1,4-bis(methoxymethyl)benzene

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:

From Dec. 16, 2020 to Jan. 19, 2021

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

July 29, 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

1,4-bis(methoxymethyl)benzene

EC Number:

229-828-2

EC Name:

1,4-bis(methoxymethyl)benzene

Cas Number:

6770-38-3

Molecular formula:

C10H14O2

IUPAC Name:

1,4-bis(methoxymethyl)benzene

Test material form:

liquid

Test animals

Species:

mouse

Strain:

ICR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: SPF (Beijing) biotechnology CO., LTD.
- Age at study initiation: 45~50 days when arrival, 52~57 days at the commencement of each animal's dosing.
- Weight at study initiation: At the beginning of acclimation, the male mice body weight range was 31.04~33.82g and the female mice body weight range was 24.11~28.92g.
- Assigned to test groups randomly: [no/yes, under following basis: ]
- Fasting period before study:
- Housing:Animals were housed in plastic cages (L29.0×W18.0×H16.0cm) lied on shelf (L140.0×W43.0×H150.0cm) in the SPF grade barrier system (Room D102). There were 7 cages per layer, and 5 layers per rack. During the acclimatization, the male animals were housed one per cage because they were aggressive each other, and the female animals were housed 3 or 5 per cage; during the test, the male animals were housed one per cage and the female animals were housed 5 per cage.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.3~23.7℃ (target value 20~25℃)
- Humidity (%): 45~61% (target value 40%~70%)
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: According to the results of the test item solubility, the test item could be dissolved in corn oil. Moreover, corn oil is also accredited by OECD Guideline for Testing of Chemicals, 474 (July 29, 2016).
- Concentration of test material in vehicle: 40, 45, 80, 90 mg/mL
- Amount of vehicle (if gavage or dermal): 15 ml
- Type and concentration of dispersant aid (if powder):
- Lot/batch no. (if required): 20200821
- Purity:

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: 8.002g and 9.001g test items for the first administration and 8.004g and 9.004g test items for the second administration were weighed using the electronic balance (M-15) and transferred them into the calibrated 50ml glass container, respectively. Corn oil was added into the container to the calibrated 50ml scale line and discontinuously homogenate using a homogenate machine for at least 1 minute to obtain the uniform test item solution in vehicle. Then the test item solutions of the other concentrations were prepared by dilution.

Duration of treatment / exposure:

24 hours

Frequency of treatment:

All groups were administered for twice in the afternoon on each day and the interval of each administration was approximately 24 hours.

Post exposure period:

nearly 19 hours

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

400 mg/kg bw/day

Remarks:

female

Doses / concentrations 2

Dose / conc.:

800 mg/kg bw/day

Remarks:

female

Doses / concentrations 3

Dose / conc.:

1 600 mg/kg bw/day

Remarks:

female

Doses / concentrations 4

Dose / conc.:

450 mg/kg bw/day

Remarks:

male

Doses / concentrations 5

Dose / conc.:

900 mg/kg bw/day

Remarks:

male

Doses / concentrations 6

Dose / conc.:

1 800 mg/kg bw/day

Remarks:

male

No. of animals per sex per dose:

5 males and 5 females

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide monohydrate (CP), 50 mg/kg.bw

Examinations

Tissues and cell types examined:

After administration, general symptom observations (cage-side observation and/or observation animals with holding) of all test animals were made following the order of administration and recorded. During the dosing period, the animals were observed once on the day of the first administration, and twice on the day of the second administration. All animals were observed and weighed prior to sacrifice.

Details of tissue and slide preparation:

DETAILS OF SLIDE PREPARATION:
All animals were sacrificed by cervical dislocation approximately 19 hours after the second administration. Bone marrow from sternum was harvested and mixed with a drop of fetal calf serum on a side of a slide. Then the slides were smeared. After air-dried, the slides were fixed in methanol for 10 minutes, and stained with Giemsa’s solution for 30 minutes, flushed with distilled water and air dried.

METHOD OF ANALYSIS:
The slides were analyzed by the manual method using microscopy. All slides were coded randomly so that the manual scorer was unaware for all treatment conditions. 4000 polychromatic erythrocytes cells (PCE) were scored per animal for the number of micronucleated polychromatic erythrocytes (MNPCE) and the incidence of micronuclei was shown in permillage. At the same time, the PCE in 500 red blood cells (RBC) were scored and the ratio between PCE and RBC was calculated for each animal.
Observed under immersion objective, both PCE and NCE (normochromatic erythrocytes) are anucleate cells. After Giemsa staining, PCE shows bice and NCE shows light jacinth.
Identification of micronuclei:
Circle is the main form, and oval, ring, hemicycle is found occasionally. Outline of micronuclei is clear and flat. The staining of Micronuclei should be same as the nucleus of an adjacent nucleated cell. Usually, the upper limit is half of a diameter of an erythrocyte. PCE with two or more micronuclei is counted as one MNPCE.

Evaluation criteria:

If the incidence of MNPCE in at least one of the treatment groups shows statistically significant increase compared to the concurrent NG group (P＜0.05) and is outside the 95% control limits of historical negative control distribution, at the same time, the increase is dose-related or very clear in one dose level, the results will be deemed as positive.
The result is evaluated as negative if none of the treatment groups exhibits a statistically significant increase in the incidence of MNPCE compared to the concurrent NG group and all results are within the 95% control limits of historical negative control distribution.
Where the test result is not clearly negative or positive, the data would be evaluated by expert judgment and/or further investigations of the existing experiments completed. In some cases, analyzing more cells or performing a repeat experiment using modified experimental conditions could be useful. In cases even after further investigations, the data will preclude making a conclusion that the test item produces either positive or negative results, then the study will therefore be conclude as equivocal.

Statistics:

In the validated excel (2007) [OECD Mammalian erythrocyte micronucleus test-the statistical results table for the test data 1 (1.0)]. Before each administration and sampling, the mean and standard deviation of the body weight in each group were calculated; the ratio between PCE and RBC of each animal was calculated, and the mean and standard deviation of the ratios in each group were calculated at the same time, that in the treated groups should be no less than 20% of NG group. The incidence of MNPCE of each animal was calculated, and the mean and standard deviation in each group were calculated at the same time, then the data were evaluated with two tailed t-test method in Excel (2007) to observe if there were statistical significant differences in the treated groups and CP group as compared to the value of NG group.

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

The results of body weight: During the observation period in this test, no significant difference in body weight of male and female mice in all treated groups compared with the concurrent NG group (P>0.05).
The results of symptom observations: all the males in 1800mg/kg.bw group and all the females in 1600 mg/kg.bw appeared accelerated respiration on the day after each administration and recovered next morning after each administration, but no animal died during observation period. At the same time, no animal appeared toxic symptom and death in the other groups during the observation period.
The result of analysis of slides: The frequencies of micronucleated PCE for male mice were 1.4±0.4‰ in NG group, 1.5±0.5‰ in 450mg/kg.bw group, 1.3±0.5‰ in 900mg/kg.bw group, 1.4±0.5‰ in 1800mg/kg.bw group and 21.0±4.0‰ in CP group, and the frequencies of micronucleated PCE for female mice were 1.4±0.4‰ in NG group, 1.4±0.4‰ in 400mg/kg.bw group, 1.4±0.5‰ in 800mg/kg.bw group, 1.6±0.8‰ in 1600mg/kg.bw group and 20.6±2.9‰ in CP group. The results of t-test showed that no statistically significant difference in the frequency of micronucleated PCE was found in the treated groups as compared with the concurrent NG group (P>0.05). At the same time, a statistically significant difference in the frequency of micronucleated PCE was found in CP group as compared with the concurrent NG group (P<0.01). Furthermore, the ratios between PCE and RBC in all treated groups were more than 20 percent of the ratio in the concurrent NG group.

Applicant's summary and conclusion

Conclusions:

It was considered that the test item was not to induce the increase of the frequencies of micronucleated PCE in mice.

Executive summary:

This study was conducted to detect the possibility that test item could induce the increase in the frequency of micronucleated PCE in mice, in order to provide genotoxicity related data for the test item. The method was designed to be compatible with the requirements of OECD Guideline 474.

Under the conditions of this study, the results of micronucleus test were negative. Thus, it was considered that the test item was not to induce the increase of the frequencies of micronucleated PCE in mice.