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Administrative data

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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guidelineopen allclose all
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

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
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

Test animals

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

Administration / exposure

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:
--
Doses / concentrationsopen allclose all
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

Examinations

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

Results and discussion

Test resultsopen allclose all
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.

Applicant's summary and conclusion

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.