α,α,α-trichlorotoluene

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: other: Salmonella and Escherichia strains: gene mutation; Bacillus strains: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The authors tested the mutagenic activity of benzotrichloride with a bacterial reverse mutation test by following a methodology similar to the OECD guideline 471 and with a recombination assay performed according to Kada, Tutikawa and Sadaie (1972). GLP status of the study is not specified. Minor deviations to the guideline observed. Hence, this study should be considered reliable with restrictions, a Klimisch 2.c study, comparable to a guideline study with acceptable restrictions.

Data source

Reference

Reference Type:

publication

Title:

Mutagenicity of benzotrichloride and related compounds

Author:

Yasuo K., Fujimoto S., Katoh M., Kikuchi Y. and Kada T.

Year:

1978

Bibliographic source:

Mutation Research, 58, 143-150

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

for the Salmonella and Escherichia strains

Deviations:

yes

Remarks:

no positive control, number of concentrations unclear and cytotoxicity not checked

Principles of method if other than guideline:

Recombination assay performed with Bacillus strains was performed according to Kada T., Tutikawa K. and Sadaie Y. (1972) (Mutation Research 16: 165-174)

GLP compliance:

not specified

Type of assay:

other: Salmonella and Escherichia strains: bacterial reverse mutation assay (e.g. Ames test) ; Bacillus strains: recombination assay

Test material

Test material information

Constituent 1

Reference substance name:

α,α,α-trichlorotoluene

EC Number:

202-634-5

EC Name:

α,α,α-trichlorotoluene

Cas Number:

98-07-7

Molecular formula:

C7H5Cl3

IUPAC Name:

α,α,α-trichlorotoluene

Constituent 2

Reference substance name:

trichloromethylbenzene

IUPAC Name:

trichloromethylbenzene

Details on test material:

- Name of test material (as cited in study report): BTC, benzotrichloride
- Supplier: Tokyo Kasei Co. Ltd

No more data available

Method

Target gene:

No data reported but histidine gene is the common target gene of the Salmonella test.

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 98

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 100

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 1535

Species / strain 4

Species / strain / cell type:

E. coli WP2

Additional strain / cell type characteristics:

other: one strain try hcr, another B/r try

Species / strain 5

Species / strain / cell type:

other: Bacillus subtilis M45 (rec -) and H17 (rec +)

Metabolic activation:

with and without

Metabolic activation system:

Rat liver microsome fraction S-9 prepared as indicated by Ames et al.

Test concentrations with justification for top dose:

- For Salmonella TA 98 and TA1535: 0.5 and 1.0 µmoles/plates
- For Salmonella TA 100 strain: 1.0 and 2.0 µmoles/plates
- For Escherichia WP2 try hcr strain: 0.25, 0.51, 1.02, 1.53, 2.05 and 2.56 µmoles/plate
- For Escherichia WP2 B/r try strain: 0.51, 1.02, 2.05, 2.56, 3.07 and 4.09 µmoles/plate
- For the Bacillus strains: 1.5, 2.6 and 3.6 µmoles/disk

Vehicle / solvent:

- Solvent used: DMSO

No more data available

Controls

Untreated negative controls:

other: see overall remarks

Negative solvent / vehicle controls:

other: see overall remarks

True negative controls:

not specified

Positive controls:

no

Details on test system and experimental conditions:

Bacterial reverse mutation assay (Salmonella and Escherichia strain):

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Expression time (cells in medium): 20 minutes
- Selection time (if incubation with a selection agent): 48 hours



Recombination assay (Bacillus strains):

DURATION
- Expression time (cells in growth medium): 18 hours, then difference in inhibition zone lenghts was measured

No further data

Evaluation criteria:

No data reported

Statistics:

No data

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

see table 1 in remarks on results

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

other: see overall remarks

Untreated negative controls validity:

other: see overall remarks

Positive controls validity:

not specified

Test results 2

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

see table 1 in remarks on results

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

other: see overall remarks

Untreated negative controls validity:

other: see overall remarks

Positive controls validity:

not specified

Test results 3

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

see table 1 in remarks on results

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

other: see overall remarks

Untreated negative controls validity:

other: see overall remarks

Positive controls validity:

not specified

Test results 4

Species / strain:

other: E. coli WP2 try hcr

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

without metabolic activation: slight activity; with metabolic activation see table 1 in remarks on results

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Test results 5

Species / strain:

other: E. coli WP2 B/r try

Metabolic activation:

with

Genotoxicity:

other: 3-fold increase in comparison to the control at 2.05 µmol/plate

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Test results 6

Species / strain:

other: E. coli WP2 B/r try

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Test results 7

Species / strain:

other: Bacillus subtilis M45 and H17

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

see table 2 in remarks on results

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Any other information on results incl. tables

Table 1: Results for the bacterial reverse mutation assay with metabolic activation (i.e. rat liver microsome fraction S-9)

Strain	Concentration (µmoles/plate)	# revertant colonies
Salmonella TA 98	0	54
	0.5	152
	1.0	213.0
Salmonella TA 100	0	133
	1.0	1234
	2.0	1224
Salmonella TA 1535	0	4
	0.5	16
	1.0	50
E. coli WP2 hcr	0	21
	0.5	115
	1.0	349

Table 2: Results for the Bacillus recombination assay without metabolic activation

Concentration (µmoles/plate)	Inhibition for Bacillus subtilis (mm)
	H17 Rec+	M45 Rec-
1.5	0	1.5
2.6	0	5.0
3.6	2.0	8.5

- Results for the time dependence of active metabolites study

Benzotrichloride can be hydrolysed by non enzymatic reactions forming benzoic acid. Hence the stability of benzotrichloride was studied to characterize its mutagenic metabolite. The mutagenic activity of benzotrichloride versus incubation time attained a peak value after 20 minutes incubation and the activity gradually decreased probably because of the killing effect. This may indicate that the whole process consisting of enzymatic reaction for the test substance, incorporation of metabolite into bacteria and production of mutagenic DNA damage may have taken place before plating.

When S9 mix and benzotrichloride were pre-incubated for various periods after which bacteria were added and the mixture incubated for 20 minutes before plating, the mutagenic activity of the test substance for E. coli WP2 hcr decreased to a control level within 3 minutes of the pre-incubation time. It therefor seemed that the mutagenic metabolite may be further quickly metabolized or easily decomposed withput enzyme.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
positive with metabolic activation for the reversion assay
positive without metabolic activation for the recombination assay

The authors tested the mutagenic activity of benzotrichloride with (1) a bacterial reverse mutation test by following a methodology similar to the OECD guideline 471 and (2) with a recombination assay performed according to Kada (1972). Under the test conditions, the mutagenicity of benzotrichloride was clearly positive in the reverse mutation assay with metabolic activation system.Furthermore the mutagenicity was also positive in the recombination assay for Bacillus subtilis M45 and H17 without metabolic activation.

Executive summary:

The authors tested the mutagenic activity of benzotrichloride (CAS n° 98-07-7) with (1) a bacterial reverse mutation test by following a methodology similar to the OECD guideline 471 and (2) with a recombination assay performed according to Kada, Tutikawa and Sadaie (1972) (Mutation Research 16: 165-174).

For the bacterial reverse mutation test following strains were used: Salmonella TA 98, TA 100, TA 1535 and Escherichia coli WP2 try hcr and WP2 try B/r. All Salmonella strains are assumed to have been tested with and without the metabolic activation system (i.e. rat liver S9 mix) and the E. coli strains were tested both with and without the metabolic activation system. For Salmonella strains TA 98 and TA 1535 reported tested concentrations are 0.5 and 1.0 µmoles/plate, while for Salmonella TA 100 1.0 and 2.0 µmoles/plate was tested. The concentrations used to test the E. coli WP2 try hcr strain were 0.25, 0.51, 1.02, 1.53, 2.05 and 2.56 µmoles/plate, while for E. coli WP2 B/r try 0.51, 1.02, 2.05, 2.56, 3.07 and 4.09 µmoles/plate of the test substance were tested. However for all tested strains, it does not appear very clear whether more concentrations were tested and if only positive results were reported. The number of revertant colonies were counted and compared to the control to assess the mutagenicity of the test substance.

For the recombination assay the mutagenic potential of the test substance was tested with Bacillus subtilis M45 (rec-) and H17 (rec+)with and without metabolic activation system at concentrations of 1.5, 2.6 and 3.6 µmoles/plate. The assessment of the mutagenicity is based on the inhibition of the growth zone.

Finally for all these experiments the test substance was dissolved in DMSO and solvent controls were performed (study is unclear on this point for some strains) while no positive controls were executed.

Under the test conditions, the mutagenicity of benzotrichloride was clearly positive in the reverse mutation assay with metabolic activation system to Salmonella TA 98, TA 100, TA 1535 and E. coli WP2 try hcr. Slight mutagenic activity was observed for E. coli WP2 try hcr without metabolic activation and the peak value for E. coli WP2 B/r try with metabolic activation was about 3-fold the control level. Furthermore the mutagenicity of the test substance was also considered positive in the recombination assay for Bacillus subtilis M45 and H17 without metabolic activation.

Thus considering the overall study, benzotrichloride may be considered a potent mutagen with metabolic activation. Therefore, further testing would be required to clarify the situation.

Some details about the method and results are missing (e.g. unclear number of concentrations used and uncertainty if solvent controls were performed for all strains). However, this study is similar to the OECD guideline 471 with minor deviations, then this study should be considered as reliable with restrictions, a Klimisch 2.c study, comparable to a guideline study with acceptable restrictions.