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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:
05 August 2009 - 08 April 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Compliant to GLP and testing guideline; 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 gene mutation assay

Test material

Constituent 1
Reference substance name:
Post crystallization of ammonium sulphate aqueous phase products resulting from ammoniac neutralisation of sulphuric acid waste waters formed during methylmethacrylate synthesis
IUPAC Name:
Post crystallization of ammonium sulphate aqueous phase products resulting from ammoniac neutralisation of sulphuric acid waste waters formed during methylmethacrylate synthesis
Details on test material:
- Physical state: brown black liquid
- Lot/batch No.: 01/07/09
- Expiration date of the lot/batch: 01/07/2011
- Storage conditions of test material: at room temperature
- Purity/Impurities: not applicable (complex composition)

Method

Target gene:
Histidine operon
Species / strain
Species / strain / cell type:
S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
312.5, 625, 1250, 2500 and 5000 μg/plate for all the strains in the first experiment,
312.5, 625, 1250, 2500, 3750 and 5000 μg/mL for all the strains in the second experiment.
with and without S9 mix.
Vehicle / solvent:
Vehicle: water for injections
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
Migrated to IUCLID6: sodium azide; 9-aminoacridine; 2-nitrofluorene; mitomycin C; 2-ahthramine
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar
The preliminary test and all experiments were performed according to the direct plate incorporation mehod.

DURATION
- Preincubation period: 60 minutes
- Exposure duration: 48 or 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.
Statistics:
Not applicable.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium, other: TA 1535, TA 1537, 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:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
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:
not applicable
Positive controls validity:
valid
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):
positive with metabolic activation
negative without metabolic activation

The test item showed a mutagenic activity in the bacterial reverse mutation test with the TA 98 Salmonella typhimurium strain, in the presence of a metabolic activation system.
Executive summary:

The potential of the test item to induce reverse mutation was evaluated 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.

A preliminary toxicity test was performed to define the dose-levels of the test item 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 was dissolved in water for injections. 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.

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 in the preliminary test, the highest selected 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 all the strains in the first experiment,

.           312.5, 625, 1250, 2500, 3750 and 5000 µg/mL for all the strains in the second experiment.

 

No precipitate was observed in the Petri plates when scoring the revertants at any dose-levels. No noteworthy toxicity was noted at any dose-levels in any tester strains. A slight increase in the number of revertants was noted in the TA 102 strain in the second experiment (1.8-fold the vehicle control value). Since this increase did not exceed the threshold of 2-fold the vehicle control value and was not observed in the first experiment, it was not considered as biologically relevant. The test item did not induce any other noteworthy increase in the number of revertants.

 

Experiments with S9 mix

The selected treatment-levels were:

.           312.5, 625, 1250, 2500 and 5000 µg/plate for all the strains in the first experiment,

.           312.5, 625, 1250, 2500, 3750 and 5000 µg/mL for all the strains in the second experiment.

 

No precipitate was observed in the Petri plates when scoring the revertants at any dose-levels. No noteworthy toxicity was noted at any dose-levels in any tester strains. Slight increases in the number of revertants were noted in the TA 102 strain in the first experiment (up to 1.9-fold the vehicle control value). Since these increases did not exceed the threshold of 2-fold the vehicle control value and were not observed in the second experiment, they were not considered as biologically relevant. Noteworthy increases in the number of revertants, exceeding the threshold of 2-fold the vehicle control value (up to 2.9-fold the vehicle control) were noted in the TA 98 strain in both experiments. The test item did not induce any other noteworthy increase in the number of revertants.

Under the experimental conditions of this study, the test item showed a mutagenic activity in the bacterial reverse mutation test with the TA 98 Salmonella typhimurium strain, in the presence of a metabolic activation system.

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