Carbamic acid, [(1S,2S)-3-chloro-2-hydroxy-1-(phenylmethyl)propyl]-, methyl ester

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

other information

Study period:

03/10/95 to 06/16/95

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed in compliance with EPA and FDA Good Laboratory Practice (GLP) Guidelines. Complies with U.S. federal regulation codes (FDA, 21 CFR 58.10; EPA-TSCA, 40 CFR 792.10; EPA-FIFRA, 40 CFR 160.10).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: Complies with U.S. federal regulation codes (FDA, 21 CFR 58.10; EPA-TSCA, 40 CFR 792.10; EPA-FIFRA, 40 CFR 160.10).

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Chlorohydrine

IUPAC Name:

Chlorohydrine

Details on test material:

Test Article: Chlorhydrine
Physical Description: white fluffy solid
Date Received: 02/21/95

Method

Target gene:

Tester Strains

The tester strains used were the Salmonella typhimurium histidine auxotrophs TA98, TA100, TA1535, TA1537, and TA1538 as described by Ames et al (1975The specific genotypes of these strains are shown below:

TESTER STRAIN GENOTYPES
Histidine Mutation: hisG46 ; hisC3076 ; hisD3052 ; TA1535 ; TA1537 ; TA1538 ; TA100 ; TA98.
Additional Mutations: LPS; Repair ; R Factor ; rfa ; uvrB ; - ; rfa ; uvrB ; +R.

In addition to a mutation in the histidine operon, the tester strains contain two additional mutations which enhance their sensitivity to some mutagenic compounds. The rfa wall mutation results in the loss of one of the enzymes responsible for the synthesis of part of the lipopolysaccharide barrier that forms the surface of the bacterial cell wall. The resulting cell wall deficiency increases permeability to certain classes of chemicals such as those containing large ring systems (i.e. benzo (a) pyrene) that would otherwise be excluded by a normal intact cell wall. The second mutation, a deletion of the uvrB gene, results in a deficient DNA excision repair system which greatly enhances the sensitivity of these strains to some mutagens. Since the uvrB deletion extends through the bio gene, all of the tester strains containing this deletion also require the vitamin biotin for growth. Strains TA98 and TA100 also contain the R-factor plasmid, pKMl0l, which further increases the sensitivity of these strains to some mutagens. The mechanism by which this plasmid increases sensitivity to mutagens has been suggested to he by modifying an existing bacterial DNA repair polymerase complex involved with the mismatch-repair process. Tester strains TA98, TA1537, and TA1538 are reverted from histidine dependence (auxotrophy) to histidine independence (prototrophy) by frameshift mutagens. TA1535 is reverted by base substitution mutagens and TA100 is reverted by mutagens which cause both frameshift and base substitution mutations.

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium, other: hisG46 ; hisC3076 ; hisD3052.

Additional strain / cell type characteristics:

other: LPS; Repair ; R Factor.

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1538

Additional strain / cell type characteristics:

other: rfa ; uvrB

Species / strain 3

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: rfa ; uvrB ; +/-R

Metabolic activation:

with and without

Metabolic activation system:

exogenous metabolic system (S9)

Test concentrations with justification for top dose:

The doses tested in the mutagenicity assay were selected based on the results of a dose range finding study using tester strain TA100 and ten doses of test article ranging from 5,000 to 6.67 µg per plate, one plate per dose, both in the presence and absence of S9 mix.

The assay was conducted with six doses of test article in both the presence and absence of S9 mix along with concurrent vehicle and positive controls using three plates per dose. The doses tested in the mutagenicity assay were 5,000, 3,330, 1,000, 667, 333, and 100 µg per plate in both the presence and absence of S9 mix.

Controls

Negative solvent / vehicle controls:

yes

Remarks:

Appropriate vehicle controls will be plated for all strains in the presence and absence of S9 mix. Vehicles compatible with this test system include but will not be limited to: Deionized H2O, dimethylsulfoxide, ethanol, and dimethylformamide.

Positive controls:

yes

Remarks:

2-aminoanthracene (CAS #613-13-8), 2-nitrofluorene (CAS #607-57-8), sodium azide (CAS #26628-22-8), ICR-191 (CAS #1707-45-0).

Positive control substance:

sodium azide

Remarks:

Migrated to IUCLID6: 2-nitrofluorene

Results and discussion

Any other information on results incl. tables

Under the conditions of the study, Chlorohydrine did not cause a positive increase in the number of revertants per plate for any of the tester strains either in the presence or absence of S9 mix.

Applicant's summary and conclusion

Conclusions:

Based on these results, it can be concluded that Chlorohydrine per se, and any of the formed metabolites is not mutagenic in the Ames test under the described experimental conditions.

Executive summary:

MUTAGENICITY TEST ON CHLORHYDRINE IN THE SALMONELLA/MAMMALIAN-MICROSOME REVERSE MUTATION ASSAY (AMES TEST) WITH A CONFIRMATORY ASSAY

Study performed in compliance with EPA and FDA Good Laboratory Practice (GLP) Guidelines. Complies with U.S. federal regulation codes (FDA, 21 CFR 58.10; EPA-TSCA, 40 CFR 792.10; EPA-FIFRA, 40 CFR 160.10).

MATERIALS

Tester Strains

The tester strains used were the Salmonella typhimurium histidine auxotrophs

TA98, TA100, TA1535, TA1537, and TA1538 as described by Ames et al (1975).The specific genotypes of these strains are shown below:

TESTER STRAIN GENOTYPES

Histidine Mutation: hisG46 ; hisC3076 ; hisD3052 ; TA1535 ; TA1537 ; TA1538 ; TA100 ; TA98.

Additional Mutations: LPS; Repair ; R Factor ; rfa ; uvrB ; - ; rfa ; uvrB ; +R.

In addition to a mutation in the histidine operon, the tester strains contain two additional mutations which enhance their sensitivity to some mutagenic compounds. The rfa wall mutation results in the loss of one of the enzymes responsible for the synthesis of part of the lipopolysaccharide barrier that forms the surface of the bacterial cell wall. The resulting cell wall deficiency increases permeability to certain classes of chemicals such as those containing large ring systems (i.e. benzo (a) pyrene) that would otherwise be excluded by a normal intact cell wall. The second mutation, a deletion of the uvrB gene, results in a deficient DNA excision repair system which greatly enhances the sensitivity of these strains to some mutagens. Since the uvrB deletion extends through the bio gene, all of the tester strains containing this deletion also require the vitamin biotin for growth. Strains TA98 and TA100 also contain the R-factor plasmid, pKMl0l, which further increases the sensitivity of these strains to some mutagens. The mechanism by which this plasmid increases sensitivity to mutagens has been suggested to he by modifying an existing bacterial DNA repair polymerase complex involved with the mismatch-repair process. Tester strains TA98, TA1537, and TA1538 are reverted from histidine dependence (auxotrophy) to histidine independence (prototrophy) by frameshift mutagens. TA1535 is reverted by base substitution mutagens and TA100 is reverted by mutagens which cause both frameshift and base substitution mutations.

TEST CONCENTRATIONS

The doses tested in the mutagenicity assay were selected based on the results of a dose range finding study using tester strain TA100 and ten doses of test article ranging from 5,000 to 6.67 µg per plate, one plate per dose, both in the presence and absence of S9 mix. The assay was conducted with six doses of test article in both the presence and absence of S9 mix along with concurrent vehicle and positive controls using three plates per dose. The doses tested in the mutagenicity assay were 5,000, 3,330, 1,000, 667, 333, and 100 µg per plate in both the presence and absence of S9 mix.

Vehicle Controls

Appropriate vehicle controls will be plated for all strains in the presence and absence of S9 mix. Vehicles compatible with this test system include but will not be limited to: Deionized H2O, dimethylsulfoxide, ethanol, and dimethylformamide.

Positive Controls

2-aminoanthracene (CAS #613-13-8), 2-nitrofluorene (CAS #607-57-8), sodium azide (CAS #26628-22-8), ICR-191 (CAS #1707-45-0).

METHODS

Chlorohydrine was evaluated for mutagenic activity in the Ames test. A standard plate incorporation assay in the presence and absence of an exogenous metabolic system (S9) was performed according to OECD guidelines and under GLP. Five Salmonella typhimurium tester strains (TA98, TA100, TA1535, TA1537, and TA1538) were employed. The activity of the S9-mix and the responsiveness of the tester strains were verified by including appropriate controls for each experiment.

Chlorohydrine was dissolved in dimethylformamide (DMF). The doses tested in the definitive assay were selected based on the results of a dose range finding study using tester strain TA100 and ten doses of test article ranging from 5000 to 6.67 µg per plate, one plate per dose, both in the presence and absence of S9 mix. No cytotoxicity was observed with tester strain TA100 in either the presence or absence of S9 mix.

In the definitive assay, the doses tested were 100 to 5,000 µg per plate in both the presence and absence of S9 mix. A confirmatory assay was also run.

RESULTS

Under the conditions of the study, Chlorohydrine did not cause a positive increase in the number of revertants per plate for any of the tester strains either in the presence or absence of S9 mix.

CONCLUSION

Based on these results, it can be concluded that Chlorohydrine per se, and any of the formed metabolites is not mutagenic in the Ames test under the described experimental conditions.