Butanal, reaction products with aniline

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Butanal, reaction products with aniline was investigated in a Salmonella/microsome tests (Ames tests). Result: negative, no evidence of mutagenic activity of butanal, reaction products with aniline was seen (with and without mutagenic activation). Additional, butanal, reaction products with aniline was evaluated as negative in an in-vitro MNT and also in a V79/HGPRT test.

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:

other: scientifically acceptable and well documented

Principles of method if other than guideline:

The condensation product of acroleins with aromatic bases was examined for mutagenic activity in four histidine-dependent auxotrophs of Salmonella typhimurium strains TA 98, TA 100, TA 1535 and TA 1537, using pour-plate assays. The procedures used complied with the OECD Guidelines for testing of chemicals (1983) and the EPA Toxic Substances Control Act Test Guidelines (1985). Each test, in each strain, was conducted twice.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

no data

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Cultures of the histidine-dependent strains of Salmonella typhimurium were derived from cultures provided by Prof. Bruce Ames, University of California. The characteristics of the indiVidual strains are as follows:
TA 1535 - contains a histidine missense mutation but is also deficient in a DNA repair system (uvr B) and has a defective lipopolysaccharide coat on the cell wall. It is reverted by many agents causing base-pair substitutions, but is not sensitive to frameshift mutagens.
TA 100 - is the same as TA 1535 but contains a resistance transfer factor conferring ampicillin resistance and increasing sensitivity to some mutagens (plasmid pKM 101). In addition to base-pair substitutions, it is also able to detect certain frameshift mutagens.
TA 1537 - bears a histidine frameshift mutation. Like TA 1535, it is defective in a DNA repair system and lipopolysaccharide coat. It is sensitive to agents causing frameshift mutations involving insertion or deletion of a single base-pair.
TA 98 - contains another histidine frameshift mutation. Again it has a defective DNA repair system and lipopolysaccharide coat but also contains the pKM 101 plasmid. It is reverted by agents causing deletion of two adjacent base-pairs (double frameshift mutations), but not by simple alkylating agents causing base-pair substitutions.

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix

Test concentrations with justification for top dose:

Test 1: 50, 158, 500, 1580, 5000 µg/plate
Test 2: 50, 158, 500, 1580, 5000 µg/plate

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

other: 2-aminoanthracene

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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

The studies, which were conducted in the absence and presence of an activating system derived from rat liver (S-9 mix), employed a

range of levels of the test material from 50 to 5000 ug per plate, selected following a preliminary toxicity test in strain TA 98.

All tests included solvent (dimethyl sulphoxide) controls with and without S-9 mix.

No increases in reversion to prototrophy were obtained with any of the four bacterial strains at the test material levels tested, either in the presence or absence of S-9 mix.

Conclusions:

Interpretation of results: negative

Executive summary:

The condensation product of acroleins with aromatic bases was examined for mutagenic activity in four histidine-dependent auxotrophs of Salmonella typhimurium, strains TA 98, TA 100, TA 1535 and TA 1537, using pour-plate assays. The procedures used complied with the OECD Guidelines for testing of chemicals (1983) and the EPA Toxic Substances Control Act Test Guidelines (1985).

Each test, in each strain, was conducted on two separate occasions.

The studies, which were conducted in the absence and presence of an activating system derived from rat liver (S-9 mix), employed a range of levels of the test material from 50 to 5000 ug per plate, selected following a preliminary toxicity test in strain TA 98.

All tests included solvent (dimethyl sulphoxide) controls with and without S-9 mix.

No increases in reversion to prototrophy were obtained with any of the four bacterial strains at the test material levels tested, either in the presence or absence of S-9 mix.

Marked increases in the number of revertant colonies were induced by the known mutagens benzo[a]pyrene, 2-nitrofluorene, 2-aminoanthracene, 9-aminoacridine and sodium azide when examined under similar conditions.

It was concluded that the condensation product of acroleins with aromatic bases was devoid of mutagenic activity under the conditions of the test.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: chromosome aberration

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:

other: OECD Guideline for the testing of chemicals 487 In vitro mammalian cell micronucleus test OECD (2010)

Principles of method if other than guideline:

Butanal, reaction products with aniline was examined for mutagenic activity in the micronucleus test in vitro. Two independent assays were performed, the first assay conducted with a treatment time of 4 hours (pulse treatment), consisting of one experiment in the absence and one experiment in the presence of an extrinsic metabolizing system (S9 mix). In the second assay, an experiment without S9 mix was performed with treatment time extended to 24 hours (continuous treatment).

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Target gene:

no data

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Additional strain / cell type characteristics:

other: The cells have a stable karyotype with a modal chromosome number of 22

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Exp: 1: 10, 20, 40, 60, 80, 100 µg/ml (without S9) 4h treatment
Exp: 1: 10, 20, 40, 60, 80, 100 µg/ml (with S9) 4 h treatment
Exp: 2: 1, 2.5, 5, 10, 20, 30, 40, 50 µg/ml (without S9) 24 h treatment

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: vinblastine

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: Without S9 mix, limiting cytotoxicity was reached at a concentration of 40 µg/mL (4 hours treatment) or 20 µg/mL (24 hours treatment). With S9 mix (4 hours treatment), limiting cytotoxicity was observed at a concentration of 60 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

The results of the solvent control confirmed the spontaneous micronucleus frequency which is characteristic for V79 cells.

Appropriate positive control items (mitomycin C, vinblastine sulfate, cyclophospha-mide) showed the expected increase in micronucleus frequencies which demon-strates the ability and the sensitivity of the test system to detect cytogenetic damage.

No precipitates could be observed up to the highest concentration used (100 µg/mL).

In the first experiment without S9 mix (pulse treatment), 80 % cytotoxicity (100 % – RICC) was observed at a concentration of 40 µg/ml. At this concentration, cytotoxicity evident from a 64 % decrease of the PI was observed. In the second experiment without S9 mix (continuous treatment), 41 % cytotoxicity (100 % – RICC) was noted starting at 20 μg/mL. A 27 % decrease of the PI was observed at this concentration. Higher concentrations were excluded from evaluation for micronuclei due to excessive cytotoxicity. Based on these results, 40 µg/mL (pulse treatment) or 20 µg/mL (continuous treatment) were selected as the highest test concentrations scored for micronuclei in the experiments without S9 mix.

In the initial experiment with pulse treatment in the presence of S9 mix, 63% cytotoxicity (100 % RICC) occurred at 60 µg/mL. 68 % cytotoxicity (determined by PI) was noted at this concentration. Based on these results, 60 µg/mL was selected as the highest test concentration for pulse treatment with S9 mix scored for micronuclei.

The micronucleus test showed no biologically relevant increase in the frequency of micronucleus containing V79 cells treated with Butanal, reaction products with aniline in the absence (both pulse and continuous treatment) or in the presence of S9 mix (pulse treatment) up to cytotoxic concentrations.

Conclusions:

Interpretation of results: negative

Executive summary:

Butanal, reaction products with aniline was examined for mutagenic activity in the micronucleus test in vitro. In this study, two independent assays were performed, the first assay conducted with a treatment time of 4 hours (pulse treatment), consisting of one experiment in the absence and one experiment in the presence of an extrinsic metabolizing system (S9 mix). In the second assay, an experiment without S9 mix was performed with treatment time extended to 24 hours (continuous treatment).

As a rule the highest concentration tested should either be cytotoxic or correspond to the solubility limit of the test item (microscopically visible precipitates). Solid test items showing neither cytotoxicity nor precipitation should be tested up to 10E-2 mol/L or 5 mg/mL (OECD guideline 487).

The negative control (dimethyl sulfoxide) and appropriate positive controls with known mutagens (mitomycin C, vinblastine sulfate, cyclophosphamide) demonstrated the suitability and sensitivity of the test system.

In both assays precipitates of the test item could not be observed.

In the experiments without S9 mix, limiting cytotoxicity was reached at a concentration of 40 µg/mL (4 hours treatment) or 20 µg/mL (24 hours treatment). In the experiment with S9 mix (4 hours treatment), limiting cytotoxicity was observed at a concentration of 60 µg/mL.

The micronucleus was negative in V79 cells treated with Butanal, reaction products with aniline in the absence (4 hours or 24 hours treatment) or in the presence of S9 mix.

Reference 3

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:

The study was performed to investigate the potential of Butanal, reaction products with aniline to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster. The treatment time was 4 h in the first and second experiment with and without 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 concentrations in µg/mL
period mix
Experiment I
4 hours - 0.6 1.3 2.5 5.0 10.0 p 15.0 p 20.0 p
4 hours + 5.0 10.0 20.0 40.0 80.0 p 120.0 p 160.0 p
Experiment II
4 hours - 1.0 2.0 3.0 4.0 5.0 6.0 8.0 10.0
4 hours + 6.0 12.0 24.0 36.0 48.0 60.0 72.0 p
p = precipitation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: First experiment: 5.0 µg/mL without metabolic activation and at 80.0 µg/mL with metabolic activation. Second experiment: 4.0 µg/mL without metabolic activation and at 60.0 µg/mL with metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

No relevant and/or reproducible increase in mutant colony numbers/10E6 cells was observed in the main experiments up to the maximum concentration. The mutation frequency remained well within the historical range of solvent controls, the induction factor did not reach or exceed the threshold of three.

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. No significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in any of the experimental groups.

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

Conclusions:

Interpretation of results: negative

Executive summary:

The study was performed to investigate the potential of Butanal, reaction products with aniline to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster.

The treatment time was 4 h in the first and second experiment with and without metabolic activation.

The maximum concentration of the pre-experiment (2800 µg/mL) was equal to a molar concentration of approximately 10 mM. The concentration range of the main experiments was limited by the cytotoxicity of the test item.

No substantial and/or 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, Butanal, reaction products with aniline was negative in this HPRT assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In all in-vitro genetic toxicity assays (Ames test, MNT, V79/HGPRT test), butanal, reaction products with aniline was negative.

Justification for selection of genetic toxicity endpoint
The results of the Ames tests, the chromosome aberration test and the HPRT test were considered

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

In all in-vitro genetic toxicity assays (Ames test, MNT, V79/HGPRT test), butanal, reaction products with aniline was negative. According to CLP classification criteria (Regulation (EC) No 1272/2008) a classification is not justified.