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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)

Data source

Reference
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
1-Chloro-6-Hydroxyhexane
IUPAC Name:
1-Chloro-6-Hydroxyhexane
Constituent 2
Chemical structure
Reference substance name:
6-chlorohexan-1-ol
EC Number:
217-925-2
EC Name:
6-chlorohexan-1-ol
Cas Number:
2009-83-8
Molecular formula:
C6H13ClO
IUPAC Name:
6-chlorohexan-1-ol
Test material form:
liquid: viscous
Details on test material:
Sample ID APCI000640
Chemical Name 1-chloro-6-hydroxyhexane
Physical State liquid
CAS # 2009-83-8
EU # 217-925-2
Manufacturer Air Products and Chemicals, Inc.
Batch # / Lot # 141125-1

Method

Target gene:
uvrB-
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Strains Genotype Type of mutations indicated
TA1537 his C 3076; rfa-; uvrB-: frame shift mutations
TA98 his D 3052; rfa-; uvrB-;R-factor
TA1535 his G 46; rfa-; uvrB-: base-pair substitutions
TA100 his G 46; rfa-; uvrB-;R-factor
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
Strain Genotype Type of mutations indicated
WP2uvrA trp-; uvrA-: base-pair substitution
Metabolic activation:
with and without
Metabolic activation system:
S9-Mix
Test concentrations with justification for top dose:
The test item was tested using the following method. The maximum concentration was 5000 μg/plate (the maximum recommended dose level). Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.
Vehicle / solvent:
dimethyl sulphoxide
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Untreated negative controls:
no
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Untreated negative controls:
no
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Untreated negative controls:
no
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Untreated negative controls:
no
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene (2AA)
Details on test system and experimental conditions:
The five strains of bacteria used, and their mutations, are defined above.
All of the Salmonella strains are histidine dependent by virtue of a mutation through the histidine
operon and are derived from S. typhimurium strain LT2 through mutations in the
histidine locus. Additionally due to the "deep rough" (rfa-) mutation they possess a faulty
lipopolysaccharide coat to the bacterial cell surface thus increasing the cell permeability to
larger molecules. A further mutation, through the deletion of the uvrB- bio gene, causes an
Report Envigo Study Number: TJ05YB
Page 13
inactivation of the excision repair system and a dependence on exogenous biotin. In the
strains TA98 and TA100, the R-factor plasmid pKM101 enhances chemical and UV-induced
mutagenesis via an increase in the error-prone repair pathway. The plasmid also confers
ampicillin resistance which acts as a convenient marker (Mortelmans and Zeiger, 2000). In
addition to a mutation in the tryptophan operon, the E. coli tester strain contains a uvrA- DNA
repair deficiency which enhances its sensitivity to some mutagenic compounds. This
deficiency allows the strain to show enhanced mutability as the uvrA repair system would
normally act to remove and repair the damaged section of the DNA molecule (Green and
Muriel, 1976 and Mortelmans and Riccio, 2000).
The bacteria used in the test were obtained from:
• University of California, Berkeley, on culture discs, on 04 August 1995.
• British Industrial Biological Research Association, on a nutrient agar plate, on 17 August 1987

All of the strains were stored at approximately -196 °C in a Statebourne liquid nitrogen
freezer, model SXR 34.
In this assay, overnight sub-cultures of the appropriate coded stock cultures were prepared in
nutrient broth (Oxoid Limited; lot number 1758279 10/20) and incubated at 37 °C for
approximately 10 hours. Each culture was monitored spectrophotometrically for turbidity
with titres determined by viable count analysis on nutrient agar plates.

The test item was immiscible in sterile distilled water at 50 mg/mL but was fully miscible in
dimethyl sulphoxide at the same concentration in solubility checks performed in-house.
Dimethyl sulphoxide was therefore selected as the vehicle.

The test item was tested using the following method. The maximum concentration was
5000 μg/plate (the maximum recommended dose level). Eight concentrations of the test item
(1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate) were assayed in triplicate against each
tester strain, using the direct plate incorporation method.
Without Metabolic Activation
0.1 mL of the appropriate concentration of test item, solvent vehicle or appropriate positive
control was added to 2 mL of molten, trace amino-acid supplemented media containing
0.1 mL of one of the bacterial strain cultures and 0.5 mL of phosphate buffer. These were
then mixed and overlayed onto a Vogel-Bonner agar plate. Negative (untreated) controls
were also performed on the same day as the mutation test. Each concentration of the test
item, appropriate positive, vehicle and negative controls, and each bacterial strain, was
assayed using triplicate plates.
With Metabolic Activation
The procedure was the same as described previously except that following the
addition of the test item formulation and bacterial culture, 0.5 mL of S9-mix was added to the
molten, trace amino-acid supplemented media instead of phosphate buffer.
Incubation and Scoring
All of the plates were incubated at 37 ± 3 °C for approximately 48 hours and scored for the
presence of revertant colonies using an automated colony counting system. The plates were
viewed microscopically for evidence of thinning (toxicity).

Test for Mutagenicity: Experiment 2 – Pre-Incubation Method
The second experiment was not performed because the OECD 471 test guideline permits
non-repetition of the experiment when a clear, positive response is obtained in the first
experiment. Therefore, a second, confirmatory experiment was not required.
Evaluation criteria:
There are several criteria for determining a positive result. Any, one, or all of the following
can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and
Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester
strain (especially if accompanied by an out-of-historical range response (Cariello and
Piegorsch, 1996)).
A test item will be considered non-mutagenic (negative) in the test system if the above
criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the
data generated will prohibit making a definite judgment about test item activity. Results of
this type will be reported as equivocal.
Statistics:
Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05)
for those values that indicate statistically significant increases in the frequency of revertant
colonies compared to the concurrent solvent control.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Prior to use, the master strains were checked for characteristics, viability and spontaneous
reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and
the S9-mix used in the experiment was shown to be sterile. The test item formulation was
also shown to be sterile. These data are not given in the report.
Results for the negative controls (spontaneous mutation rates) are presented in Table 1 and
were considered to be acceptable. These data are for concurrent untreated control plates
performed on the same day as the Mutation Test.
The individual plate counts, the mean number of revertant colonies and the standard
deviations, for the test item, positive and vehicle controls, both with and without metabolic
activation, are presented in Table 2 and Table 3.

The maximum dose level of the test item was selected as the maximum recommended dose
level of 5000 μg/plate. There was no visible reduction in the growth of the bacterial
background lawn at any dose level, either in the presence or absence of metabolic activation
(S9-mix), in the first mutation test (plate incorporation method). No test item precipitate was
observed on the plates at any of the doses tested in either the presence or absence of S9-mix.
The test item induced substantial increases in the frequency of TA1535 revertant colonies in
both the presence and absence of S9-mix with a clear, dose-related response noted at the
upper dose levels. The individual revertant counts at the statistically significant dose levels
exceeded the in-house untreated/vehicle control counts for the bacterial strain with a
maximum fold increase over the concurrent vehicle controls of 5.5 times noted in the absence
of S9-mix and 6.1 in the presence of S9-mix at 5000 μg/plate. A smaller response was also
noted to TA100 at 5000 μg/plate in both the absence and presence of S9-mix, although these
increases did not achieve a twofold increase over the concurrent vehicle controls and the
individual revertant colony counts were within the in-house untreated/vehicle control counts
for the bacterial strain.
The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the
normal range. All of the positive control chemicals used in the test induced marked increases
in the frequency of revertant colonies, both with or without metabolic activation. Thus, the
sensitivity of the assay and the efficacy of the S9-mix were validated.

Applicant's summary and conclusion

Conclusions:
Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with Chlorohexanol using the Ames plate incorporation method at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system. The dose range was 1.5 to 5000 μg/plate. Chlorohexanol was considered to be mutagenic under the conditions of this test.
Executive summary:

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with chlorohexanol using the Ames plate incorporation method at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system. The dose range was 1.5 to 5000 μg/plate. Chlorohexanol was considered to be mutagenic under the conditions of this test.