Reaction mass of 5-[(Disubstituted-dihydrocarbopolycyclyl)diazenyl]-6-hydroxy-4-methyl-2-substituted-1-propyl-1,2-dihydropyridine-3-carbonitrile and 1-Butyl-5-[(disubstituted-dihydrocarbopolycyclyl)diazenyl]-6-hydroxy-4-methyl-2-substituted-1,2-dihydropyridine-3-carbonitrile

Genetic toxicity: in vitro

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Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

"Ninth Addendum to OECD Guidelines for Testing of Chemicals". Section 4, No. 471:
"Bacterial Reverse Mutation Test", adopted July 21,1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: "Commission Directive 2000/32/EC, L1362000, Annex 4D", dated May 19, 2000

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Japanese Guidelines

Version / remarks:

"Kanpoan No. 287 - Environment Protection Agency"
"Eisei No. 127 - Ministry of Health & Welfare"
"Heisei 09/10/31 Kikyoku No. 2 -- Ministry of International Trade & Industry"

Deviations:

no

Principles of method if other than guideline:

Guidelines followed

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Reference

Name:

Unnamed

Type:

Constituent

Test material form:

solid

Details on test material:

Batch: P2/05 UL
Appearance: light orange crystalline powder
Expiration date: 31.05.2011
Storage: at room temperature

Specific details on test material used for the study:

Identity: FAT 41039/A
Batch No.: P2/05 UL
State of the substance: Solid
Colour: Light orange
Purity: Content of organic pigments: >99 %; 97 % main components
Solubility in water: Approximately < 0.001 g/L at room temperature
Solubility in vehicle: Soluble in: DMF (ca. 0.15 %), ethanol (< 0.01 %), toluol (ca. 0.05 %), aceton (ca. < 0.05 %)
Stability in solvent: 7 days at room temperature in water, saline polyethylene glycol and carboxymethylcellulose 1 day at room temperature in vaseline and FCA
Storage:At room temperature
Expiration Date: May 31, 2011

Method

Target gene:

histidine auxotrophs

Species / strain

Species / strain / cell type:

bacteria, other: Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 Escherichia coli WP2 uvrA

Metabolic activation system:

S9 liver fractions of rats induced with phenobarbital/beta-naphthoflavone

Test concentrations with justification for top dose:

Pre-Experiment/Experiment I: 3; 10, 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment II: 33; 100; 333; 1000; 2500; and 5000 µg/plate

Vehicle / solvent:

Solvent: Ethanol- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative non-toxicity to the bacteria.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-NOPD: for TA 1537, TA 98 without S9; 2-aminoanthracene (2-AA): for TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA with S9

Details on test system and experimental conditions:

METHOD OF APPLICATION:; in agar (plate incorporation)

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

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

Results and discussion

Test results

Key result

Species / strain:

other: Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

exception in strain TA 1537 with metabolic activation in experiment I at 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Discussion of Results:

- The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments, with the exception in strain TA 1537 with metabolic activation in experiment I, reduced background growth was observed at 5000 µg/plate.

- No toxic effects, evident as a reduction in the number of revendants, occurred in the test groups with and without metabolic activation with the exception in strain TA 1537 with metabolic activation in experiment I, a reduction in the number of revendants was observed at 5000 µg/plate.

- No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with FAT 41039/A at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). - There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

- Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies.

Applicant's summary and conclusion

Conclusions:

FAT 41039/A did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used in this bacterial reverse mutation assay.

Executive summary:

FAT 41039/A was assessed for its potential to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration and the controls were tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment/Experiment I: 3; 10, 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment II: 33; 100; 333; 1000; 2500; and 5000 µg/plate

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments, with the exception in strain TA 1537 with metabolic activation in experiment I, reduced background growth was observed at 5000 µg/plate. No toxic effects, evident as a reduction in the number of revendants, occurred in the test groups with and without metabolic activation with the exception in strain TA 1537 with metabolic activation in experiment I, a reduction in the number of revendants was observed at 5000 µg/plate. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with FAT 41039/A at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.