Registration Dossier

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:
25 June 1993 to 29 September 1993
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP methodology followed and O.E.C.D. n° 471 (1983) and E.E.C. 84/449 - annex V - method B14 (1984) used to performed the experiment.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1993
Report Date:
1993

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
Use of Sodium Azide (NaA) instead of Methyl Methane Sulfonate (MMS) as positive control on TA100 without metabolc activation. The test article appearance was not lquid to viscous as mentioned in the signed protocol.
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
semi-solid (amorphous): gel
Remarks:
migrated information: paste
Details on test material:
- Identification: FAT 92267/A.
- Identification for the study: 01255 G3 002.
- Dispensary code n°: 515-93.
- Presentation: Light yellow paste.
- Batch: 3.86.
- Packaging: Plastic flask.- Quantity received: 100 gr.
- Date of receipt: 7 July 1993.
- Storage: Room temperature.
- Expiry date: August 1994.
- Purity: 81.1% was taken into account for test article formulation and tested dose levels.

Method

Target gene:
Salmonella typhimurium histidine (his) reversion system.
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 1538
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 from liver of rats treated with AROCLOR 1254.
Test concentrations with justification for top dose:
- Preliminary study only on strain TA 100: 6173/1235/247/49/10 microgr./plate corresponding to active ingredient concentrations of 5000/1000/200/40 and 8 microgr/plate.
- Main study number 1 (in all strain): 6173/1235/247/49/10 microgr./plate corresponding to active ingredient concentrations of 5000/1000/200/40 and 8 microgr/plate.
- Main study number 2 (in all strain): 6173/3087/1543/772/386 microgr./plate corresponding to a active ingredient concentrations of 5000/2500/1250/625/313 microgr/plate.
Vehicle / solvent:
- Identification: Absolute ethanol
- Supplier: Prolabo
- Batch number: 92113 and 92350.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Absolute ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
ethylmethanesulphonate
other: 2-Aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: In agar (plate incorporation)

NUMBER OF REPLICATIONS: Triplicate

All the preparations were used on the day of formulation.

Treatment design:
After treatment each tube of molten agar contains:
- 2 ml of molten agar
- 100 microl. of bacterial culture
- 50 or 100microl. of the test article formulated depending on the vehicle used
- 500 microl. of S9 Mix (if appropriate)

The mixture will be poured onto VB medium constituing an agar upper layer. The petri dishes will be incubated at 37°C +/1°C for 48 hours minimum.


Evaluation criteria:
The assay will usually be considered valid if the following criteria are met :
- The mean negative control counts fall within the normal range.
- The positive control chemicals induce clear increases in revertant numbers.
confirming discrimination between different strains, and an active Sc Mix preparation
- No more than 5 % of the plates in the assay are lost through contamination or some other unforeseen event.

The test article will be considered to be clearly mutagenic If :
- The number of revendants in presence of the test article is greater than or equal to twice the number of revertants in the negative control at one dose level (the upper dose before toxicity) or more with a dose correlation
- The positive trends/effects are reproducible.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Toxicity was observed at 1235 and 6173 microgr./plate.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Toxicity was observed at 1235 and 6173 microgr./plate.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

- PRELIMINARY STUDY:

A precipitate appeared during incorporation of the test article solutions at concentrations of 6173 and 1235 microgr./plate.

- MAIN STUDY N°1:

A precipitate was noted during incorporation at 1235 and 6173 microgr./plate. On a few occasion the colonies were counted manually instead of automatically.

*Without metabolic activation:

Signs of toxicity were observed at 6173 microgr./plate in all the tester strains and at 1235 microgr./plate in TA1535, TA 1537 and TA

1538 only.

For all tested concentrations, the mean number of colonies was less than twice the mean number of colonies in the negative control except with TAI 537 at 6173 microgr/plate where severe toxicity was noted. In this case, the increase of the mean number of colonies cannot be interpreted as the result of a mutagenic activity.

All data were acceptable and no positive increase in the number of revertants per plate containing the test article was observed with any of the tester strains. All criteria for a valid study were met.

*With metabolic activation:

Signs of toxicity were noted in all the tester strains at 6173 microgr./plate.

For all tested concentrations, the mean number of colonies was less than twice the mean number of colonies in the negative control with each of the tester strains.

All data were acceptable and no positive increase in the number of revertants per plate containing the test article was observed with any of the tester strains. All criteria for a valid study were met.

- MAIN STUDY N°2:

From the results obtained in the first study, a closer range of doses near to the top limit dose were used in this study : 386 / 772 / 1543 / 3087 and 6173 microgr./plate in both the presence and absence of metabolic activation system (corresponding to active ingredient concentrations of 313, 625, 1250, 2500 and 5000 microgr./plate respectively).

The highest dose-level corresponded approximately to an active ingredient concentration of 5000 microgr./plate which is the required maximum dose-level.

A precipitate was constantly noted after the incorporation at 1543 / 3087 and 6173 microgr/plate,

Precipitation was also occasionally seen at lower dose levels. As a result of this precipitate, colonies were systematically counted manually at 6173 microgr./plate and on a few occasions only at 3087 and 1543 microgr./plate.

*Without metabolic activation:

Toxicity was observed in all the tester strains at the two highest dose-levels except with TA98 at 3087 microgr./plate.

For all concentrations, the mean number of colonies was less than twice the mean number of colonies in the negative control with each of the tester strains.

All data were acceptable and no positive increase in the number of revendants per plate containing the test article was observed with any of the tester strains. All criteria for a valid study were met.

*With metabolic activation:

Toxicity was observed in all the tester strains at the highest dose-level of 6173 microgr./plate.

For all concentrations, the mean number of colonies was less than twice the mean number of colonies in the negative control with each of the tester strains.

All data were acceptable and no positive increase in the number of revertants per plate containing the test article was observed with any of the tester strains. All criteria for a valid study were met.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Under the experimental conditions employed, It can be concluded that the test article, FAT 92267/A did not cause an increase in the number of revendants per plate of any tester strains used TA98, TAI00, TA 1535, TAI537 and TA 1538 either in presence or absence of a metabolic activation system.
In both the two Independent studies performed.
Executive summary:

The experiment was performed following GLP methodology and the OECD Guideline 471 (Bacterial Reverse Mutation Assay) was used.

The mutagenic potential of FAT 92267/A was evaluated using Salmonella typhimurium in the absence and presence of a liver microsomal system.

The test article (FAT 92267/A) was tested on 5 strains of Salmonella typhimurium (TA98, TA 100, TA 1535, TA 1537, TA 1538), with and without metabolic activation.

A range of sub-toxic concentrations was determined in a preliminary study on the strain TA 100 without metabolic activation.

The 5 concentrations (6173, 1235, 247, 49 and 10 microgr./plate corresponding to active ingredient concentrations of 5000, 1000, 200, 40 and 8 microgr./plate respectively) were tested in triplicate on the strains mentioned above with and without metabolic activation.

The results were confirmed in a second independent study, using a closer range near to the top limit dose (6173, 3087, 1543, 772 and 386 microgr./plate corresponding to active ingredient concentrations of 5000, 2500, 1250, 625 and 313 microgr./plate respectively).

A negative control (vehicle) and a positive control (specific standard mutagen) were included in each study.

Conclusion:

Under the experimental conditions employed, it can be concluded that the test article, FAT 92267/A, did not cause an increase in the number of revertants per plate of any tester strains used TA 98, TA 100, TA 1535, TA 1537 and TA 1538 either in presence or absence of a metabolic activation system in both the two independent studies performed.