L 165

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:

October 29, 2003 - November 20, 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study has been performed in accordance with OECD 471 (1987), EU Method B.13/14 (2000) and according to GLP principles.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

(1987)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

(2000)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Details on test material:

- Name of test material (as cited in study report): Leuchtstoff L 165
- Physical state: white solid

Method

Target gene:

- S. typhimurium: Histidine gene

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone

Test concentrations with justification for top dose:

- Experiment 1:
Preliminary test, TA98 and TA100:
Without and with S9-mix: 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate
Main study, TA1535, TA1537, TA102:
Without and with S9-mix: 33, 100, 333, 1000, 2500 and 5000 µg/plate
- Experiment 2, all strains:
Without and with S9-mix: 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate

Vehicle / solvent:

- Solvent used: DMSO
- Justification for choice of solvent: 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:

other: see section "Any other information on results incl. tables"

Details on test system and experimental conditions:

METHOD OF APPLICATION:
- Experiment 1: in agar (plate incorporation)
- Experiment 2: pre-incubation

DURATION
- Exposure duration: 48 hour

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain.

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies.

OTHER EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was determined.

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 TA 102) 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.

Results and discussion

Test results

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: See section "any other information on results incl. tables"

RANGE-FINDING/SCREENING STUDIES:
- Precipitation: no data
- No toxicity or mutagenicity was observed up to and including the top dose of 5000 µg/plate

COMPARISON WITH HISTORICAL CONTROL DATA:
- The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. Only the positive control value was exceeded in strain TA1537 with S9-mix in experiment 2, only indicating the sensitivity of the strains.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- No visible reduction of the background growth was observed up to and including the top dose of 5000 µg/plate, in both experiments
- Toxic effects, evident as a reduction in the number of revertants, were observed with and without metabolic activation in strain TA102 at 1000 µg/plate and above in experiment 1.
- No toxicity or mutagenicity was observed up to and including the top dose of 5000 µg/plate in experiment 2

Any other information on results incl. tables

The test substance precipitated in the overlay agar at the following concentrations (µg/plate):

Strain	Experiment 1		Experiment 2
	Without S9-mix	With S9-mix	Without S9-mix	With S9-mix
TA1535	2500, 5000	5000	2500, 5000	1000 – 5000
TA1537	2500, 5000	1000 – 5000	1000 – 5000	1000 – 5000
TA98	-	-	1000 – 5000	333 – 5000
TA100	-	-	1000 – 5000	333 – 5000
TA102	1000 – 5000	1000 – 5000	333 – 5000	333 – 5000

- means: no visible precipitation observed

The undissolved particles had no influence on the scoring

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

The mutagenic activity of the substance was evaluated in accordance with OECD 471 (1987), EU Method B.13/14 (2000) and according to GLP principles. Based on the results of this study it is concluded that the substance is not mutagenic in the Bacterial Reverse Mutation Assay.

Executive summary:

The mutagenic activity of the substance was evaluated in accordance with OECD 471 (1987), EU Method B.13/14 (2000) and according to GLP principles. The test was performed in two independent experiments, at first a direct plate assay was performed and secondly a preincubation assay, both in the absence and presence of S9-mix. Adequate negative and positive controls were included. The 5 bacterial strains tested were S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102.

In both experiments, the substance did not induce a significant increase in the number of revertant colonies in any of the 5 strains at any concentration, up to 5000 µg/plate, neither in the presence nor in the absence of metabolic activation. Based on the results of this study it is concluded that the substance is not mutagenic in the Bacterial Reverse Mutation Assay.