Dihexyl ether

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

August 2018 to December 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

This study was performed for the purpose of notification in another region requesting this study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

2016

Deviations:

yes

Remarks:

temp. value (18.1°C - 25.2°C) and rel. humidity value (max. of 77%) outside the expected ranges were recorded occasionally times during the study. These differences were considered not to adversely affect the results or integrity of the study.

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

Dihexyl ether

EC Number:

203-987-8

EC Name:

Dihexyl ether

Cas Number:

112-58-3

Molecular formula:

C12H26O

IUPAC Name:

1-(hexyloxy)hexane

Test material form:

liquid

Test animals

Species:

mouse

Strain:

NMRI

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services Germany GmbH (Address: Sandhofer Weg 7, Sulzfeld D-97633, Germany)
- Age at study initiation: Approximately 7 or 8 weeks (preliminary experiments) or 9 weeks (main test) at the treatment
- Weight at study initiation: 36.5 – 40.3 g (males, preliminary experiment I.)
29.0 – 32.0 g (females, preliminary experiment I.)
36.2 – 38.5 g (males, preliminary experiment II.)
30.0 – 31.5 g (females, preliminary experiment II.)
30.5 – 32.6 g (females, preliminary experiment III.)
26.1 – 29.4 g (females, main test)
The weight variation did not exceed ± 20 percent of the mean weight/sex at the start of the treatment.
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: Type II polypropylene/polycarbonate cages
- Diet (e.g. ad libitum): ssniff(R) SM R/M "Autoclavable complete diet for rats and mice ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.1-25.2°C (target: 22 ± 3°C)
- Humidity (%): 32-77% (target: 30-70%)
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: August 22, 2019 To: September 12, 2019

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: Olive oil
- Amount of vehicle (if gavage or dermal): 5 ml/kg bw

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The necessary amount of the test item was weighed into a calibrated volumetric flask or other appropriate glass container; the required volume of vehicle was added and mixed using a magnetic stirrer to obtain homogenous formulations. The concentrations of the test item formulations were selected to assure the same dosing volumes in mice for all dose levels (the treatment volume was 5 mL/kg bw in case of the negative control (in the preliminary toxicity test II. and III.) and the test item treated groups in the preliminary experiments; the treatment volume was 5 mL/kg bw in case of the negative control and the test item treated groups and 10 mL/kg bw in case of the positive control group in the main study). All the dose levels were expressed in terms of the weight of the test item in the formulations.

Duration of treatment / exposure:

14 days

Frequency of treatment:

once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

500 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

1 000 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

2 000 mg/kg bw/day

No. of animals per sex per dose:

5
High dose group: 5+2 (Two additional mice were dosed in the High dose group to replace any animal which may die before the scheduled sacrifice time or serve as additional sample in case of borderline results. One additional animal was used for evaluation, because one animal was found dead in the High dose group before necropsy (Day 15). Bone marrow smears were also prepared from the other additional mouse, but those slides were not evaluated.)

Control animals:

yes

Positive control(s):

Cyclophosphamide at 60 mg/kg bw (10 ml/kg bw) at one occasion by the ip route

Examinations

Tissues and cell types examined:

Bone marrow

Details of tissue and slide preparation:

Bone marrow was obtained from two exposed femurs of each surviving mouse immediately after sacrifice. The bone marrow was flushed into a sterile centrifuge tube from of each pair of femurs with foetal bovine serum (5 mL) using a syringe and needle. After mixing, the cell suspension was concentrated by a gentle centrifugation and the supernatant was discarded. Smears of the cell pellet were made on standard microscope slides (3 slides / animal). (Note:Bone marrow smears were prepared from the replacement mice. The bone marrow smears of one of the additional animal of High dose group was scored as it was necessary for the proper evaluation of the study. The bone marrow smears of the other additional animal were not scored. However, all the processed slides will be archived in the raw data.) Slides were air-dried at room temperature for approximately 24 hours. Dried slides were fixed in methanol for a minimum of 5 minutes and allowed to air-dry. Slides were stained with 10 % Giemsa solution for 18 minutes then thoroughly rinsed with distilled water, and then air-dried at room temperature overnight. After staining, coverslips were mounted on them.

Evaluation criteria:

Criteria for Identification of Micronucleated Erythrocytes:

- A bluish mauve strongly coloured uniform round or oval particle in the cell.
- The particle should be large enough for the colour to be recognisable, and it should be located inside the cells. Areas with micronucleus-like particles outside the cells should not be used for analysis.
- During focusing, the particle should stay uniform in colour /light refraction and shape within a large interval and focus in the same plane as the erythrocyte.
- The unit of damage is deemed to be the cell, and therefore cells with two or more micronuclei will be counted as single micronucleated cells.

The Micronucleus Test was considered valid if it met the following criteria:

- the frequency of micronucleated polychromatic erythrocytes found in the negative (vehicle) control was within the range of historical laboratory control data,
- the positive control item produced biologically relevant increase in the number of micronucleated polychromatic erythrocytes,
- each treated and control group included at least 5 analysable animals.

Statistics:

Kruskal-Wallis Non Parametric ANOVA test at the Test Site

Results and discussion

Test results

Sex:

female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Minor clinical signs related to treatment were observed considered to be treatment related. Increased in the liver weights by 44.2% in the High dose were observed, clearly demonstrating a systemic dose related effect as proof of systemic exposure.

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: up to 2000 mg/kg bw/d
- Solubility: soluble in vehile up to 400 mg/ml
- Clinical signs of toxicity in test animals:
- Evidence of cytotoxicity in tissue analysed:
- Rationale for exposure:
- Harvest times:
- High dose with and without activation:
- Other:

RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels (for Cytogenetic or SCE assay):
- Induction of micronuclei (for Micronucleus assay):
- Ratio of PCE/NCE (for Micronucleus assay):
- Appropriateness of dose levels and route:
- Statistical evaluation:

Applicant's summary and conclusion

Conclusions:

Di-n-hexyl ether test item was shown to have significant systemic exposure, it did not cause statistically or biologically significant increases in the frequency of micronucleated polychromatic erythrocytes in mice. Thus, there was no evidence of any genotoxic activity of the test item under the conditions of this study.

Executive summary:

An in vivo mammalian erythrocyte micronucleus test was conducted according to OECD guideline 474 (Varga-Kanizsai B; 2019). NMRI mice received daily oral doses of 500, 1000 or 2000 mg/kg bw for 14 days. The highest dose level was chosen based on the results of preliminary studies. Minor clinical signs related to treatment were observed which were considered to be treatment related. Increaseds in the liver weights by 44.2% in the High dose were observed, clearly demonstrating a systemic dose related effect as proof of systemic exposure. Treatment with the test item did not affect the ratios of polychromatic erythrocytes among total erythrocytes. No statistically significant or biologically relevant increases in the frequency of micronucleated polychromatic erythrocytes (MNPCE) were seen for test item treated mice in any dose groups at any sampling time when compared to the corresponding negative (vehicle) control values. In conclusion, there was no evidence of any genotoxic activity of the test item under the conditions of this study.