1,3-dichlorobenzene

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

OECD TG 471 (Shimazu, 1983): Not mutagenic 

 

OECD TG 476 (Wollny, 2015): Not mutagenic 

 

The in vitro gene mutation studies conducted were not conclusive for classification due to deviations noted. Therefore the classification of the test substance required the use of an additional in vivo study. The results from the in vitro (mamalian cells) OECD TG 476 combined with the results from the in vitro study were sufficient to rule out classification of the substance as mutagenic according to the CLP Regulation (EC) No 1272/2008. 

Link to relevant study records

Reference

Reference 1

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:

comparable to guideline study with acceptable restrictions

Remarks:

The study deviates from the TG by not using one of the following strains E. coli WP2 uvrA, or E. coli WP2 uvrA (pKM101), or S. typhimurium TA102.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

Rats were injected with PCB instead of Aroclor 1254. The S. thyphimurium strains, TA98, TA1538, TA1537, TA100 and TA 1535 were used instead of TA1535, TA1537 (or TA97a or TA97), TA98, and TA100 , and E.coli WP2 strains or S. typhimurium TA102.

Principles of method if other than guideline:

The mutagenicity of chlorobenzene compounds in Salmonella typhimurium (strains TA98, TA1538, TA1537, TA100 and TA1535) was examined.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

his

Species / strain / cell type:

other: TA 98, TA 100, TA 1535, TA 1537, TA 1538

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

0.02, 0.04, 0.08, 0.16, 0.32, 0.64, 1.28, 2.56 µL

Vehicle / solvent:

- Vehicle used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: N-Ethyl-N´-nitro-N-nitrosoguanidine (ENNG), 2-nitrofluorene (2-NF), 9-aminoacridine (9-AA) and 2-aminoanthracene (2-AA)

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation
NUMBER OF REPLICATIONS: all test were performed in duplicate and repeated at least 3 times

Evaluation criteria:

Colonies of his+ revertants were counted after incubation, and chemicals inducing more than twice the number of revertant colonies on the control plate were considered as mutagenic.

Statistics:

Not performed

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Vehicle controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

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

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'. Remarks: TA 98, TA 100, TA 1535, TA 1537, TA 1538

Mutagenicity of 1,3-dichlorobenzene in S.typhimurium TA 98, TA 1538, TA 1537, TA100, and TA 1535

 

 

Dose per plate µL	His+ revertants/plate
	TA98		TA 1538		TA 1537		TA 100		TA 1535
	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
0.02	30 ± 3	29 ± 4	24 ± 4	24 ± 5	8 ± 2	6 ± 2	180 ± 22	228 ± 22	36 ± 5	32 ± 4
0.04	28 ± 3	33 ± 7	22 ± 3	18 ± 3	13 ± 3	9 ± 2	172 ± 16	196 ± 17	31 ± 4	38 ± 6
0.08	30 ± 5	24 ± 3	21 ± 3	22 ± 3	10 ± 2	9 ± 2	135 ± 25	190 ± 15	32 ± 5	30 ± 3
0.16	21 ± 3	29 ± 5	17 ± 2	30 ± 6	8 ± 2	8 ± 2	142 ±18	186 ±18	29 ± 4	24 ± 3
0.32	23 ± 4	22 ± 3	25 ± 5	20 ± 2	9 ± 2	11 ± 4	154 ± 13	177 ±16	22 ± 3	28 ±4
0.64	17 ± 2	25 ± 4	21 ± 4	31 ± 5	10 ± 3	9 ± 2	156 ±18	181 ± 17	28 ± 5	24 ± 3
1.28	15 ± 3 *	20 ± 3	0*	2± 8*	3 ± 3*	5 ± 3*	155 ± 9	164 ±14	6 ± 8*	17 ± 3
2.56	0*	0*	0*	0*	0*	0*	0*	42 ±22*	0*	0*

 

 

 

 

Conclusions:

Under the conditions of the study, the test item was not mutagenic and not classified according to CLP Regulation (EC) 1272/2008.

Executive summary:

The mutagenicity of 1,3-dichlorobenzene was examined in Salmonella typhimurium (strains TA98, TA, 1538, TA 1537, TA 100, and TA 1535). The test was carried out with and without liver microsomal activation.

1,3-dichlorobenzene showed no mutagenic activity and toxicity at higher doses.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Principles of method if other than guideline:

This in vitro assay was performed to assess the potential of 1,3-dichlorobenzene to induce gene mutations by means of two independent HPRT experiments using the Chinese hamster cell line V79. The treatment time was 4 hours in the first experiment with and without metabolic activation. In the second experiment the cells were exposed to the test item for 24 hours without metabolic activation and 4 hours with metabolic activation.

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

HPRT locus

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Additional strain / cell type characteristics:

other: the cells have a stable karyothype with a modal chromosome number of 22

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

exposure S9
period mix
concentrations in µg/mL
Experiment I
4 hours - 5.9 11.8 23.5 47.0 94.0 188.0
4 hours + 5.9 11.8 23.5 47.0 94.0 188.0
Experiment II
24 hours - 5.9 11.8 23.5 47.0 94.0 141.0
4 hours + 5.9 11.8 23.5 47.0 70.5 94.0

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: Without metabolic activation: EMS; ethylmethane sulfonate; With metabolic activation: DMBA; 7,12-dimethylbenz(a)anthracene

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: Relevant cytotoxic effects were observed at 187.5 µg/mL and above following 24 hours treatment without metabolic activation. No relevant cytotoxicity occurred after 4h treatment with and without metabolic activation.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: other: Chinese hamster lung fibroblasts (V79)

Remarks:

Migrated from field 'Test system'.

The test item 1,3-dichlorobenzene, CAS 541-73-1 was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster.

The study was performed in two independent experiments, using identical general experimental procedures.

In the first experiment the treatment period was 4 hours with and without metabolic activation. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.

The main experiments were evaluated at the following concentrations:

exposure S9

period mix

concentrations in µg/mL

Experiment I

4 hours - 11.8 23.5 47.0 94.0 188.0

4 hours + 5.9 11.8 23.5 47.0 94.0

Experiment II

24 hours - 5.9 11.8 23.5 47.0 94.0

4 hours + 11.8 23.5 47.0 70.5 94.0

Phase separation occurred in experiment I at 94.0 µg/mL and above without metabolic activation and at 94.0 µg/mL with metabolic activation. In experiment II phase separation was observed at 94.0 µg/mL and above with and without metabolic activation.

Relevant cytotoxic effects, indicated by a relative cloning efficiency I or a relative cell density at first subcultivation of less than 50% in both parallel cultures, occurred in the first experiment at 94.0 µg/mL and above with and without metabolic activation. In the second experiment relevant cytotoxic effects as described above were noted at 94 µg/mL with metabolic activation. The maximum concentration of the second experiment without metabolic activation was limited by phase separation. The recommended cytotoxic range of approximately 10%-20% relative cloning efficiency or relative cell density was covered with and without metabolic activation. Compared to the pre-experiment, cytotoxicity occurred at lower concentrations in the main experiments, especially after 4 hour treatment. However, the cytotoxicity was observed at concentrations showing phase separation or at the limit of solubility. An organic phase usually is toxic on direct contact to the cells as cell membranes are damaged. As the number of cells coming into direct contact with an organic phase in culture medium is variable, cytotoxic effects in an insoluble concentration range are poorly reproducible.

No relevant and reproducible increase in mutant colony numbers/10E6 cells was observed in the main experiments up to the maximum concentration. The mutation frequency generally remained within the historical range of solvent controls. An isolated increase of the mutation frequency exceeding the threshold of three times the mutation frequency of the solvent control was noted in the first experiment, culture II with metabolic activation at 23.5 and 94.0 µg/mL. However, the threshold was not exceeded in the parallel culture or in the second experiment with metabolic activation. Consequently, the isolated increase described above was judged as biologically irrelevant.

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. A significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in the second culture of experiment I with metabolic activation. However, this increase was judged as irrelevant fluctuation as discussed above.

In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 11.1 up to 33.3 mutants per 10E6 cells; the range of the groups treated with the test item was from 5.9 up to 54.6 mutants per 10E6 cells.

EMS (150 µg/mL) and DMBA (2.2 µg/mL in experiment I and 1.1 mg/mL in experiment II) were used as positive controls and showed a distinct increase in induced mutant colonies.

Conclusions:

Under the conditions of this study , the test item is not mutagentic according to CLP Regulation (EC) No. 1271/2008.

Executive summary:

The study was performed to investigate the potential of 1,3-dichlorobenzene, CAS 541-73-1 to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster. This study was designed to be compatible with the procedures indicated by the internationally accepted guidelines and recommendations.

The assay was performed in two independent experiments. The cells were exposed to the test item for 4 hours in the first experiment with and without metabolic activation. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.

The maximum concentration of the pre-experiment (1500 µg/mL) was equal to a molar concentration of about 10 mM. The concentration range of the main experiments was limited by cytotoxicity and phase separation of the test item in culture medium.

No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments.

Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.

Therefore, 1,3-dichlorobenzene, CAS 541-73-1 is considered to be non-mutagenic in this HPRT assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Initial assessment for genetic toxicity was conducted under OECD TG 471:Bacterial Reverse Mutation Test (Shimazu, 1983). Deviations were noted in the study; the S. thyphimurium strains, TA98, TA1538,  TA1537,  TA100 and TA 1535 were used instead of TA1535, TA1537 (or TA97a or TA97), TA98, and TA100 , and E.coli WP2 strains or S. typhimurium TA102. The four main S. thyphimurium strains have GC base pairs at the primary reversion site and are known to have limited capacity to detect certain oxidising mutagens, cross-linking agents and hydrazines. Therefore the use of E.coli WP2 strains or S. typhimurium TA102 allow for the detection of such substances as the strains have an AT base pair at the primary reversion site. 

Justification for classification or non-classification

All available valid tests were negative. Therefore a classification is not justified.