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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:
12 August 2010 - O4 October 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Compliant to GLP and testing guidelines; adequate coherence between data, comments and conclusions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Details on test material:
- Name of test material: distillation residues of butyraldehyde, 2-ethylhexenal and isobutanal hydrogenation by-products
- Physical state: yellow liquid
- Purity: not indicated (complex composition)
- Lot/batch No.: LR_OXO_2010-07-12
- Date of analysis: 12 July 2010
- Expiration date of the lot/batch: July 2011
- Storage conditions of test material: at +4°C and under nitrogen gas.

Method

Target gene:
Histidine operon
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 102
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
-S9 (both experiments): 312.5, 625, 1250, 2500 and 5000 μg/plate
+S9 (1st experiment): 312.5, 625, 1250, 2500 and 5000 μg/plate
+S9 (2nd experiment): 39.06, 78.13, 156.3, 312.5, 625 and 1250 μg/plate
Vehicle / solvent:
- Vehicle used: DMSO
- Justification for choice of solvent/vehicle: test item was freely soluble in the vehicle (DMSO) at 100 mg/mL
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: sodium azide, 9-Aminoacridine, 2-Nitrofluorene, Mitomycin C (-S9); 2-Anthramine, Benzo(a)pyrene (+S9)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar

The test item was tested in a preliminary test and two mutagenicity experiments.
The preliminary test, both experiments without S9 mix and the first experiment with S9 mix were performed according to the direct plate
incorporation method. The second experiment with S9 mix was performed according to the preincubation method.

DURATION
- Preincubation period: 60 minutes
- Exposure duration: 48 to 72 hours.
Evaluation criteria:
A reproducible 2-fold increase (for the TA 98, TA 100 and TA 102 strains) or 3-fold increase (for the TA 1535 and TA 1537 strains) 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:
cytotoxicity
Remarks:
TA 1537 only
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
all but TA 98
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid.
Since the test item was freely soluble and non-toxic, the highest dose-level was 5000 µg/plate, according to the criteria specified in the international guidelines.
 
Experiments without S9 mix
The selected treatment-levels were: 312.5, 625, 1250, 2500 and 5000 µg/plate for both mutagenicity experiments.
A moderate emulsion was observed in the Petri plates when scoring the revertants at dose-levels >= 2500 µg/plate.
A moderate toxicity was observed at dose-level of 5000 µg/mL in the TA 1537 strain. No noteworthy toxicity was noted towards the other strains used.
The test item did not induce any noteworthy increase in the number of revertants, in any of the five strains.
 
Experiments with S9 mix
The selected treatment-levels were: 312.5, 625, 1250, 2500 and 5000 µg/plate for the first experiment and 39.06, 78.13, 156.3, 312.5, 625 and 1250 µg/plate for the second experiment.
A moderate emulsion was observed in the Petri plates when scoring the revertants at dose-level of 5000 µg/plate.
A strong toxicity was noted at dose-levels >= 1250 µg/plate in the TA 1537, TA 1535, TA 102 and TA 100 strains. No noteworthy toxicity was noted towards the other strains used.
 
An slight increase in the number of revertants (up to 2.4-fold the vehicle control value) was noted in the TA 98 strain in the second experiment with S9 mix. This increase exceeded the threshold of 2-fold the vehicle control value, however, it was not dose-related and not observed in the first experiment. Consequently, this increase was considered not to be biologically significant.
The test item did not induce any noteworthy increase in the number of revertants, in any of the other 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 with/without S9

The test item, distillation residues of butyraldehyde, 2-ethylhexenal and isobutanal hydrogenation by-products, did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.
Executive summary:

The objective of this study was to evaluate the potential of the test item, distillation residues of butyraldehyde, 2-ethylhexenal and isobutanal hydrogenation by-products (batch No. LR_OXO_2010-07-10), to induce reverse mutation in Salmonella typhimurium.

 

The study was performed according to the international guidelines (OECD 471 and Commission Directive No. B13/14) and in compliance with the principles of Good Laboratory Practice.

 

Methods

A preliminary toxicity test was performed to define the dose-levels of distillation residues of butyraldehyde, 2-ethylhexenal and isobutanal hydrogenation by-products to be used for the mutagenicity study. The test item was then tested in two independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver post‑mitochondrial fraction (S9 fraction) of rats induced with Aroclor 1254.

 

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

 

Five strains of bacteria Salmonella typhimurium: TA 1535, TA 1537, TA 98, TA 100 and TA 102 were used. Each strain was exposed to at least five dose-levels of the test item (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 item distillation residues of butyraldehyde, 2-ethylhexenal and isobutanal hydrogenation by-products 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.

 

with S9 mix

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

.           5 µg/plate of Benzo(a)pyrene (BAP): TA 100 strain,

.           10 µg/plate of 2-Anthramine (2AM): TA 102 strain.


Results

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

Since the test item was freely soluble and non-toxic, the highest dose-level was 5000 µg/plate, according to the criteria specified in the international guidelines.

 

Experiments without S9 mix

The selected treatment-levels were: 312.5, 625, 1250, 2500 and 5000 µg/plate for both mutagenicity experiments.

A moderate emulsion was observed in the Petri plates when scoring the revertants at dose-levels >= 2500 µg/plate.

A moderate toxicity was observed at dose-level of 5000 µg/mL in the TA 1537 strain. No noteworthy toxicity was noted towards the other strains used.

The test item did not induce any noteworthy increase in the number of revertants, in any of the five strains.

 

Experiments with S9 mix

The selected treatment-levels were: 312.5, 625, 1250, 2500 and 5000 µg/plate for the first experiment and 39.06, 78.13, 156.3, 312.5, 625 and 1250 µg/plate for the second experiment.

A moderate emulsion was observed in the Petri plates when scoring the revertants at dose-level of 5000 µg/plate.

A strong toxicity was noted at dose-levels >= 1250 µg/plate in the TA 1537, TA 1535, TA 102 and TA 100 strains. No noteworthy toxicity was noted towards the other strains used.

 

An slight increase in the number of revertants (up to 2.4-fold the vehicle control value) was noted in the TA 98 strain in the second experiment with S9 mix. This increase exceeded the threshold of 2-fold the vehicle control value, however, it was not dose-related and not observed in the first experiment. Consequently, this increase was considered not to be biologically significant.

The test item did not induce any noteworthy increase in the number of revertants, in any of the other strains.

 

Conclusion

Under our experimental conditions, the test item, distillation residues of butyraldehyde, 2-ethylhexenal and isobutanal hydrogenation by-products

(batch No. LR_OXO_2010-07-10), did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.