Blue MGi 1037-Na (notification presscake)

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:

June 14, 2000 - June 29, 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Directive 92/69/EEC, B.14

Version / remarks:

1992

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Method

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:

Type and composition of metabolic activation system:
- source of S9 : liver microsomal fraction
- method of preparation of S9 mix : Livers of 8-12 weeks old male rats, strain Wistar which received three applications of 80 mg/kg bw Phenobarbital i.p. dissolved in aqua deionised and beta-naphthoflavone orally dissolved in corn oil. The livers were prepared 24 h after the last treatment.
S9 liver microsomal fraction was also obtained and used from hamsters. The livers of 7-8 weeks old male Syrian golden hamsters.
- concentration or volume of S9 mix and S9 in the final culture medium : 15% final concentration (rat S9) or 30% (hamster S9)

Test concentrations with justification for top dose:

Concentration range in the main test (with metabolic activation): 33 - 5000 µg/plate
Concentration range in the main test (without metabolic activation): 33 - 5000 µg/plate

Vehicle / solvent:

Solvent: Bidistilled water

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without S9 mix, TA1535, TA100

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-Nitro-o-phenylene-diamine

Remarks:

without S9 mix, TA1537, TA98

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: methyl methane sulfonate

Remarks:

without S9 mix, TA 102

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

with S9 mix, TA1535, TA100, TA1537, TA98, TA102

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

congo red

Remarks:

with hamster S9 mix only, TA 98

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments : 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance adde in agar (plate incorporation, experiment I), pre-incubation (experiment II)

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 30 min at 30°C
- Exposure duration/duration of treatment: 48 h at 37°C

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: reduction in number of revertants

Evaluation criteria:

A test item is considered positive if either a dose related increase in the number of revertants or a biologically relevant and reproducible increase for at least one test concentration is induced. A test item producing neither a dose related increase in the number of revertants nor a biologically relevant and reproducible positive response at any one of the test points is considered non-mutagenic in this system. A biologically relevant response is described as follows: A test item is considered mutagenic if in strains TA 98, TA 100, and TA 102 the number of reversions is at least twice as high and in strains TA 1535 and TA 1537 at least three times higher as compared to the spontaneous reversion rate. Also, a dose-dependent increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test item regardless whether the highest dose induced the above described enhancement factors or not.

Statistics:

No statistical evaluation

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:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

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:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 97

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

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:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

STUDY RESULTS
- see attachement
Slight toxic effects, evident as a reduction in the number of revertants, were observed without metabolic activation in strains TA 1535 (33 µg/plate) and TA 98 (10 µg/plate) and with metabolic activation in strain 1537 (333 and 1000 µg/plate) in experiment I, and at 5000 µg/plate with metabolic activation in strain TA 1537 in experiment II. The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used.

HISTORICAL CONTROL DATA
- see attachment

Applicant's summary and conclusion

Conclusions:

BLUE MGi 1037 is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

Executive summary:

A study according OECD TG 471 was performed to investigate the potential of BLUE MGi 1037 to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and TA 102.

The assay was performed in two independent experiments, both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

33; 100; 333; 1000; 2500; and 5000 µg/plate.

Slight toxic effects, evident as a reduction in the number of revertants, were observed without metabolic activation in strains TA 1535 (33 µg/plate) and TA 98 (10 µg/plate) and with metabolic activation in strain 1537 (333 and 1000 µg/plate) in experiment I, and at 5000µg/plate with metabolic activation in strain TA 1537 in experiment II.

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with BLUE MGi 1037 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 and 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.

Therefore, BLUE MGi 1037 is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.