracen	0.5 µg/plate	444
	TA-98	Benzo(a)pyrene	10 µg/plate	652
	WP2uvrA	2-aminoanthracen	10 µg/plate	292
	WP2uvrA	Benzo(a)pyrene	10 µg/plate	88
		S-9 mix	500 µl	0
		p-chlorbenzotrichlorid TTR	0.8 µg/plate	0

Table 3: Surviving fraction (i.e. ratio of number of colonies from solvent control plates and from plates with test compound) obtained using the solvent control with the Salmonella strain TA 100

		Surviving fraction
		Without metabolic activation	With metabolic activation
Test compound	0 µg/plate	1.0	1.0
	0.8 µg/plate	1.0	1.0
	4 µg/plate	1.0	1.0
	20 µg/plate	0.9	1.0
	60 µg/plate	0.00	0.8
	100 µg/plate	0.00	0.7
	500 µg/plate	0.00	0.00

Applicant's summary and conclusion

Conclusions:

Mutagenic

Executive summary:

The authors tested the mutagenicity of 4-chloro-benzotrichloride (CAS n° 5216 -25 -1) in the absence and presence of a metabolizing system with a methodology similar to the OECD guideline 471. The Salmonella typhimurium strains TA 100, TA 1535, TA 1537, TA 1538 and TA 98 and the Escherichia coli strain WP2uvrA were used and the metabolizing system was obtained from rat liver homogenates. The number of revertants per plate was estimated and was a basis for comparison with solvent controls.

Based on the range-finding/screening test, 500 µg/plate was chosen as top dose level for the mutagenicity test and the test compound proved to be toxic to most of the bacterial strains at 20 or 100 µg/plate according to observed diminuation or even complete absence of the bacterial lawn.

In the mutagenicity test, seven different doses ranging from 0.16 to 500 µg/plate were used (3 replicates per dose) and the strains were exposed for 48 to 72 h. Control plates without mutagen showed the described number of spontaneous revertant colonies reported in literature and all the positive control compounds gave the expected increase in the number of revertant colonies.

Furthermore the mutagenicity test showed a dose dependant increase in the number of revertant colonies with the S. typhimurium strain TA 98 in absence of the metabolic activation system (S-9 mix) after exposure to the test compound. In the presence of the metabolic activation system, treatment with the test substance resulted in a relevant increase in the number of revertant colonies in the S. typhimurium strain TA 98. Also slightly increased numbers of revertant colonies were obtained in the absence and presence of the metabolic activation system for S. typhimurium strains TA 100 and TA 1537. Considering the overall data, thus, 4 -chloro-benzotrichloride can be considered mutagenic with and without metabolic activation system for the tested strains.