Reaction mass of 2-hydroxy-4-(methylthio)butanoic acid and 2-hydroxy-4-(methylthio)butanoic acid dimer

Genetic toxicity: in vitro

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 April 14 to July 5, 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study conducted according to OECD guideline 471 without any deviation.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-hydroxy-4-(methylthio)butyric acid

EC Number:

209-523-0

EC Name:

2-hydroxy-4-(methylthio)butyric acid

Cas Number:

583-91-5

Molecular formula:

C5H10O3S

IUPAC Name:

2-hydroxy-4-(methylthio)butanoic acid

Details on test material:

- Name of test material (as cited in study report): HMTBA (Rhodimet AT88), 2-Hydroxy-4-methylthiobutanoic acid
- Physical state: brown viscous liquid
- Analytical purity: 89.8%
- Impurities (identity and concentrations): water content: 9.1%, ammonium sulfate: 1.5%
- Purity test date: 22 March 2010
- Lot/batch No.: 7809307
- Date received: 23 March 2010
- Expiration date of the lot/batch: 03 November 2010
- Storage condition of test material: at room temperature

Method

Target gene:

Not applicable

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not applicable

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 102

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver post-mitochondrial fraction (S-9), obtained from Molecular Toxicology Incorporated, USA

Test concentrations with justification for top dose:

- Range-finder experiment: 10, 100, 500, 1000, 2500 and 5000 µg/plate (with and without S-9) in TA 98, TA 100 and TA 102 strains;
- Experiments 1 and 2: 312.5, 625, 1250, 2500 and 5000 µg/plate (with and without S-9)
All the concentrations and dose-levels were expressed as active item, taking into account the purity of the test item (89.8%).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethylsulphoxide (DMSO)
- Justification for choice of solvent/vehicle: The test item was freely soluble in the vehicle (DMSO) at 100 mg/mL.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: See table 1

Details on test system and experimental conditions:

METHOD OF APPLICATION: Plate incorporation method; as the results of Experiment 1 were negative, treatments in the presence of S-9 in experiment 2 included a pre-incubation step (incubation for 1 hour at 37°C).

DURATION
After plating, the plates were inverted and incubated at 37°C in the dark for 48 to 72 h.

SELECTION AGENT (mutation assays): histidine

NUMBER OF REPLICATIONS:
Range-finding test: single plate
Main experiments: triplicate plates

DETERMINATION OF CYTOTOXICITY
- Method: decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.

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:

None

Results and discussion

Test results

Test results 1

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

- Range-finder experiment: No precipitate was observed in the Petri plates when scoring the revertants at any dose-levels. No noteworthy toxicity was noted towards the three strains used, either with or without S9 mix.
Since the test item was freely soluble and non-toxic, the highest selected dose-level was 5000 µg/plate.

- Experiments 1 and 2: No precipitate was observed in the Petri plates when scoring the revertants at any dose-levels. No noteworthy toxicity was noted towards all the strains used, either with or without S9 mix.
Slight increases in the number of revertants exceeding the threshold of 2-fold the vehicle control value (up to 2.5-fold) were induced in the TA 98 strain in the first experiment without S9 mix. Since these increases were neither dose-related, nor reproducible (not observed in the second experiment), they were considered as non-biologically relevant.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Under the test conditions, HMTBA (Rhodimet AT88) is not considered as mutagenic in this bacterial system according to the criteria of the Annex VI to the Directive 67/548/EEC and CLP Regulation(EC) N° (1272-2008). 

Executive summary:

In a GLP study performed according to OECD guideline 471, HMTBA (Rhodimet AT88) was tested for mutagenicity using Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102 with the plate incorporation and preincubation methods in the presence and absence of metabolic activation system (S-9 mix). In range-finder test, using TA98, TA100 and TA102 tested at concentrations between 10 and 5000 µg/plate with and without S-9, the test item was freely soluble and non-toxic; therefore the selected treatment-levels were: 312.5, 625, 1250, 2500 and 5000 µg/plate for both mutagenicity experiments with and without S9 mix. The positive controls induced the appropriate responses in the corresponding strains. HMTBA (Rhodimet AT88) showed no substantial increases in revertant colony numbers over control count obtained with any of the tester strains at any concentrations in either presence or absence of S-9. Under the test conditions, HMTBA (Rhodimet AT88) is not considered as mutagenic in this bacterial system according to the criteria of the Annex VI to the Directive 67/548/EEC and CLP Regulation(EC) N° (1272-2008).