Reaction mass of L-isoleucine and octadecan-1-ol and ethanesulphonic acid and docosan-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:

03/2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

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-isoleucine and octadecan-1-ol and ethanesulphonic acid and docosan-1-ol

EC Number:

931-105-8

Cas Number:

1156505-34-8

Molecular formula:

C26-30H56-64O5NS

IUPAC Name:

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

Test material form:

solid: flakes

Method

Target gene:

see report

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535

Additional strain / cell type characteristics:

not applicable

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 97

Remarks:

a

Additional strain / cell type characteristics:

not applicable

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

not applicable

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

not applicable

Species / strain 5

Species / strain / cell type:

S. typhimurium TA 102

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S-9

Test concentrations with justification for top dose:

test article was diluted in ethanol 95% and tested at the following concentation per plate: 5mg, 1.6mg, 0.5mg, 0.16mg, 0.05mg and 0.016mg/plate. The concentrations tested were based on the OECD 471 recommended concentrations for non-cytotoxic compounds that are not soluble at 5 mg/plate. The highest concentration tested (2.0mg/plate) was not completely soluble.

Vehicle / solvent:

Ethanol

Controls

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

Positive controls:

yes

Positive control substance:

sodium azide

mitomycin C

other: 2-aminoflurone and 2-aminoanthracene

Details on test system and experimental conditions:

Broth Culture preparation:
Commercial culture disc were used to inoculate nutrient broth for testing. The cultures were incubated
at 37C for 10-14 hrs on an orbital shaker until when measured spectrochemically at 660nm, an abs
orbance reading of approximately 1.0 to 2.0 was obtained. Validation data of the cultures showed ab
sorbance readings int he above range resulted in concentrations of approximately 10^9 CFU/mL.
Strain genotype verification
the culture disc lot numbers used were checked for presence of appropriate strain genotype c
haracteristics. These tests included verification of the following:
- presense of uvrB mutation
- presence or absence of R-factor plasmid
-presence of rfa mutation
requirement for histidine
The uvrB mutation was verified by demonstrating UV sensitivity (lack of repair system). The R-factor
was checked by determining sensitivity or resistance to ampicillin (0.08% in 0.02 NaOH. The pres
ense of the rfa mutation was verified by demonstrating sensitivity to crystal violet (0.1% in water) on
nutrient agar plates. The histidine requirement was assured by plating onto minimal glucose agar
plates spread with 0.1mL of 0.5 mM biotin and both with and without 0.1 M histidine.

Metabolic activation system
The S-9 activation system was used to screen for the presense of mutagens from byproducts of the
test sample. Rat liver S-9 homogenate was obtained from Moleculat Toxicology Inc. The homoenate
was kept frozen at <-60C upon receipt. Plates requiring activation contained approximately 20microL
rat liver S-9 per plate. When working with soft agar, the plates did not exceed 47C
Top agar preparation
Aliquots of top agar were melted and maintained at 45C. Each 100 mL aliquot of top agar was fortified
with 5-10 mL of 0.5mM biotin and 0.%mM histidin prior to use.
Plate incorporation tests;
the test sample, solvent control and chemical controls were tested both with and without S-9 activa
tion. Sterile 13x100 mm test tubes were transferred to a waterbath held at 45. two mL aliquots of top
agar were transferred to each test tube. Three replicates of each of the materials were prepared and
the test organis and materials were added as follows:
three replicates for the solvent control with 100 microL solvent control.
three replicates for the each sample concentration with 100 microL test organism plus 100microL
sample
Three test tubes for each chemical control with 100 microL test organism plus 10 microL chemical control. Each replicate requiring S-9 activation had 0.5mL ot the prepared S-9 mix added. The replicates were vortexed, poured onto MGPA plates, swirled to form an even layer and allowed to solidify. The plates were incubated for growth at 37C for 48-72 hrs.

Spot tests
the sample was also analysed using spot method on plates with and without the activation system. T
wo mL aliquots of the top agar mixture and 0.1 mL of the appropriate test organism was added to min
imal glucose agar plates. The plates were allowed to harden then 10 microL of the sample was added
as a spot on the surface of the plate. The plates were incubated for growth of the organisms at 37C
for 48-72 hrs. only the highest sample concentration was tested using the spot method.
Chemical control materials
sodium azide,
mitomycin-C,
4-nitro-0-phenylene-diamine (NPD),
2-aminofluorene (2AF)
The chemical controls were tested using the plate incorporation method only.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

for all concentrations tested

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

for all concentrations tested

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

for all concentrations tested

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

for all concentrations tested

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 97

Remarks:

a

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

for all concentrations tested

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

The sample concentration tested did not meet the criteria for a potential mutagen

Executive summary:

The AMES test is used to determine the potential mutagenic activity of the test sample. The test sample

did not produce a two-fold increase in the number of revertants or a clear dose related response in

any of the five tester strains. The spot plates showed a clear zone at the inoculation site indicating that

the sample was toxic to the bacteria but was not surrounded by a ring of increased reversion which

indicates the sample was not mutagenic. In summary, the sample concentration tested did not meet the

criteria for a potential mutagen