1,2,4-trichlorobenzene

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation results in bacteria:

- Ames test: Not mutagenic to S. thyphimurium strains TA 1535, TA 1537, TA 98 and TA 100 (OECD TG 471 / GLP / WoE 2) (Haworth, 1983)

- Ames test: Not mutagenic ((Q)SAR the Mutagenicity (Ames test) model (SarPy/IRFMN) / WoE 2) (Paul, 2022)

Link to relevant study records

Reference

Reference 1

Endpoint:

genetic toxicity in vitro

Remarks:

Type of genotoxicity: other: EU Risk Assessment

Type of information:

other: EU Risk Assessment

Adequacy of study:

other information

Reliability:

other: EU Risk Assessment

Rationale for reliability incl. deficiencies:

other: no reliability is given as this is a summary entry for the EU RAR

Reason / purpose for cross-reference:

reference to other study

Principles of method if other than guideline:

EU Risk Assessment

GLP compliance:

not specified

Type of assay:

other: EU Risk Assessment

EU Risk Assessment (2003):

 

Genotoxicity testing of 1,2,4-trichlorobenzene with in vitro mammalian systems have produced predominantly negative results. However, 1,2,4-trichlorobenzene produced positive results in a DNA repair assay on hepatocyte primary culture (Shimada et al 1983 Final report: December 5, 1983. NTIS/OTS 0291. Doc. ID: FYI-AX-0284-0291IN)

Reference 2

Endpoint:

genetic toxicity in vitro

Remarks:

Type of genotoxicity: other: EU Risk Assessment

Type of information:

other: EU Risk Assessment

Adequacy of study:

other information

Reliability:

other: EU Risk Assessment

Rationale for reliability incl. deficiencies:

other: no reliability is given as this is a summary entry for the EU RAR

Reason / purpose for cross-reference:

reference to other study

Principles of method if other than guideline:

EU Risk Assessment

GLP compliance:

not specified

Type of assay:

other: EU Risk Assessment

EU Risk Assessment (2003):

 

Several genotoxicity studies performed on microbial test organism were summarized in the EU Risk Assessment. Some of these studies were reliable studies with restrictions or reliability was not assignable because references were only cited as secondary literature.

Genotoxicity testing with several test organisms (including several strains of Salmonella thyphimurium) have generally yielded negative results, with or without metabolic activation.

However, Matsui et al. (Matsui et al.1989.Water Sci Technol 21: 875-887) tested 1,2,4-TCB for its effect on DNA repair in aBacillus subtilis recassay. 1,2,4-TCB showed activity that was described as strongly DNA damaging potential after S9 activation.

Reference 3

Endpoint:

genetic toxicity in vitro, other

Remarks:

(Q)SAR

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Guideline:

other: REACH guidance on QSARs R.6

Version / remarks:

May 2008

Principles of method if other than guideline:

- Software tool(s) used including version: The Mutagenicity (Ames test) model (SarPy/IRFMN) v. 1.0.7
- Model(s) used: VEGA Mutagenicity (AMES test) SARpy/IRFMN Model
- Model description: see field: Attached justification
- Justification of QSAR prediction: see field Attached justification
- Reference: Honma, M. et al, 2019. Improvement of quantitative structure–activity relationship (QSAR) tools for predicting Ames mutagenicity: outcomes of the Ames/QSAR International Challenge Project Environ. Mutagenesis. 2019(34) 3–16.

Type of assay:

other: (Q)SAR

Specific details on test material used for the study:

SMILES: C1=CC(=C(C=C1Cl)Cl)Cl

Metabolic activation:

not applicable

Test concentrations with justification for top dose:

N/A - QSAR

Key result

Species / strain:

other: QSAR classification scheme

Metabolic activation:

not applicable

Genotoxicity:

other: QSAR classification scheme

Remarks:

QSAR classification scheme

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The predictions are adequate for use alone or in a weight-of-evidence approach, concluding that 1,2,4- trichlorbenzene is not mutagenic to bacteria during an OECD 471 test (Ames test). This is a required endpoint under the REACH Regulation.

Conclusions:

Under the conditions of the model employed, the test item is predicted not to be mutagenic to bacteria during an OECD TG 471 (AMES test).

Executive summary:

The test item was assessed using VEGA Mutagenicity (Ames test) SARpy/IRFMN Model version 1.0.7 where the SMILES code was the input for the model.

For further information on the model and prediction please refer to the QRMF and QPRF.

The study indicates non-mutagenicity for this substance.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study with acceptable deviations

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

Only 4 strain tested instead of 5. Positive control: 2-aminoantracene was only indicator of efficiency of S9-mix. Individual plate reading not reported.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

his

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

microsomal enzyme reaction mix (S9-mix) prepared from Sprague-Dawley rats and male Syrian hamsters

Test concentrations with justification for top dose:

0.0, 3.3, 10.0, 33.3, 100.0, 333.3 ug/plate

Vehicle / solvent:

- Vehicle/solvent used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (2-AA): in all strains in presence of rat and hamster S-9. 4-Nitro-o-phenylenediamine (NOPD): on TA98, without S-9. Sodium azide (SA) : on TA100 and TA 1535, without S-9. 9-aminoacridine (9AAD) was tested on 1537, without S-9.

Remarks:

The actual concentration for each positive control chemical used for each strain and activation condition was selected by the individual laboratory based on dose-response curves generated at the beginning of the testing program (see table 1)

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 48h
- Selection time (if incubation with a selection agent): 48h
- Concentration of S9 mix: 10% for both Hamster S9 mix and Rat S9 mix

SELECTION AGENT (mutation assays): L- histidine

NUMBER OF REPLICATIONS: 2 trial per strain and 3 dishes per dose

DETERMINATION OF CYTOTOXICITY
- Method: other: viability on complete medium and reduced numbers of revertant colonies per plate and/or thinning or absence of the bacterial lawn

Evaluation criteria:

A positive response was indicated by a reproducible, dose-related increase, whether it be twofold over background or not.
An equivocal response was defined as an increase in revertants which was not dose-related, not reproducible, or was of insufficient magnitude to support a determination of mutagenicity. A negative response was obtained when no increase in revertant colonies is observed following chemical treatment.

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: To select the dose range for the mutagenetic assay, the test chemicals were checked for toxicity to TA 100 up to a concentration of 10 mg/plate or the limit of solubility, both in the presence and absence of S-9 mix. If toxicity was not apparent in the preliminary toxicity determination, the highest dose tested was 10 mg/plate; otherwise the upper limit of solubility was used. If toxicity was observed, the doses of test chemical were chosen so that the high dose exhibited some degree of toxicity. Occasionally, in the earlier tests, the high dose was greater than 10 mg/plate.

Remarks on result:

other: all strains/cell types tested

Remarks:

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

TA 100

TA 1535

TA 1537

TA 98

Dose

NA

RLI

HLI

NA

RLI

HLI

NA

RLI

HLI

NA

RLI

HLI

0.0

99±5.2

113±2.0

115±8.7

20±1.0

13±3.0

13±0.6

17±1.5

14±1.9

12±2.7

37±3.5

35±2.3

37±4.7

3.3

104±4.0

107±5.5

124±2.0

16±1.5

15±2.5

13±2.9

18±3.8

10±1.5

18±5.2

38±6.4

34±2.3

31±0.9

10.0

86±6.7

115±9.5

132±5.8

18±4.2

9±0.0

10±2.7

23±2.7

11±1.2

23±3.7

28±1.2

36±4.8

35±2.7

33.3

61±10.1 s

123±2.2

124±7.3

19±1.2

13±3.2

11±1.7

11±1.7

9±0.3

17±0.7

24±1.7

29±0.9

43±2.3

100.0

t

112±6.7

100±6.9

6±6.0s

9±1.8

12±2.7

9±1.8

8±1.7

17±4.0

24±1.3

34±4.1

38±6.4

333.3

t

114±9.4

21±21.5s

t

13±0.7s

6±1.5s

5±2.3

7±1.2

0±0.0s

17±2.5

34±3.7

9±8.5s

POS

625±28.8

460±15.3

963±104.3

444±23.0

305±3.2

268±34.8

308±8.0

158±10.6

244±5.8

850±18.0

288±3.0

1258±130.5

Table 1 Mutagenic responses of Salmonella strains TA100, TA 1535, TA 1537, and TA 98 (mean±SEM) to 1,2,4-trochlorobenzene.

 Abbreviations:NA, not activated; RLI, rat liver S-9, Aroclor1254 induced; HLI, hamster liver S-9, Aroclor1254 induced, t=complete clearing background, POS, positive control

Conclusions:

negative with metabolic activation
negative without metabolic activation

Executive summary:

Haworth (1983)

 

Ames test was performed with the Salmonella typhimurium strains TA1535, TA1537, TA98, and TA100 at 0, 3.3, 10.0, 33.3, 100.0, and 333.3µg 1,2,4-TCB/plate with and without S-9 from Aroclor 1254 induced rat and hamster livers using the preincubation procedure. Test doses were chosen following checks for toxicity: in the absence of toxicity a maximum of 10 mg/plate was used.

The test resulted was negative with and without metabolic activation.

The test was conducted according to OECD guideline 471 with deviations (Only 4 strains tested instead of 5. Positive control: 2-aminoantracene was only indicator of efficiency of S9-mix. Individual-plate-readings were not reported).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Table 7.6.1/1 : Summary of genotoxicity data:

 

Test n°	Test Guideline / Reliability	Focus	Strains  / cells tested	Metabolic activation	Test concentration	Statement
1 (1983)	Ames Test (OECD 471, GLP) WoE, rel.2	Gene mutation	S. thyphimurium TA 1535, TA 1537, TA 98, TA 100,	-S9 +S9	Tested up to 333 µg/plate (up to cytotoxic concentration)	-S9: not mutagenic + S9: not mutagenic
2 (12022)	Ames Test (QSAR) WoE, rel.2	Gene mutation	S. thyphimurium	-S9 +S9	-	-S9: not mutagenic + S9: not mutagenic

Gene mutation Assay (Test n° 1-2)

A bacterial reverse mutation assay (Ames test) was performed with the substance (Test n°1). This study was used to conclude on the potential of the substance to induce gene mutation in bacteria. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains with any dose of test material, either in the presence or absence of metabolic activation. The test indicates that the substance does not induce gene mutations in bacteria whereas all positive control chemicals (with and without metabolic activation) induced significant increase of colonies. 

As E. coli WP2 uvrA, or E. coli WP2 uvrA (pKM101), or S. typhimurium TA102 strains was not evaluated in this study, it was not possible to conclude on the cross-linking potential of the substance. Therefore, a (Q)SAR assessment was performed using the VEGA Mutagenicity (Ames test) SARpy/IRFMN Model where the SMILES string was used to determine the results (Test n°2). No evidence of mutagenicity was noted for the substance in the model predictions. Though not explicit on strains included the (Q)SAR predictions for this group of chemicals was based on substances that both did and did not have data on S. thyphimurium TA 102 and E.coli WP2 strains, all of which were negative.  The model, therefore, as it pertains to coverage of effects to all strains and the modes of actions has coverage for this substance type, based on evaluation of the results and nearest neighbours.  Evaluation of the training and test sets was conducted. Moreover, based on structure, these specific modes of actions identified by S. thyphimurium TA 102 and E.coliWP2 strains are unlikely to occur.

Based on the weight-of-evidence the substance is therefore considered as non-mutagenic according to the Ames test. 

Justification for classification or non-classification

Harmonised classification

The substance has no harmonised classification according to the Regulation (EC) No. 1272/2008 (CLP). 

 

Self Classification

Based on information available no additional self-classification is proposed regarding mutagenicity according to the CLP and to the GHS.