2-chlorobenzaldehyde

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Tests for gene mutations with 2-chlorobenzaldehyde in the bacterial reverse mutation assay with the test strains S. typhimurium TA97, TA98, TA100, TA104, TA1535, TA1537, TA1538 and E coli WP2uvrA/pkm101 gave negative results in the presence or absence of a metabolic activation systems. Negative results were also obtained in two strains of Saccharomyces cerevisiae (XV185-14C and D7) without metabolic activation.
The test item was negative in an in vitro micronucleus assay and an in vitro assay for structural chromosomal aberrations. The test item did not induce DNA error-prone repair (umo-test) in vitro, but was positive in an in vitro assay for the induction of aneuploidy and polyploidy.

The parent compound (CS, [(2-chlorophenyl)methylene]malonitrile) was negative in micronucleus tests in vivo in mice.

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:

year of publication: 1992

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Sufficently documented literature data

Principles of method if other than guideline:

Bacterial reverse mutation assay (Ames test), preincubation assay

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium, other: TA 97, TA 98, TA 100, TA 1535, TA 1537

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

0 (control), 3.3, 10.0, 33.0, 100.0, 200.0, 333.0, 500.0, 666.0 µg/plate

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: without metabolic activation: sodium azide (TA1535 and TA100), 9-aminoacridine (TA97 and TA1537), 4-nitro-o-phenylenediamine (TA 98 and 1538); with metabolic activation: 2-aminoanthracene (all strains)

Details on test system and experimental conditions:

20 min preincubation procedure

Species / strain:

S. typhimurium, other: TA 97, TA 98, TA 100, TA 1535, TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

It was stated in the text, that maximum concentrations of 10000 µg/plate are only tested if substances were not toxic. As 2-chlorobenzaldehyde was only tested up to 666 µg/plate it can be concluded, that it was toxic at higher concentrations. But, no results of the testing for cytoxicity were presented in the publication.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

2-Chlorobenzaldehyde was not mutagenic under the conditions tested.

Executive summary:

2-Chlorobenzaldehyde was not mutagenic in the bacterial reverse mutation assay (Ames test, test strains: Salmonella typhimurium TA 97, TA 98, TA 100, TA 1535, TA 1537) with and without metabolic activation (S9 -mix).

This well documented and reliable (RL2) study has been selected as key study for the evaluation of gene mutations.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

The parent compound (CS, [(2-chlorophenyl)methylene]malonitrile) was negative in micronucleus tests in vivo in mice.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

The study results of the source compound were considered applicable to the target compound.

Reason / purpose for cross-reference:

read-across source

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

not specified

Negative controls validity:

not specified

Conclusions:

The test item did not induce micronuclei in vivo under the current test conditions.Therefore, the test item was not genotoxic (negative).

Executive summary:

The study used as source investigated in vivo mammalian somatic cell cytogenicity. The study results of the source compound were considered applicable to the target compound.

Mice were treated with single intraperitoneally doses of 10, 20, or 40 mg/kg bw CS. No induction of micronuclei in vivo was observed, even in the lethal concentration range.

The study has been judged to be reliable with restriction (RL2) due to some shortcomings, e.g. missing data on substance purity, housing conditions. The study was selected as key study for 2 -chlorobenzaldehyde.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

2-Chlorobenzaldehyde was tested negative for gene-mutagenic properties in Salmonella typhimurium in the presence and absence of a metabolising system derived from rat liver homogenate (S9-mix). In a well documented literature study (Zeiger et al., 1992)), which has been selected as key study, test item concentrations up to 666 µg/plate gave negative results in test strains TA 97, TA 98, TA 100, TA 1535 and TA 1537. These data are supported by two other well documented publications (Nestmann et al., 1980 and Rietveld et al., 1983): test item concentrations up to 2000 µg/plate did not result in gene mutations in the test strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538. Additional, up to 3500 µg/plate did not induce gene mutations in TA 100 in the presence of metabolic activation. The publication of Ohkubo et al. (1996), which has been judged not to be reliable (publication in Japanese, only tables on results in English), reports negative results with 2-chlorobenzaldehyde in the test strains TA 98, TA 100, TA 104 and E. coli WP2uvrA/pkM101. None of these studies is in accordance with existing guidelines. Especially, test concentrations are below the highest recommended test concentration of 5000 µg/plate. This is probably due to toxicity, as described in the key study. Due to the congruent negative findings in all studies in several test strains (covering all test strains recommended in OECD TG 471) in the presence and absence of metabolic activation, it is concluded that 2-chlorobenzaldehyde does not induce gene mutations in the bacterial reverse mutation assay.

2-Chlorobenzaldehyde did not induce micronuclei in a micronucleus assay in V79 Chinese hamster cells, which were exposed for 3 h to up to 150 µM. 2-Chlorobenzaldehyde was only slightly cytotoxic even at the highest concentration tested (Ziegler-Skylakakis et al., 1989). 2-Chlorobenzaldehyde did not induce structural chromosomal aberrations in concentrations up to 37.5 µM in a cytogenetic assay in vitro (V79 Chinese hamster cells, 20 h exposure duration) (Bauchinger und Schmid, 1992). Exposure of V79 Chinese hamster cells for 20 h to up to 37.5 µM resulted in a significant increase in the number of aneuploid and polyploid cells (Schmid and Bauchinger, 1991). None of these studies, which all have been judged to be reliable with restrictions, is in accordance with existing guidelines. The data on the induction of aneuploidy and polyploidy in V79 Chinese hamster cells at concentrations up to 37.5 µM seem to contradict the negative results for the induction of micronuclei in V79 Chinese hamster cells at concentrations up to 150 µM. However, the different exposure durations have to be considered. Furthermore, the high incidence of polyploid cells may have prevented the detection of the aneuploidogenic effect in the micronucleus assay.

According to the guidance provided in the Annex VI of the Directive 67/548/EEC that substances, which show positive results only in one or more in vitro mutagenicity assays should normally not be classified, further investigations using in vivo assays would be indicated. Considering the findings obtained with the riot control agent [(2-chlorophenyl)methylene]malonitrile (CS), which rapidly hydrolyses to 2-chlorobenzaldehyde and malononitrile in vivo and in vitro, it will be discussed whether an in vivo micronucleus assay seems to be appropriate to confirm the in vitro findings for 2-chlorobenzaldehyde.

The mutagenic effects of CS and 2-chlorobenzaldehyde were investigated in some experiments in parallel: CS induced micronuclei in V79 Chinese Hamster cells after 3 h of exposure in concentrations of 19 µM to 75 µM. Cytotoxic effects (reduced survival index) were obvious in all dose groups (Ziegler-Skylakakis et al., 1989). CS also induced aneuploid and polyploid V79 Chinese hamster cells after 20 h of exposure in concentrations up to 37.5 µM. The extend of aneuploidy and polyploidy was lesser than with 2-chlorobenzaldehyde (Schmid and Bauchinger, 1991). Additionally, CS induced clastogenic effects (chromatide type aberrations and sister chromatide exchanges) in V79 Chinese hamster cells (Bauchinger and Schmid, 1992). Further investigations revealed that CS induces micronuclei in vitro which contain whole chromosomes (Miller und Nüsse, 1993; Nüsse et al., 1992; Nüsse et al., 1996). The induction of micronuclei in vitro is probably due to an interaction of CS with the spindle fibers (Salassidis et al., 1991). No induction of micronuclei in vivo was observed in rats after a single teatment with CS (Wild et al., 1983; Grawé et al., 1997).

To summarize, both substances - CS and 2-chlorobenzaldehyd - induce aneuploidy and polyploidy in vitro. CS induces structural chromosomal aberrations in vitro and micronuclei in vitro, but not in vivo. 2-Chlorobenzaldehyde was tested negative for chromosomal aberrations and micronuclei in vitro. Induction of micronuclei by 2-chlorobenzaldehyde in vivo was not investigated.

CS is mainly decomposed to 2-chlorobenzaldehyde and malononitrile. Only a smaller amount – about 10% – is reduced to 2-chlorobenzyl malononitrile (see chapter 7.1). Malononitrile is further degraded to the non-genotoxic agent cyanide. No data exist on the genotoxic effects of 2-chlorobenzyl malononitrile. A conservative assumption would be that the genotoxic activity of CS is mainly due to the formation of 2-chlorobenzaldehyde.

CS was negative in the tests for micronuclei in vivo. Although the bone marrow is very well supplied with blood, in these studies it did not induce toxic effects in the bone marrow, and uncertainty remains whether the target organ was reached by the substance. However, CS was tested up to lethal concentrations and based on a comparison of LD50 values it can deduced that a micronucleus assay in vivo with 2-chlorobenzaldehyde would have to use similar dose ranges. Therefore a similar internal dose of 2-chlorobenzaldehyde is expected from testing 2-chlorobenzaldehyde in vivo as was available in the CS studies. It is therefore assumed that 2-chlorobenzaldehyde would also not induce toxic effects in the bone marrow, and the same uncertainties in interpretation would prevail. As no new/improved information is expected from testing 2-chlorobenzaldehyde in vivo, the genetic potential fo 2-chlorobenzaldehyde is evaluated by read-across technique based on the data on CS. Based on these data it is concluded that 2 -chlorobenzaldehyde would not induce micronuclei in vivo.

Short description of key information:

Tests for gene mutations with 2-chlorobenzaldehyde in the bacterial reverse mutation assay with the test strains S. typhimurium TA97, TA98, TA100, TA104, TA1535, TA1537, TA1538 and E coli WP2uvrA/pkm101 gave negative results in the presence or absence of a metabolic activation systems. Negative results were also obtained in two strains of Saccharomyces cerevisiae (XV185-14C and D7) without metabolic activation.
The test item was negative in an in vitro micronucleus assay and an in vitro assay for structural chromosomal aberrations. The test item did not induce DNA error-prone repair (umo-test) in vitro, but was positive in an in vitro assay for the induction of aneuploidy and polyploidy.
The parent compound (CS, [(2-chlorophenyl)methylene]malonitrile) was negative in micronucleus tests in vivo in mice.

Tests for gene mutations with 2-chlorobenzaldehyde in the bacterial reverse mutation assay with the test strains S. typhimurium TA97, TA98, TA100, TA104, TA1535, TA1537, TA1538 and E coli WP2uvrA/pkm101 gave negative results in the presence or absence of a metabolic activation systems. Negative results were also obtained in two strains of Saccharomyces cerevisiae (XV185-14C and D7) without metabolic activation.
The test item was negative in an in vitro micronucleus assay and an in vitro assay for structural chromosomal aberrations. The test item did not induce DNA error-prone repair (umo-test) in vitro, but was positive in an in vitro assay for the induction of aneuploidy and polyploidy.
The parent compound (CS, [(2-chlorophenyl)methylene]malonitrile) was negative in micronucleus tests in vivo in mice

Justification for classification or non-classification

2-Chlorobenzaldehyde was negative in in vitro bacterial reverse mutation assays, an in vitro micronucleus assay and an in vitro assay on structural chromosomal aberrations. The test item induced polyploidy and aneuploidy in V79 Chinese hamster cells in vitro. Based on the negative findings in in vivo micronucleus assays with the riot control agent [(2-chlorophenyl)methylene]malonitrile (CS), which rapidly hydrolyses to 2-chlorobenzaldehyde and malononitrile in vivo and in vitro, it is concluded that 2-chlorobenzaldehyde has not to be classified for mutagenicity according to Annex VI of the Directive 67/548/EEC and REGULATION (EC) No 1272/2008.