Pentyl 2-cyano-(3-(6-methoxybenzothiazol-2-ylimino)-2,3-dihydro-1H-isoindol-1-ylidene)acetate

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:

key study

Study period:

From Sep. 13, 2000 to Jan. 21, 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: EEC Directive 92/69, L 383 A, Annex B 14

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study report): FC 84508

Method

Target gene:

Not applicable

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:

S. typhimurium TA 102

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

Plate incorporation test:
a: without metabolic activation:
50,160, 500, 1600 and 5000 µg/plate
b: with metabolic activation:
50,160, 500, 1600 and 5000 µg /plate

Preincubation test:
a: without metabolic activation:
16, 50, 160, 500, 1600 and 5000 µg/plate
b: with metabolic activation:
16, 50, 160, 500, 1600 and 5000 µg /plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

mitomycin C

other: 2-aminoanthrecene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) and preincubation

DURATION
- Preincubation period: 20 to 30 min at approx. 30 °C
- Exposure duration: 48 h at approx. 37 °C

NUMBER OF REPLICATIONS: Three

DETERMINATION OF CYTOTOXICITY
- Method: Microscopic thinning of the bacterial lawn and at least halving of the number of spontaneously occurring mutants compared to the corresponding solvent control value

Evaluation criteria:

The test item was considered positive if (a) at least a 2-fold increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control at complete bacterial background lawn
(b) a dose-related increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control in at least two to three concentrations of the test compound at complete bacterial background lawn.

If the test substance does not achieve either of the above criteria, it is considered to show no evidence of mutagenic activity in this system

Statistics:

According to the OECD guideline 471, a statistical analysis of the data was not mandatory

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

at cytotoxic concentrations without dose-dependency

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 6

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Visible precipitation of the test compound on the plates was observed at 500 µg/plate and above.
- Other confounding effects: Because of heavy precipitation of the test compound the bacterial lawn could only be evaluated at the dose level of 1600 µg /plate and lower concentrations in the plate incorporation test.

COMPARISON WITH HISTORICAL CONTROL DATA: Yes

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Toxicity: In the plate incorporation test toxicity was observed in a dose range of 1600 µg/plate and above in the absence and in the presence of metabolic activation. In the preincubation test toxicity was not observed either with or without metabolic activation.

Any other information on results incl. tables

Sterility checks and control plates:

Sterility of S9-mix and the test compound were indicated by the absence of contamination on the test material and S9-mix sterility check plates. Control plates (background control and positive controls) gave the expected number of colonies, i.e. values were within the laboratory's historical control range.

Applicant's summary and conclusion

Conclusions:

Under the test conditions, the test substance is considered to be mutagenic in Salmonella typhimurium strain TA 98 in the presence of exogenous metabolic activation.

Executive summary:

A study was conducted to determine the mutagenic potential of test substance according to OECD Guideline 471, EPA OPPTS 870.5100 and EU method B.14. in compliance with GLP.

 

Salmonella typhimurium strains TA 100, TA 1535, TA 1537, TA 98 and TA 102 were used in the mutagenicity assay. Two independent mutagenicity studies were conducted (one plate incorporation test and one preincubation test), each in the absence and in the presence of a metabolizing system derived from a rat liver homogenate. For both studies, the test substance was suspended in DMSO, and each bacterial strain was exposed to 5 dose levels, in the preincubation test to 6 dose levels. Visible precipitation of the test compound on the plates was observed at 500 µg/plate and above.

Because of heavy precipitation of the test compound the bacterial lawn could only be evaluated at the dose level of 1600 µg /plate and lower concentrations in the plate incorporation test.

 

The concentrations for the plate incorporation test were 50, 160,500, 1600 and 5000 µg/plate. Because of toxicity in the plate incorporation test dose levels from 16 to 5000 µg/plate were chosen for the preincubation test. Control plates without mutagen showed that the number of spontaneous revertant colonies was within the historical control range. All the positive control compounds showed the expected increase in the number of revertant colonies.

 

In the plate incorporation test toxicity was observed in a dose range of 1600 µg/plate and above in the absence and in the presence of metabolic activation. In the preincubation test toxicity was not observed either with or without metabolic activation.

 

In the presence of metabolic activation, treatment of the bacterial strains with test substance resulted in relevant and dose-dependent increases in the number of revertant colonies with the strain TA 98.

 

Under the test conditions, the test substance is considered to be mutagenic in Salmonella typhimurium strain TA 98 in the presence of exogenous metabolic activation.