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Genetic toxicity: in vitro

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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 1998-02-06 to 1998-03-23
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP lab following OECD guideline

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Butylal
- Physical state: colorless liquid
- Analytical purity: >99.99%
- Impurities (identity and concentrations): Formaldehyde (0.0038% w/w), water (not detected), n butanol (not detected).
- Composition of test material, percentage of components: Formaldehyde (0.0038% w/w), water (not detected), n butanol (not detected), butylal (>99.99% w/w)
- Lot/batch No.: E971205
- Expiration date of the lot/batch: No expiry date
- Container: one smoked glass flask
- Storage condition of test material: at room temperature and protected from light

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
None
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
S. typhimurium TA 102
Details on mammalian cell type (if applicable):
None
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
None
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 mix (see Point 1 under "Any other information on materials and methods incl. table")
Test concentrations with justification for top dose:
The test substance was dissolved in the vehicle at a concentration
. of 50 mg/ml for the preliminary toxicity test and the first mutagenicity experiment,
. of 50, 10 and 5 mg/ml for the second mutagenicity experiment.
The preparations were made immediately before use.
Vehicle / solvent:
The vehicle was dimethylsulfoxide (DMSO), batch Nos. K22294850 549 and I743850 731 (Merck Clévenot, 77500 Chelles, France).

Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Six known mutagens, dissolved in dimethylsulfoxide (except for mitomycin C which is dissolved in distilled water), were used to check the sensitivity of the test system. Please refer to Table 1 of "Any other information on materials and methods incl. tabl
Details on test system and experimental conditions:
Treatment
The experiments were performed according to:
. direct plate incorporation method (preliminary toxicity test, both experiments without S9 mix, first experiment with S9 mix): test substance solution (0.05 to 0.1 ml), S9 mix (0.5 ml) when required and bacterial suspension (0.1 ml) were mixed with 2 ml of overlay agar (containing traces of the relevant aminoacid and biotin and maintained at 45°C). After rapid homogenization, the mixture was overlaid onto a Petri plate containing minimum medium.
. preincubation method (second experiment with S9 mix): test substance solution (0.05 to
0.1 ml), S9 mix (0.5 ml) and bacterial suspension (0.1 ml) were incubated for 60 minutes at 37°C before adding the overlay agar and pouring onto the surface of a minimum agar plate.

After 48 to 72 hours of incubation at 37°C, revertants were scored with an automatic counter (Artek counter, model 880, O.S.I., 75015 Paris, France).

Preliminary toxicity test
To assess the toxicity of the test substance to the bacteria, six dose-levels (one plate/dose-level) were tested in the TA 98, TA 100, TA 102 and WP2 uvrA strains, with and without S9 mix.
The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.

Mutagenicity experiments
In two independent experiments, five dose-levels of BUTYLAL (three plates/dose-level) were tested on each strain, with or without S9 mix.

In each experiment, the following controls were included using triplicate plates:
. vehicle controls: each bacterial tester strain treated with the vehicle,
. positive controls: each bacterial tester strain treated with appropriate reference mutagens.

The sterility of the S9 mix was checked before the beginning and at the end of each experiment and was found to be satisfactory.
Evaluation criteria:
Treatment of results
In each experiment, for each strain and for each experimental point, the number of revertants per plate was scored. The individual results and the mean number of revertants, with the corresponding standard deviation and ratio (mutants obtained in the presence of the test substance/mutants obtained in the presence of the vehicle), are presented in a table.

Acceptance criteria
This study would be considered valid since the following criteria are fully met:
.the number of revertants in the vehicle controls is consistent with our historical data
.the number of revertants in the positive controls is higher than that of the vehicle controls and is consistent with our historical data.

Evaluation criteria
A reproducible two-fold increase in the number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationship was considered as a positive result. Reference to historical data, or other considerations of biological relevance may also be taken into account in the evaluation of the data obtained.

Results and discussion

Test resultsopen allclose all
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:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
determined in the preliminary test
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium 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
Remarks:
determined in the preliminary test
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
determined in the preliminary test
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
PRELIMINARY TOXICITY TEST
The test substance was freely soluble in the vehicle (DMSO) at 50 mg/ml.
Consequently, with a maximum dose volume of 100 µl/plate, the dose-levels were 10, 100, 500, 1000, 2500 and 5000 µg/plate.
No precipitate was observed in the Petri plates when scoring the revertants at all dose-levels.
Without S9 mix, slight to marked toxicity was observed in the TA 98 strain at dose-levels ≥ 500 µg/plate. In the TA 100 strain, moderate to total toxicity was induced at the same dose-levels. At dose-levels ≥ 100 µg/plate, slight to total an slight to moderate toxicity were noted in the TA 102 and WP2 uvrA strains, respectively.
With S9 mix, slight toxicity was observed in the TA 98 and WP2 uvrA strains, at 5000 µg/plate and at dose-levels ≥ 2500 µg/plate, respectively.
Moderate to total toxicity was induced in the TA 100 strain at dose-levels ≥ 1000 µg/plate and in the TA 102 strain at dose-levels  500 µg/plate.

MUTAGENICITY EXPERIMENTS
The number of revertants of the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid.
Since the test substance was toxic in the preliminary test, the choice of the highest dose-level was based on the level of toxicity, according to the criteria specified in the international guidelines.
No precipitate was observed in the Petri plates when scoring the revertants at all dose-levels.

Experiments without S9 mix:
The selected treatment levels were as follows:
• 62.5, 125, 250, 500 and 1000 µg/plate, for the TA 1535 strain in both experiments and for the TA 1537 strain in the second experiment,
• 31.25, 62.5, 125, 250 and 500 µg/plate, for the 5 remaining strains in the first experiment and for the 4 remaining strains in the second experiment.

In the TA 1535 strain, slight to moderate toxicity was observed at dose-levels ≥ 500 µg/plate in the first experiment. In the second experiment with this strain as well as with the TA 1537 strain, only slight toxicity was noted at 1000 µg/plate. Slight toxicity was noted at 500 µg/plate in the TA 98 strain (first experiment) and in the TA 102 strain (second experiment). In the first experiment with the TA 102 strain, moderate toxicity was observed at dose-levels ≥ 250 µg/plate.
In both the TA 100 and WP2 uvrA strains, slight toxicity was induced at dose-levels ≥ 250 µg/plate, in the first experiment only.
No increase in the number of revertants, in comparison with the vehicle control values, was observed in all tester strains in both experiments.

Experiments with S9 mix:
The selected treatment levels were as follows:
• 31.25, 62.5, 125, 250 and 500 µg/plate, for the TA 102 strain in the first experiment as well as for the TA 1535, TA 1537 and TA 98 strains in the second experiment,
• 62.5, 125, 250, 500 and 1000 µg/plate, for the TA 100 strain in both experiments, for the TA 1535 strain in the first experiment and for the TA 102 strain in the second experiment,
• 312.5, 625, 1250, 2500 and 5000 µg/plate, for the TA 1537, TA 98 strains in the first experiment and for the WP2 uvrA strain in both experiments.

Slight toxicity was observed in the TA 1535 and TA 100 strains at 1000 µg/plate in the first experiment. In the second experiment (preincubation method), slight toxicity was induced in the TA 1535 strain at 500 µg/plate and moderate to total toxicity was noted in the TA 100 strain at dose-levels  500 µg/plate.
Slight to marked toxicity was observed in both TA 1537 and TA 98 strains at dose-levels ≥ 1250 µg/plate in the first experiment. With the preincubation method, slight toxicity was noted at 500 µg/plate in these two tester strains.
In the TA 102 strain, slight toxicity was observed at 1000 µg/plate (second experiment).
Slight to moderate toxicity was induced in the WP2 uvrA strain in the first experiment at dose-levels ≥ 1250 µg/plate. In the second experiment with this strain, slight to marked toxicity was observed at dose-levels ≥ 625 µg/plate.

The test substance did not induce any significant increase in the number of revertants, in both experiments, in any of the six strains.

Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Under the experimental conditions, the test substance BUTYLAL does not show mutagenic activity in the bacterial reverse mutation test on Salmonella typhimurium and Escherichia coli.
Executive summary:

The objective of this study was to evaluate the potential of the test substance BUTYLAL to induce reverse mutation inSalmonella typhimuriumandEscherichia coli.

 

 

Methods 

A preliminary toxicity test was performed to define the dose-levels of BUTYLAL to be used for the mutagenicity study. The test substance was then tested in two independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254.

 

Both experiments were performed according to the direct plate incorporation method except the second with S9 mix, which was performed according to the preincubation method (60 minutes,).

 

Five strains of bacteriaSalmonella typhimurium: TA 1535, TA 1537, TA 98, TA 100 and
TA 102 and one strain ofEscherichia coli: WP2 uvrA were used. Each strain was exposed to five dose-levels of the test substance (three plates/dose-level). After 48 to 72 hours of incubation at, the revertant colonies were scored.

The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.

 

The test substance BUTYLAL was dissolved in dimethylsulfoxide (DMSO).

 

The dose-levels of the positive controls were as follows:

 

without S9 mix:

. 1 µg/plate of sodium azide (NaN3): TA 1535 and TA 100 strains,

. 50 µg/plate of 9-Aminoacridine (9AA): TA 1537 strain,

. 0.5 µg/plate of 2-Nitrofluorene (2NF): TA 98 strain,

. 0.5 µg/plate of mitomycin C (MMC): TA 102 strain,

. 2 µg/plate of N-ethyl-N-nitro-nitrosoguanidine (ENNG):WP2 uvrA strain.

 

with S9 mix:

. 2 µg/plate of 2-Anthramine (2AM): TA 1535, TA 1537, TA 98 and TA 100 strains,

. 10 µg/plate of 2-Anthramine (2AM): TA 102 andEscherichia coliWP2 uvrA strains.

 

 

Results 

The test substance was freely soluble in the vehicle at 50 mg/ml.

Consequently, with a maximum dose volume of 100 µl/plate, the dose-levels for the preliminary toxicity test were 10, 100, 500, 1000, 2500 and 5000 µg/plate.

Since the test substance was toxic in the preliminary test, the choice of the highest dose-level was based on the level of toxicity, according to the criteria specified in the international guidelines.

No precipitate was observed in the Petri plates when scoring the revertants at all dose-levels.

 

Experiments without S9 mix:

The selected treatment levels were as follows:

·      62.5, 125, 250, 500 and 1000 µg/plate, for the TA 1535 strain in both experiments and for the TA 1537 strain in the second experiment,

·      31.25, 62.5, 125, 250 and 500 µg/plate, for the 5 remaining strains in the first experiment and for the 4 remaining strains in the second experiment.

 

Slight to moderate toxicity was observed in all tester strains, depending on the dose-levels.

 

No increase in the number of revertants, in comparison with the vehicle control values, was observed in all tester strains in both experiments.

 

Experiments with S9 mix:

The selected treatment levels were as follows:

·      31.25, 62.5, 125, 250 and 500 µg/plate, for the TA 102 strain in the first experiment as well as for the TA 1535, TA 1537 and TA 98 strains in the second experiment,

·      62.5, 125, 250, 500 and 1000 µg/plate, for the TA 100 strain in both experiments, for the TA 1535 strain in the first experiment and for the TA 102 strain in the second experiment,

·      312.5, 625, 1250, 2500 and 5000 µg/plate, for the TA 1537, TA 98 strains in the first experiment and for the WP2 uvrA strain in both experiments.

 

Slight to total toxicity was observed in all tester strains, depending on the dose-levels and the experimental conditions.

The test substance did not induce any significant increase in the number of revertants, in both experiments, in any of the six strains.

The number of revertants of the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid.

 

Conclusion

 Under the experimental conditions, the test substance BUTYLAL does not show mutagenic activity in the bacterial reverse mutation test on Salmonella typhimurium and Escherichia coli.