6-chlorohexan-1-ol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

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.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

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

Target gene:

uvrB-

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

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.

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.

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Chlorohexanol is not clastogenic or aneugenic in the bone marrow, micronucleus test of male and females mice up to a dose of 1000 and 1500 mg/kg, respectively (the maximum tolerated dose in accordance with current regulatory guidelines) under the experimental conditions described in this report.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 11, 2017 - Jun 13, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The in-vivo study was conducted in the context of a registration outside EU.

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

31 May 2008

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

adopted 29 July 2016

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: Source: Sponsor; Batch: 141125
- Purity, including information on contaminants, isomers, etc.: 98.86%
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature

Species:

mouse

Strain:

NMRI

Details on species / strain selection:

NMRI mice (SPF) were used as the test system. These mice are recommended by international guidelines (e.g. OECD, EC).

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: 6-8 weeks old at the start of treatment
- Weight at study initiation: The body weights of the mice at the start of the treatment in the main study were within 20% of the sex mean. The mean body weights were for males 35.3 ± 1.9 g and for females 26.2 ± 1.2 g and the range was for males 31 – 39 g and for females 23 - 28 g.
- Assigned to test groups randomly: The mice were identified by a unique number on the tail written with a marker pen. The animals were allocated at random to the treatment groups.
- Housing: The animals were group housed (maximum 5 animals per sex per cage) in labelled Macrolon cages (type MIII height 180 mm, length 380 mm and width 220 mm) containing sterilized sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany). Paper bedding (Enviro-dri, Wm. Lilico & Son (Wonham Mill Ltd), Surrey, United Kingdom) was provided as cage-enrichment
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C (range 18 to 24°C)
- Humidity (%): Mean relative humidity of 42 to 65% (40 to 70%)
- Air changes (per hr): Ten or greater air changes per hour with 100% fresh air (no air recirculation)
- Photoperiod: 12 hrs dark /12 hrs light

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: as described in the guidance

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: No correction was made for the purity/composition of the test item.  A solubility test was performed on visual assessment. Chlorohexanol was dissolved in corn oil. The specific gravity of corn oil is 0.9 g/mL. Chlorohexanol concentrations were dosed within 4 hours after preparation. Any residual volumed were discarded.

Duration of treatment / exposure:

48 hours

Frequency of treatment:

Two treatments were performed, administered at a 24-hour interval

Post exposure period:

--

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Vehicle control (negative control), males and females

Dose / conc.:

40 mg/kg bw/day (nominal)

Remarks:

positive control (males and females)

Dose / conc.:

250 mg/kg bw/day (nominal)

Remarks:

test item; males

Dose / conc.:

750 mg/kg bw/day (nominal)

Remarks:

test item; females

Dose / conc.:

375 mg/kg bw/day (nominal)

Remarks:

test item; females

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

test item; males

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

test item; males

Dose / conc.:

1 500 mg/kg bw/day (nominal)

Remarks:

test item; females

No. of animals per sex per dose:

Five

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamid
- Justification for choice of positive control(s): as described in test guideline.
- Route of administration: oral (gavage). The volume administered was the same as that of the test item.
- Doses / concentrations: 40 mg/kg bw

Tissues and cell types examined:

Bone marrow

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Based on the results of the dose-range finding test a full study with two sexes was performed. Since there were substantial differences in toxicity between sexes, male and female animals were used in the main study.
TREATMENT AND SAMPLING TIMES (in addition to information in specific fields): Five male and five female mice were used per sampling time in each treatment group. The animals were dosed twice with a 24-hour interval.
DETAILS OF SLIDE PREPARATION: Bone marrow was sampled 48 hours after the first dosing. The animals were sacrificed by cervical dislocation. Both femurs were removed and freed of blood and muscles. Both ends of the bone were shortened until a small opening to the marrow canal became visible. The bone was flushed with approximately 2 mL of fetal calf serum (Invitrogen Corporation, Breda, The Netherlands). The cell suspension was collected and centrifuged at 216 g for 5 min.
The supernatant was removed with a Pasteur pipette. A drop of serum was left on the pellet. The cells in the sediment were carefully mixed with the remaining serum. A drop of the cell suspension was placed on the end of a clean slide, which was previously immersed in a 1:1 mixture of 96% (v/v) ethanol (Merck, Darmstadt, Germany)/ether (Merck) and cleaned with a tissue. The slides were marked with the study identification number and the animal number. The drop was spread by moving a clean slide with round-whetted sides at an angle of approximately 45° over the slide with the drop of bone marrow suspension. The preparations were air-dried, fixed for 5 min in 100% methanol (Merck) and air-dried overnight. At least two slides were prepared per animal.
The slides were automatically stained using the "Wright-stain-procedure" in a HEMA-tek slide stainer (Hematek 3000, Siemens Healthcare, Den Haag, The Netherlands). This staining is based on Giemsa. The dry slides were automatically embedded in a 1:10 mixture of xylene (Klinipath, Duiven, The Netherlands)/pertex (Klinipath) and mounted with a coverslip in an automated cover slipper (Leica Microsystems B.V., Rijswijk, The Netherlands

Evaluation criteria:

A micronucleus test is considered acceptable if it meets the following criteria:
- The concurrent negative control data are considered acceptable when they are within the 95% control limits of the distribution of the historical negative control database.
- The concurrent positive controls should induce responses that are compatible with those generated in the historical positive control database.
- The positive control item induces a statistically significant increase in the frequency of micronucleated polychromatic erythrocytes. The positive control data will be analyzed by the Students t test (one-sided, p < 0.05) in case of homogeneous variances or by the Welch t test in case of inhomogeneous variances (one-sided, p < 0.05).

Statistics:

ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used for statistical analysis of the data. Appropriate statistical tests were selected for data evaluation.
A test item is considered positive in the micronucleus test if all of the following criteria are met:
- At least one of the treatment groups exhibits a statistically significant (one-sided, p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes compared with the concurrent negative control
- The increase is dose related when evaluated with a trend test.
- Any of the results are outside the 95% control limits of the historical control data range.

A test item is considered negative in the micronucleus test if:
-None of the treatment groups exhibits a statistically significant (one-sided, p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes compared with the concurrent negative control.
- There is no concentration-related increase when evaluated with a trend test.
- All results are within the 95% control limits of the negative historical control data range

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

see Additional information on results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

see Additional information on results

Additional information on results:

In the dose range finding study, one male and one female animal were dosed twice with 2000 mg Chlorohexanol/kg body weight. The male animal died within 25 hours after the first dosing. The female animal died within 44 hours after the first dosing. One male and three female animal were dosed twice with 1500 mg/kg body weight. The female animals had a rough coat. One animal also was lethargic, showed a hunched posture and ataxia. Due to severe toxic signs in the male animal the dose level of 1500 mg/kg body weight was considered too high for male animals. Consequently three male animals were dosed twice with 1000 mg/kg body weight. All male animals had a rough coat. One animal was also lethargic.

MICRONUCLEUS MAIN TEST: Based on the results of the dose-range finding strudy dose levels of 250, 500 and 1000 mg/kg bw (males ) and 375, 750 and 1500 mg/kg bw (females) were selceted as appropriate doses for the micronucleaus main test. Five male and five female animals were used in each treatment group.
All male animals treated with the positive control had fighting wounds.

The following clinical observations were made in the groups treated with 1000 (males) and 1500 mg (females) Chlorohexanol/kg body weight:

MORTALITY AND TOXIC SIGNS: The male animals of the groups treated with 1000 (3 males), 500 and 250 mg Chlorohexanol/kg body weight and the female groups treated with 1500 (two females), 750 and 375 mg Chlorohexanol/kg body weight and the animals of the negative and positive control groups showed no treatment related clinical signs of toxicity or mortality.
Within 2 hours after dosing all animals of the group treated with 1000 and 1500 mg/kg body weight were showed no reaction to treatment except for one female animal which had a hunched posture and a rough coat.
Within 20 hours after the first dosing all animals still showed no reaction to treatment and the female animal recovered from the treatment.
Within 2 hours after the second dosing two males treated with 1000 mg/kg body weight had a rough coat and a hunched posture. Three females dose with 1500 mg/kg body weight were lethargic, had a rough coat and a hunched posture. One animal also showed ventral recumbency.
Within 21 hours after the second dosing all animals recovered from the treatment, except for one female animal which was lethargic, showed ventral recumbency and no reaction to a stimulus. The animal was breathing slow and was comatose.

Micronucleated Polychromatic Erythrocytes:
The mean number of micronucleated polychromatic erythrocytes scored in Chlorohexanol treated groups were compared with the corresponding vehicle control group. No increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of Chlorohexanol treated animals compared to the vehicle treated animals. The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control animals was within the within the 95% control limits of the distribution of the historical negative control database.
Cyclophosphamide, the positive control item, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes in both sexes. In addition, the number of micronucleated polychromatic erythrocytes found in the positive control animals was within the 95% control limits of the distribution of the historical positive control database. Hence, all criteria for an acceptable assay were met.

Ratio Polychromatic to Normochromatic Erythrocytes: The animals of the groups, which were treated with Chlorohexanol and the negative control
showed no decrease in the ratio of polychromatic to normochromatic erythrocytes, which indicated a lack of toxic effects of this test item on the erythropoiesis. The animals of the groups treated with cyclophosphamide showed an expected decrease in the ratio of polychromatic to normochromatic erythrocytes, demonstrating toxic effects on erythropoiesis.

Conclusions:

Chlorohexanol is not clastogenic or aneugenic in the bone marrow, micronucleus test of male and females mice up to a dose of 1000 and 1500 mg/kg, respectively (the maximum tolerated dose in accordance with current regulatory guidelines) under the experimental conditions described in this report.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)