1,4-bis(methoxymethyl)benzene

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

One Ames study in 2010 shows negative to S. typhimurium (TA1535, TA1537, TA98 and TA100) and E. coli (WP2uvrA) strains.

One in Vitro Chromosomal Aberration Test in 2011 resulted that the test substance induced structural aberrations in CHL/IU cells with and without metabolic activation and induced numerical aberrations with metabolic activation.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From 2010-07-22 to 2010-09-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test) (migrated information)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

Purity: 99.80 %

Species / strain / cell type:

other: Chinese hamster lung (CHL/IU) (migrated information)

Metabolic activation:

with and without

Metabolic activation system:

Due to migration, the value was transferred to one of the current document's attachments

Test concentrations with justification for top dose:

-Cell growth inhibition study: 6.48, 13.0, 25.9, 51.9, 104, 208, 415, 830 and 1660 μg/mL (equivalent to 10 mmol/L, the highest dose to be used in the absence of toxicity according to the guideline)
- Chromosomal aberration study:
-S9 mix (short-term treatment); 0, 718, 825, 949, 1090, 1260, 1440, 1660 μg/mL
+S9 mix (short-term treatment): 0, 51.9, 104, 208, 415, 830, 1660 μg/mL
Justification for top dose: The highest dose of the test substance was set at a dose higher than IC50 because it showed a cytotoxic potential that reduced the cell growth rate to less than 50% in the absence of S9 mix when the cell growth inhibition study was conducted by the short-time treatment method. Specifically, the highest dose was set at 1,660 μg/mL followed by 6 lower doses with a common ratio of 1.15 since IC50 was found to be 1,200 μg/mL. In the presence of S9 mix, the highest dose was set at 1,660 μg/mL followed by 5 lower doses with a common ratio of 2 because the test substance did not show a cytotoxic potential that reduced the cell growth rate to less than 50%.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test substance was suspended in distilled water at 16.6 mg/mL and dissolved in DMSO and acetone at 166 mg/mL. DMSO, which is easier to handle, was selected for use as vehicle because solutions of the test substance prepared at 166 mg/mL using DMSO and acetone generated no heat or foam and showed no changes in color at room temperature for 2 hrs after preparation and DMSO is commonly used as vehicle in chromosomal aberration studies.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Without S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With S9 mix

Details on test system and experimental conditions:

Cell growth inhibition study
- Procedures:
In the study conducted by the short-time treatment method, 30 μL of solution of the test substance or vehicle mixed well with 3 mL of fresh medium was added to cells after removing pre-cultured medium in the absence of S9 mix. In the presence of S9 mix, 30 μL of solution of the test substance or vehicle added to 2.5 mL of fresh medium and mixed well with 0.5 mL of S9 mix was added to cells. In both cases, medium was removed after treatment for 6 hrs, and cells were washed 3 times with 2 mL of Ca2+- and Mg2+-free Dulbecco phosphate buffered salt solution and cultured for 18 hrs after adding 5 mL of fresh medium.
In the study conducted by the continuous treatment method, 50 μL of solution of the test substance or vehicle mixed well with 5 mL of fresh medium was added to cells after removing pre-cultured medium and treated for 24 hrs.
Regardless of whether the short-time treatment method or the continuous treatment method was used, 50 μL per culture vessel of 10 μg/mL Demecolcine solution was added 2 hrs before completing culture.
The presence or absence of precipitation of the test substance, changes in the color of medium, and corrosion of culture vessels was determined with the naked eye at the beginning and end of treatment and at the end of culture.
After completing culture, cells were exfoliated with 2 mL of 0.25 w/v% trypsin to prepare cell suspensions. Two hundred μL of cell suspension was sampled, and cells were counted using a microcell counter (CDA-500, Sysmex Corporation) after diluting with 10 mL of Cell Pack (Sysmex Corporation). The cell growth rate and 50% cell growth inhibition concentration (IC50) were calculated. IC50 was calculated using the line obtained from the cell growth rate at the lowest dose at which the cell growth rate decreases below 50% and the second lowest dose.
The remaining cell suspension was centrifuged at 1000 rpm for 5 min to recover cells and hypotonically treated for 15 min at 37C after adding 3 mL of KCl. About 0.3 mL of fixative (methanol: acetic acid = 3:1) was added to hypotonically treated cells to half-fix them. Cells were fixed using 3 mL of fixative replaced twice. Cell suspension of appropriate concentration was prepared thereafter using fixative and dropped on slides. One stained specimen was prepared per dose. After allowing them to dry spontaneously, specimens were stained for 15 min with 2 vol% Giemsa stain diluted with 1/15 mol/L phosphate buffer solution (pH 6.8).
- Observation and determination:
The presence or absence of divided cells was determined, and 50 metaphase cells were observed per dose at doses that are referred to set doses for the chromosomal aberration study to determine incidence of cells with chromosomal aberration (Structural and Numerical aberrations).

Chromosomal aberration study:
- Method: The chromosomal aberration study was conducted in the same way as the cell growth inhibition study by the short-time treatment method. Four chromosome specimens were prepared per dose (two per culture vessel). 30 μL per culture vessel of 0.01 mg/mL MMC was used as positive control in the absence of S9 mix, while 18 μL per culture vessel of 1 mg/mL CPA was used in the presence of S9 mix. Two culture vessels were used for dose.
- Doses for observation of specimens:
All specimens from the negative and positive control groups were observed. Doses of the test substance selected for observation of specimens: 1260, 1440 and 1660 μg/mL without S9 mix and 104, 208, 415, 830 and 1660 μg/mL with S9 mix
- Structural aberrations: Two hundred metaphase cells with 25±2 chromosomes (centromeres) per dose (50 per specimen) were observed. The total number of cells with structural aberrations was recorded together with the number by type of aberration. Gaps were defined as unstained areas narrower than the width of chromatid and recorded separately from structural aberrations.
- Numerical aberrations: Two hundred metaphase cells per dose (50 per specimen) were observed. The number of polyploid cells with 38 or more chromosomes was recorded.

Evaluation criteria:

The test result was considered positive if incidence of cells with structural or numerical aberrations is 10% or higher and a dose response is observed or if incidence is 5% or higher and the result is reproducible in the chromosomal aberration study and confirmatory study. Otherwise, the result was considered negative. No statistical method was employed. The D20 value (concentration at which 20% of cells show aberration) was determined for the treatment method which caused chromosomal aberrations in 5% or more cells (with the exception of treatment method that gave a negative result).

Key result

Species / strain:

other: Chinese hamster lung (CHL/IU) (migrated information)

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Cell growth inhibition test:
The IC50 value was found to be 1,200 μg/mL and 1,660 μg/mL, respectively, in the absence and presence of S9 mix by the short-term treatment method and 1,100 μg/mL by the 24-hour continuous treatment method.
Precipitation of the test material, changes in color of the medium, and erosion of culture vessels were not observed at any dose of the test material regardless of the treatment method at the start or completion of treatment or at the end of culture.
In the observation of specimens, incidence of cells with structural aberrations was 52.0% and 22.0%, respectively, at the maximum in the absence and presence of S9 mix when the short-term treatment method was used. Incidence of cells with numerical aberrations was below 5% at all doses in the absence of S9 mix by the short-term treatment method, but it increased up to 16.0% in the presence of S9 mix.

Chromosomal aberration test
- In the absence of S9 mix:
Cell growth rate and IC50: The cell growth rate was found to be 87.7, 85.8, 79.1, 75.1, 69.7, 51.7, and 14.0%, respectively, at 718, 825, 949, 1,090, 1,260, 1,440 and 1,660 μg/mL, and IC50 was calculated to be 1,400 μg/mL.
Precipitation of the test material, changes in color of the medium, and erosion of culture vessels: No precipitation of the test material, changes in color of the medium, or erosion of culture vessels was observed at any dose.
Incidence of cells with structural aberrations: Incidence of cells with structural aberrations was 2.5% and 75.0%, respectively, in the negative and positive control groups. In the test material groups, it was 2.5, 14.0 and 26.0%, respectively, at 1,260, 1,440 and 1,660 μg/mL and exceeded 10%. The test result was judged to be positive since incidence increased in a dose-dependent manner.
Incidence of cells with numerical aberrations: The test result was judged to be negative because incidence was below 5% at all doses.
D20: D20 for structural aberration was calculated to be 1.6 mg/mL.

- In the presence of S9 mix:
Cell growth rate and IC50: The cell growth rate was found to be 104.3, 98.4, 84.6, 77.6, 62.5, and 47.4%, respectively, at 51.9, 104, 208, 415, 830, and 1,660 μg/mL, and IC50 was calculated to be 1,500 μg/mL.
Precipitation of the test material, changes in color of the medium, and erosion of culture vessels: No precipitation of the test material, changes in color of the medium, or erosion of culture vessels was observed at any dose.
Incidence of cells with structural aberrations: Incidence of cells with structural aberrations was 2.0% and 24.0%, respectively, in the negative and positive control groups. In the test material groups, it was 1.0, 7.5, 7.0, 25.0, and 35.0%, respectively, at 104, 208, 415, 830 and 1,660 μg/mL and exceeded 10%. The test result was judged to be positive since incidence increased in a dose-dependent manner.
Incidence of cells with numerical aberrations: Incidence of cells with numerical aberrations was 0.0% and 0.5%, respectively, in the negative and positive control groups. In the test material groups, it was 1.0, 9.0, 13.0, 15.5, and 1.0%, respectively, at 104, 208, 415, 830 and 1,660 μg/mL and exceeded 10%. The test result was judged to be positive since incidence increased in a dose-dependent manner.
D20: D20 for structural aberration and numerical aberration was calculated to be 0.61 and 0.95 mg/mL, respectively.

Conclusions:

The test item was thought to induce both structural and numerical aberrations.

Executive summary:

An in Vitro Chromosomal Aberration Test was conducted with Cultured Chinese Hamster CHL/IU Cells according to OECD 473.

Cell growth inhibition test was conducted at 6.48, 13.0, 25.9, 51.9, 104, 208, 415, 830 and 1660 μg/mLusingshort-time treatment methodor the continuous treatment method.

Chromosomal aberration test was conducted at 718, 825, 949, 1090, 1260 1440 and 1660μg/mL in the absence of S9 mix and 51.9, 104, 208, 415, 830 and 1660μg/mLin the presence of S9 mix using short-time treatment method.

The result of chromosomal aberration test was judged to be positive by the short-term treatment method because incidence of cells with structural aberrations increased up to 26.0% and 35.0%, respectively, exceeding 10% in the absence and presence of S9 mix, and incidence increased in a dose-dependent manner. In terms of incidence of cells with numerical aberrations, the test result was judged to be negative in the absence of S9 mix by the short-term treatment method because incidence was below 5% at all doses, but it was judged to be positive in the presence of S9 mix because incidence increased up to 15.5% exceeding 10% and increases showed dos-dependency. At 1,660 μg/mL, incidence was low (1.0%) probably due to a delay in cell cycle.

The test substance induced structural aberrations in CHL/IU cells with and without metabolic activation and induced numerical aberrations with metabolic activation.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From 2010-07-27 to 2010-08-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Purity: 99.80%

Target gene:

histidine locus in several strains of Salmonella typhimurium and tryptophan locus of Escherichia coli (E. coli) strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

- Dose-finding study: 0.305, 1.22, 4.88, 19.5, 78.1, 313, 1250, 5,000 μg/plate
- Main Study 1 and Main Study 2: 5,000 μg/plate, which showed inhibitory effects on growth, followed by 2,500, 1,250, 625, 313 and 156 μg/plate
- Confirmatory study: 5,000, 3,540, 2,500, 1,770, 1,250, 884 and 625 μg/plate for TA1537 in the absence of S9 mix

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test substance was suspended in distilled water at 16.6 mg/mL and dissolved in DMSO at 50.0 mg/mL. DMSO was selected for use as vehicle because solution of the test substance prepared at 50.0 mg/mL using DMSO generated no heat or foam and showed no changes in color at room temperature for 2 hrs after preparation.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2)

Remarks:

For TA100, WP2uvrA and TA98, without S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

For TA1535, without S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Methoxy-6-chloro-9-[3-(2-chloroethyl)aminopropylamino]acridine) •2HCl (ICR-191)

Remarks:

For TA1537, without S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene (2AA)

Remarks:

For TA1535, TA1537, TA98, TA100 and WP2uvrA, with S9 mix

Details on test system and experimental conditions:

- Study methods:
Studies were conducted by the pre-incubation method in the presence and absence of S9 mix. In the dose-setting study, 3 plates were used in the negative control group, while 2 plates were used per dose in the positive control and test substance groups. In the main study, 3 plates were used per dose in the negative control, positive control and test substance groups.
- Operation procedures:
Test tubes were shaken for 20 min at 37± 0.5 °C after adding 0.1 mL of solutions of the test substance, vehicle, or positive controls, 0.5 mL of 0.1M sodium-phosphate buffer solution (pH 7.4) or S9 mix, and 0.1 mL of tester strain culture fluid. Mixture was overlaid on plates after mixing with 2 mL of soft agar. Revertant colonies formed were counted after culturing for 48 hrs at 37± 0.5 °C.
- Sterility test:
Two mL of soft agar was added to the highest-concentration solution of the test substance (0.1 mL) and S9 mix (0.5 mL) and the mixture obtained was overlaid on plates after mixing. The presence or absence of contamination was determined after culturing for 48 hrs at 37± 0.5 °C.
- Observation:
After completion of incubation, the presence or absence of precipitate of the test substance was observed with the naked eye, and whether growth was inhibited was determined under a stereoscopic microscope.
- Counting:
Revertant colonies were counted using a manual counter on plates which showed growth inhibition, while a colony analyzer (CA-11D, System Science K.K.) was used for all other plates. When it was used, counts obtained were reported as colony counts after area correction and count loss correction.

Evaluation criteria:

The result was regarded as positive if revertant colonies increased twice or more compared to the negative control and increases showed dose-dependency. Otherwise, the result was regarded as negative.

Statistics:

No statistical treatment was made.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

- Dose-finding study:
TA1537 showed 2-fold or greater increases in revertant colony counts compared to negative control in the absence of S9 mix.
All tester strains showed growth inhibition at 5000 μg/plate in both the presence and absence of S9 mix. The test material showed no precipitation in either the presence or absence of S9 mix.
- Main study 1:
No tester strain showed 2-fold or greater increases in revertant colony counts compared to negative control in either the presence or absence of S9 mix. All tester strains showed growth inhibition at 5000 μg/plate in both the presence and absence of S9 mix. The test material showed no precipitation in either the presence or absence of S9 mix.
- Main study 2:
TA1537 showed 2-fold or greater increases in revertant colony counts compared to negative control in the absence of S9 mix.
All tester strains showed growth inhibition at 5000 μg/plate in both the presence and absence of S9 mix. The test material showed no precipitation in either the presence or absence of S9 mix.
- Confirmatory study:
TA1537 did not show 2-fold or greater increases in revertant colony counts compared to negative control in the absence of S9 mix. Growth inhibition was observed at 3540 μg/plate. The test material showed no precipitation.

Conclusions:

The test item has no mutagenic potential under the conditions of the present study.

Executive summary:

An Ames test was conducted according to OECD 471. Salmonella typhimurium TA100, TA1535, TA98, TA1537, and Escherichia coli WP2uvrA were treated with the test material in the presence or absence of S9 mix.Dose-finding study, two Main study and one confirmatory studies were conducted by the pre-incubation method.

In dose-finding study, TA1537 showed 2-fold or greater increases in revertant colony counts compared to negative control in the absence of S9 mix. All tester strains showed growth inhibition at 5000 μg/plate in both the presence and absence of S9 mix. The test material showed no precipitation in either the presence or absence of S9 mix.

In Main study 1, No tester strain showed 2-fold or greater increases in revertant colony counts compared to negative control in either the presence or absence of S9 mix. All tester strains showed growth inhibition at 5000 μg/plate in both the presence and absence of S9 mix. The test material showed no precipitation in either the presence or absence of S9 mix.

In Main study 2, TA1537 showed 2-fold or greater increases in revertant colony counts compared to negative control in the absence of S9 mix. All tester strains showed growth inhibition at 5000 μg/plate in both the presence and absence of S9 mix. The test material showed no precipitation in either the presence or absence of S9 mix.

In confirmatory study, TA1537 did not show 2-fold or greater increases in revertant colony counts compared to negative control in the absence of S9 mix. Growth inhibition was observed at 3540 μg/plate. The test material showed no precipitation.

Based on these findings, it was concluded that the test item has no mutagenic potential under the conditions of the present study.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

An in vivo mammalian erythrocyte micronucleaus test in mice in 2021 shows negative that the test substance was not to induce the increase of the frequencies of micronucleated PCE in mice.

Link to relevant study records

Reference

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)

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

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

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

Dose / conc.:

400 mg/kg bw/day

Remarks:

female

Dose / conc.:

800 mg/kg bw/day

Remarks:

female

Dose / conc.:

1 600 mg/kg bw/day

Remarks:

female

Dose / conc.:

450 mg/kg bw/day

Remarks:

male

Dose / conc.:

900 mg/kg bw/day

Remarks:

male

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

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.

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.

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

Ames study: negative

In vitro Chromosomal Aberration Test: positive

In vivo mammalian micronucleus test: negative

Therefore in accordance with Regulation (EC) No. 1272/2008 Table 3.5.1, the available in vitro and in vivo data on this substance does not trigger classification for this endpoint.