AAA reaction product

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Reliability score of 1. GLP study performed in acordance to guideline with no deviations

Link to relevant study records

Reference

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:

2003-15-04 to 2003-18-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study performed in accordance to recognized testing guidelines with no deviations to study protocol

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

Principles of method if other than guideline:

NA

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Strain Target mutation Mutation type
TA 1535 hisG46 Base-pair substitution
TA 100 hisG46 Base-pair substitution
TA 98 hisD3052 Frame shift
TA 102 hisG428 Frame shift
TA 1537 hisC3076 Base-pair substitution
WP2 uvrA trp-, uvrA- Base-pair substitution

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Details on mammalian cell type (if applicable):

NA

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/beta-naphthoflavone induced rat liver S9.

Test concentrations with justification for top dose:

Concentration range in the main test (with and without metabolic activation): 33, 100, 333, 1000, 2500, 5000 µg/plate

Vehicle / solvent:

Solvent: DMSO (MERCK, D-64293 Darmstadt; purity > 99 %).
Justification: Solubility properties and non-toxicity to bacteria. No precipitation at highest tested dose level.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene, 4-nitro-o-phenylene-diamine

Details on test system and experimental conditions:

METHOD OF APPLICATION: direct plate incorporation method and the preincubation method

DURATION
- Preincubation period: none in range-finder method and plate incorporation method, 1 hour in the pre-incubation method.
- Exposure duration: at least 48 hours
- Expression time (cells in growth medium): NA
- Selection time (if incubation with a selection agent): NA
- Fixation time (start of exposure up to fixation or harvest of cells): NA

SELECTION AGENT (mutation assays): No data

NUMBER OF REPLICATIONS: triplicate plates

NUMBER OF CELLS EVALUATED: no data

DETERMINATION OF CYTOTOXICITY
- Method: inspection of background bacterial lawn of the plates

OTHER EXAMINATIONS:
- Determination of polyploidy: NA
- Determination of endoreplication: NA
- Other: NA

Evaluation criteria:

The Salmonella typhimurium and Escherichia coli reverse mutation assay was considered acceptable if it meets the following criteria:
- regular background growth in the negative and solvent control
- the spontaneous reversion rates in the negative and solvent control are in the range of our historical data
- the positive control substances should produce a significant increase in mutant colony frequencies

A mutagen response was concluded based on the following observations:
- A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
- A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
- An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
- A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

No statistical evaluation of data performed (not required)

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(> 5000 µg/plate)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(> 5000 µg/plate)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data but in accordance to OECD 471
- Effects of osmolality: No data but in accordance to OECD 471
- Evaporation from medium: No data but in accordance to OECD 471
- Water solubility: No data, but AAA reaction product stable in water for 180 days
- Precipitation: No precipitation at highest tested dose level
- Other confounding effects: NA

RANGE-FINDING/SCREENING STUDIES:

COMPARISON WITH HISTORICAL CONTROL DATA: Revertants for negative, solvent and positive controls were within the observed historical controls obtained at RCC.

ADDITIONAL INFORMATION ON CYTOTOXICITY: No data

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'. Remarks: Plate incorporation test

Initially, a pre-experiment was performed with strains TA 98 and TA 100 to evaluate the toxicity of AAA reaction product. Eight concentrations (3-5000 µg/plate) were tested for toxicity and mutation induction with three plates each. The pre-experiment is reported as part of the plate-incorporation experiment since no toxic effects were observed and 5000 µg/plate were chosen as maximal concentration. The concentration range included two logarithmic decades. The following concentrations were tested in the main experiments: 33; 100; 333; 1000; 2500; and 5000 µg/plate

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

The mutagenic potential of AAA reaction product was evaluated in salmonella typhimurium and E-coli strains with and without metabolic activation in accordance to OECD 471. AAA reaction product did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used and was therfore considered to be non-mutagenic in this reverse mutation assay.

Executive summary:

The mutagenic potential of AAA reaction product was evaluated in accordance to OECD 471. The study was performed to investigate the potential of AAA reaction product to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA. The assay was performed in two independent experiments both with and without liver microsomal activation. The concentration, including the controls, was tested in triplicate. AAA reaction product was tested at the following concentrations: 33; 100; 333; 1000; 2500; and 5000 µg/plate.

The plates incubated with the AAA reaction product showed normal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments. No toxic effects, evident as a reduction in the number of revertants, occurred with and without metabolic activation. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with AAA reaction product at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

In conclusion, AAA reaction product did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, AAA reaction product is considered to be non-mutagenic in this Salmonella Typhimurium and Escherichia coli reverse mutation assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Data from two in vitro genotoxicity studies are available:

The mutagenic potential of AAA reaction product was evaluated in salmonella typhimurium and E-coli strains with and without metabolic activation in accordance to OECD 471. AAA reaction product did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used and was therfore considered to be non-mutagenic in this reverse mutation assay.

The clastogenic potential of AAA reaction product was evaluated in vitro in V79 cells (Chinese hamster cell line) with and without metabolic activation in accordance to OECD 473. AAA reaction product did not induce structural chromosome aberrations at highest tested concentration and was therfore considered to be non-clastogenic in this assay.

Overall, based on the available in vitro data AAA reaction product does not have mutagenic nor clastogenic properties.

No further experimental testing for genetic toxicity (in vitro/in vivo) of AAA reaction product is considered scientifically justified based on exposure assessment i.e. exposure based waiving (Adaption to column 2 - Annex XI section 3 "Substance-Tailored Exposure-Driven Testing"): AAA reaction product is only used in indistrial settings under conditions with very low potential human exposure (worker). No exposure to the general population. A full description of the exposure can be found in the attached CSR (section 13). In the CSR it is concluded that for all industrial uses, very low exposure levels are identified and RCRs below xxx compared to a DNEL of xxx . Any combination of uses will result in an RCR below xxx. Workers will not be exposed at any critical level.

Justification for classification or non-classification

Based on the available in vitro data AAA reaction product does not have mutagenic nor clastogenic properties and are therefore not classified in accordane to GHS/CLP regulations.