2-[1-(ethylsulfonyl)azetidin-3-ylidene]acetonitrile

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 December 2011 to 03 January 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

As described by green and Muriel (1976)

GLP compliance:

not specified

Remarks:

Not specifed in the report

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-[1-(ethylsulfonyl)azetidin-3-ylidene]acetonitrile

EC Number:

691-838-1

Cas Number:

1187595-85-2

Molecular formula:

C7H10N2O2S

IUPAC Name:

2-[1-(ethylsulfonyl)azetidin-3-ylidene]acetonitrile

Test material form:

solid: particulate/powder

Details on test material:

Lot no: AH7-H71131-082

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: AH7-H71131-082
- Expiration date of the lot/batch: Not Specified

Method

Target gene:

Determine the potential of 3017431 and/or its metabolites to induce reverse mutations at the histidine locus in several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain WP2uvrA in the presence or absence of an exogenous mammalian metabolic activation system.

Strain Histidine mutation Mutation type
TA1537 hisC3076 Frameshift
TA98 hisD3052/R-factor* Frameshift
TA1535 hisG46 Base-pair substitutions
TA100 hisG46/R-factor* Base-pair substitutions

The Escherichia coli WP2uvrA strain detects base-pair substitutions.

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Rat liver microsomal enzymes were prepared from male Sprague Dawley rats that had been injected intraperitoneally with Aroclor 1254 at 500 mg/kg body weight.

Test concentrations with justification for top dose:

Dose-range finding test: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate.

Vehicle / solvent:

Dimethyl sulfoxide (DMSO)

Controls

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Details on test system and experimental conditions:

Test article dilutions were prepared immediately before use and delivered to the test system at
room temperature under yellow light. One-half (0.5) milliliter of S9 or Sham mix, I 00 µL of tester strain and 50 µL of vehicle or test article dilution were added to 2.0 mL of molten selective top agar at 45±2°C. After vortexing, the mixture was overlaid onto the surface of 25 mL of minimal bottom agar. When plating the positive controls, the test article aliquot was replaced by a 50 µL aliquot of appropriate positive control. After the overlay had solidified, the plates were inverted and incubated for approximately 48 to 72 hours at 37±2°C. Plates that were not counted immediately following the incubation period were stored at 2-8°C until colony counting could be conducted.

The condition of the bacterial background lawn was evaluated for evidence of test article
toxicity by using a dissecting microscope. Precipitate was evaluated by visual examination without magnification. Revertant colonies for a given tester strain and activation condition were counted either entirely by automated colony counter or entirely by hand unless the plate exhibited toxicity. Plates with sufficient test article precipitate to interfere with automated colony counting were counted manually.

Evaluation criteria:

The following criteria must be met for the mutagenicity assay to be considered valid. All
Salmonella tester strain cultures must demonstrate the presence of the deep rough mutation (rfa) and the deletion in the uvrB gene. Cultures of tester strains TA98 and TAIOO must demonstrate the presence of the pKMIOl plasmid R-factor. All WP2 uvrA cultures must demonstrate the deletion in the uvrA gene. All cultures must demonstrate the characteristic mean number of spontaneous revertants in the vehicle controls as follows (inclusive): TA98, 10 - 50; TAIOO,
80 - 240; TA1535, 5 - 45; TA1537, 3 - 21; WP2 uvrA, 10 - 60. To ensure that appropriate
numbers of bacteria are plated, tester strain culture titers must be greater than or equal to
0.3xl09 cells/rnL. The mean of each positive control must exhibit at least a 3.0-fold increase in
the number of revertants over the mean value of the respective vehicle control. A minimum of three non-toxic dose levels is required to evaluate assay data. A dose level is considered toxic if one or both of the following criteria are met: (1) A >50 % reduction in the mean number of revertants per plate as compared to the mean vehicle control value. This reduction must be accompanied by an abrupt dose-dependent drop in the revertant count. (2) At least a moderate reduction in the background lawn (background lawn code 3, 4 or 5)

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Additional information on results:

Toxicity was not observed.

Applicant's summary and conclusion

Conclusions:

All criteria for a valid study were met as described in the protocol. The results of the Bacterial
Reverse Mutation Assay indicate that, under the conditions of this study, test article 3017431 did not cause mutagenic response in any of the tester strains in either the presence or absence of Aroclor-induced rat liver S9.