Reaction mass of L-valine and ethanesulphonic acid and octadecan-1-ol and docosan-1-ol and eicosan-1-ol

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30/04/18 - 06/06/18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of L-valine and ethanesulphonic acid and octadecan-1-ol and docosan-1-ol and eicosan-1-ol

Cas Number:

2125692-22-8

Molecular formula:

C25-31H54-66NO5S

IUPAC Name:

Reaction mass of L-valine and ethanesulphonic acid and octadecan-1-ol and docosan-1-ol and eicosan-1-ol

Test material form:

solid

Method

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 97

Details on mammalian cell type (if applicable):

TA97a

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 98

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 100

Species / strain 5

Species / strain / cell type:

E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

S-9

Test concentrations with justification for top dose:

0.016 / 0.05 / 0.16 / 0.5 / 1.6 mg/plate
The concentrations tested were based on the OECD 471 highest recommended concentration for non-cytotoxic compounds.

Vehicle / solvent:

ethanol

Controls

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

sodium azide

methylmethanesulfonate

other: 4-nitro-O-phenylenediamine (NPD) / 2-aminoanthracene

Details on test system and experimental conditions:

Broth Culture Preparation: Commercial culture discs were used to make frozen working cultures. The culture discs were added to nutrient broth and incubated at 37 ± 2°C on an orbital shaker until visibly turbid. The cultures were streaked for isolation onto MGPA master plates fortified with histidine, biotin, tryptophan, and ampicillin, as appropriate. One or more colonies from the master plates were used to inoculate nutrient broth and incubated at 37 ± 2°C on an orbital shaker for 5-7 hours. Aliquots of the cultures were frozen for later use. Frozen working cultures of each strain were used to inoculate nutrient broth for testing. The cultures were incubated for 5.0-5.5 hours on an orbital shaker at approximately 100 rpm. The titers of the cultures were determined and had concentrations of approximately 10 CFU/mL or higher. The strains used for the test were checked for presence of appropriate strain phenotype characteristics.

Test Article Preparation: The test article was dissolved in warmed ethanol and tested at the following concentrations: 0.016 mg, 0.05 mg. 0.16 mg, 0.5 mg, 1.6 mg/plate. An aliquot of the solvent used was included and tested as the negative control. The concentrations tested were based on the OECD 471 highest recommended concentration for non-cytotoxic compounds.

Metabolic Activation System: The S-9 activation system was used to screen for the presence of mutagens from byproducts of the test article. Rat liver S-9 homogenate was obtained from Molecular Toxicology, Inc. The homogenate was kept frozen at < -60°C upon receipt. Plates requiring activation contained approximately 20 pL S-9 per plate. When working with soft agar the plates did not exceed 47°C.

Tod Aaar Preparation: Aliquots of top agar were melted and maintained at 45 ± 2°C. For test strains TA97a, TA98, TA1D0 and TA1535, each 100 ml aliquot of top agar was fortified with 5-10 mL of 0.5 mM biotin and 0.5 mM histidine solution prior to use. For test strain WP2, each 100 mL aliquot of top agar was fortified with 5-10 ml of 0.5 mM L-tryptophan solution.

Evaluation criteria:

The criteria for acceptance of the test and criteria for determination of a mutagen are listed below.
1) Tested strains for phenotype verification and achieved the appropriate responses.
2) All chemical controls included in the test gave the appropriate responses.
3) The reversion rates for each tester strain were within the historical ranges as outlined in the protocol. Historical data are constantly changing, as new data from acceptable tests are created. Therefore, reversion rates may differ slightly.

Criteria for a Mutaqen:
1) A reversion rate greater than 200% of the solvent control in strains TA97 and TA100. A reversion rate greater than 300% of the solvent control in strains TA98,TA1535, and WP2.
2) Demonstration of a clear dose related response when dilutions are tested.

Criteria for a Non-Mutagen:
1) A reversion rate less than or equal to 200% of the solvent control in strains TA97a and TA100. A reversion rate less than or equal to 300% of the solvent control in strains TA98, TA1535, and WP2.
2) No dose related response when dilutions are tested.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 97

Remarks:

97a

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Species / strain:

E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

The test article concentrations did not produce a two-fold or three-fold increase in the number of revertants or produce a clear dose related response in any of the 5 tester strains. The spot tests showed no zone of increased reversion or of toxicity. In summary, the test article concentrations tested against the five strains did not meet the criteria for a potential mutagen.

Executive summary:

The bacterial reverse mutation assay (Ames test) is used to determine the potential mutagenic activity of a test article by exposing a large number of the test organisms to dilutions of the test article in agar plates. The agar plates are monitored for growth of revertants (organisms mutating to the wild type) which are counted and used to estimate the mutagenic potential of the test article.

The Ames test employs several histidine auxotrophic (His-) strains of Salmonella typhimurium, which require the amino acid histidine for growth, and a tryptophan auxotrophic (Trp-) strain of Escherichia coli, which requires tryptophan for growth. The test detects mutations which cause the bacterial strains to revert to histidine or tryptophan independent (His+ or Trp+) bacteria. These revertants are detected by their ability to grow in the absence of an external source of histidine or tryptophan, respectively. The assay uses S. typhimurium tester strains TA97a, TA98, TA100, and TA1535, and £ co//test strain WP2, which were selected to detect various types of mutagens. The test is performed both with and without metabolic activation using an S-9 activation system. The S-9 activation system is designed to simulate mammalian liver enzyme systems and is used to detect substances which undergo metabolic activation from non-mutagenic forms.

All test method acceptance criteria were met. The test procedure(s) listed above were followed without deviation.

Results: The results are calculated using a validated computer program. Manual calculations may differ slightly due to rounding. All results greater than 300 colony forming units (CPU) are considered estimates.

The test article concentrations did not produce a two-fold or three-fold increase in the number of revertants or produce a clear dose related response in any of the 5 tester strains. The spot tests showed no zone of increased reversion or of toxicity. In summary, the test article concentrations tested against the five strains did not meet the criteria for a potential mutagen.