4,4'-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with [(dimethylamino)methyl]phenol and piperazine

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

This study was conducted between 05 September 2016 and 23 January 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Translation from Japanese Study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

2014

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

Partial Revision of Testing Methods Concerning New Chemical Substances
(Notification No. 1221-1, PSEHB, MHLW•, No. 1 of 20151209, MIB, METI; No. 1512211, EPB, MOE; dated December 21, 2015)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

4,4'-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with [(dimethylamino)methyl]phenol and piperazine

EC Number:

500-429-8

EC Name:

4,4'-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with [(dimethylamino)methyl]phenol and piperazine

Cas Number:

159034-96-5

Molecular formula:

(C15 H16 O2 . C9 H13 N O . C4 H10 N2 . C3 H5 Cl O)x

IUPAC Name:

4,4'-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with [(dimethylamino)methyl]phenol and piperazine Amine adduct with epoxy resin~

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

Test substance
Name 4,4' -Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with [(dimethylamino) methyl] phenol and piperazine
Other name THEMIS
CAS-No. 159034-96-5
Lot No. 160715
Molecular weight Mm 3,000; Mw 11,000
Purity 100.0%
Solubility in water Unknown
Melting point 120°C to140°C
Appearance Powder, pale pink
Storage conditions Stored at room temperature (actual temperature: 19.6°C to 25.8°C, acceptable range: 10°C to 30 °C) and protected from light in tight container.
Stability of the test substance The test substance is swelled in water, and is softened at 100”C or more.
Handling precautions Wear safety glasses, a mask, and rubber gloves.

Method

Species / strain

Species / strain / cell type:

Chinese hamster lung (CHL/IU)

Details on mammalian cell type (if applicable):

- Cell line: CHL/IU (derived from the lungs of female Chinese hamsters)
- Source: DS Pharma Biomedical Co., Ltd.
- Date of purchase : August 2, 2016
- Specificity of cell line:
Modal chromosome number (2n): 25
Doubling time: 15.2 hours
Mycoplasma infection: Negative
- Number of passages:
At purchase: 14
At freezing for stock: 17
At treatment: 19 to 25 (4 to 25 days after thawing)
- Storage:
The cells were mixed with a culture medium containing DMSO (one-tenth volume of the final volume). They were subdivided into tubes in 1 mL aliquots, and were frozen on August 9, 2016 and then stored in liquid nitrogen. The cells were thawed and cultured for use in the experiments during the study.
- Culture conditions:
Plate: Plastic plate (diameter: 6 cm and 10 cm)
Temperature: 37°C
CO2 concentration: 5%
Humidity: Under moist atmosphere
Incubator: Programmable incubator
- Culture medium
MEM and MEM culture medium were purchased and prepared before and during the study. They were used in common with other studies

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

- source of S9
Kikkoman Biochemifa Company
- method of preparation of S9 mix
S9 was prepared from the livers of 7-week-old male Sprague-Dawley rats treated with phenobarbital and 5,6-benzoflavone.

Test concentrations with justification for top dose:

cell growth inhibition test: 1, 0.5, 0.25, 0.125, 0.0625, and 0.0313 mg/mL,
chromosomal aberration test (main test 1): 0.7, 0.6, 0.5, 0.45, 0.4, 0.35, 0.3, 0.25, 0.2, 0.15, and 0.1 mg/mL,
chromosomal aberration test (main test 2): 0.3, 0.25, 0.2, 0.15, 0.1, 0.075, 0.05, 0.025, 0.0125, and 0.00625 mg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Physiological saline (abbr.: saline)
- Justification for choice of solvent/vehicle:
In the results of the solvent selection test, the test substance suspended at 20 mg/mL in saline and at 200 mg/mL in acetone but did not dissolve or suspend at 200 mg/mL in dimethyl sulfoxide (abbr.:DMSO). Exothermic reaction, discoloration and foaming were not observed in the preparation. Therefore, saline was selected as the vehicle for the test substance and was used as the negative control substance in this study.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

mitomycin C

Details on test system and experimental conditions:

Preparation of test substance formulations and positive control substance solutions
Preparation of test substance formulations
(l) The test substance was weighed [cell growth inhibition test and chromosomal aberration test (main test 1): 20 mg, chromosomal aberration test (main test 2): 14 mg] and suspended in saline by ultrasonication and vortex mixing.
(2) The final volume of the formulation was adjusted with saline [cell growth inhibition test: 10 mL, chromosomal aberration test (main test 1): 25 mL, chromosomal aberration test (main test 2): 40 mL] to prepare the highest test substance formulation of 10-fold concentration of the final concentration [cell growth inhibition test: 2 mg/mL, chromosomal aberration test (main test l): 0.8 mg/mL, chromosomal aberration test (main test 2): 0.35 mg/mL].
(3) A portion of the highest test substance formulation was serially diluted with saline to prepare each test substance formulation of 10-fold concentration of the final concentration.
(4) All the procedures, including those for weighing the test substance and dilution of the formulations, were performed under UV-cut light at room temperature. The test substance formulations were used just after preparation. The storage period after preparation to the time of use was as follows;
Cell growth inhibition test: 15 to 35 minutes
Chromosomal aberration test (main test 1): 20 to 35 minutes
Chromosomal aberration test (main test 2): 20 to 30 minutes

Preparation of positive control substance solutions
Preparation of MMC solution
(1) MMC in a 2-mg vial was dissolved in 5 mL of water.
(2) It was serially diluted with saline to prepare 100-fold concentrations of the prescribed doses for each treatment condition (short-term treatment assay: 10 µg/mL, continuous treatment assay: 5 µg/mL).
(3) The solution was prepared on February 8, 2016 (expiration date: January 31, 2017), subdivided into tubes in 0.5 mL aliquots and stored in a freezer (actual temperature: -25.9°C to -21.7°C, acceptable range: -40°C to -15°C).
(4) The frozen stock was thawed and used on the experimental day.

Preparation of BP solution
(1) BP was weighed for 45 mg and dissolved in 30 mL of DMSOto prepare 1.5 mg/mL solution (100-fold concentration of the final concentration for treatment).
(2) The solution was prepared on February 8, 2016 (expiration date: February 7, 2017) subdivided into tubes in 0.5 mL aliquots and stored in a freezer (actual temperature: -25.9°C to -21.7°C, acceptable range: -40°C to -15°C).
(3) The frozen stock was thawed and used on the experimental day.

CELL GROWTH INHIBITION TEST
- Treatment conditions
-S9 mix assay: Short-term treatment assay (6-hour treatment and 18-hour recovery) without S9 mix
+S9 mix assay: Short-term treatment assay (6-hour treatment and 18-hour recovery) with S9 mix
24-hour assay: Continuous treatment assay (24-hour treatment) without S9 mix

- Test substance concentrations
-S9 mix assay: 3.13, 6.25, 12.5, 25, 50, 100, and 200 µg/mL
+S9 mix assay: 3.13, 6.25, 12.5, 25, 50, 100, and 200 µg/mL
24-hour assay: 3.13, 6.25, 12.5, 25, 50, 100, and 200 µg/mL

- Rationale for concentration selection
The preliminary test was performed at 20, 200, and 2000 µg/mL in each treatment condition before conducting the cell growth inhibition test. One plate was used per concentration for each treatment condition. Cell treatment was conducted as described below. The tested plates were observed by an inverted phase-contrast microscope to determine cell densities after the treatment. The cell density was presented as the relative percentage (%) to the negative control plate.
Based on the results, 200 µg/mL at which the cell growth inhibition was predicted to be more than 50% as the highest concentration and the concentrations described above were selected for the cell growth inhibition test.

- Cell treatment
(1) A 5 mL portion of the 4E+3 cells/mL cell suspension was placed onto a plastic plate (diameter: 6 cm) and incubated for 3 days.
(2) After the MEM culture medium was removed from the plate, the test substance formulation, S9 mix and MEM culture medium were added to the plate. In the negative control group, saline was used in place of the test substance formulation. Two plates were used per concentration for each treatment condition. In addition, the number of cells at the beginning of the treatment was measured using untreated 2 plates by the method above.
(3) Cells were freated for 6 hours in the short-term treatment assays and for 24 hours in the continuous treatment assay.
(4) In the short-term treatment assay, the surface of the cells were washed three times with MEM after 6-hour treatment, and the cells were incubated in 5 mL of fresh MEM culture medium for additional 18 hours.
(5) At the beginning and end of the treatment, all plates were examined macroscopically for possible precipitation of the test substance.

- Measurement of cell growth index (RPD)
(1) The cells were washed with phosphate buffer saline [abbr.: PBS (-)].
(2) The cells were treated with 0.25% trypsin-EDTA at 37°Cfor 5 minutes.
(3) The MEM culture medium was added to the plate, and the cells were dissociated by pipetting.
(4) The number of cells was measured with a cell counter.
(5) The population doubling (PD) was calculated from the following formula.
PD = log [(Number of cells at the end of culture) / (Number of cells at the beginning of the treatment)] / log 2
(6) The relative population doubling (RPD) was calculated from the following formula. RPD (%) = (PD in the test substance treatment group) / (PD in the negative control group) x 100
The RPD value was regarded as 0% when PD value of the test substance treatment group was less than 0.

- Calculation of 50% cell growth inhibition concentration
The IC50 was calculated in the -S9 mix, +S9 mix, and 24-hour assays. The IC50 value was calculated from a liner equation derived from the two data points showing cell growth indices higher and lower than 50%, but closest to 50%.

CHROMOSOMAL ABERRATION TEST
- Treatment conditions
-S9 mix assay, +S9 mix assay, and 24-hour assay

- Test substance concentrations
Main test 1
In the cell growth inhibition test, the IC50 were 27.8, 51.9, and 25.0 µg/mL in the -S9 mix, +S9 mix, and 24-hour assays, respectively.
Based on these results, the concentrations described below were set for the chromosomal aberration test (main test 1).
-S9 mix assay: 10, 15, 20, 25, 30, 35, 40, and 45 µg/mL
+S9 mix assay: 10, 20, 30, 40, 50, 60, 70, and 80 µg/mL
24-hour assay: 10, 15, 20, 25, 30, 35, 40, and 45 µg/mL

Main test 2
As a result of the chromosomal aberration test (main test 1), RPD value was 41.8% at 10 µg/mL in the -S9 mix assay and was 31.1% at 10 µg/mL in the 24-hour assay.
Because the accepted concentrations of the test substance treatment group for the metaphase analysis were less than three in the -S9 mix and 24-hour assays, the chromosomal aberration test (main test 2) was conducted at the concentrations described below;
-S9 mix assay: 1.25, 2.5, 5, 7.5, 10, 15, 20, 25, 30, and 35 µg/mL
24-hour assay: 0.625, 1.25, 2.5, 5, 10, 15, 20, 25, 30, and 35 µg/mL

- Positive control substance concentrations
-S9 mix assay: MMC at 0.1 µg/mL
+S9 mix assay: BP at 15 µg/mL
24-hour assay: MMC at 0.05 µg/mL

- Rationale for positive control substance concentration selection
These positive control substances are known to induce chromosomal aberrations at the concentrations described in the section above.

- Preparation of specimens
(1) Two hours before the end of culture, colcemid was added to the medium in each plate at a final concentration of 0.1 µg/mL to accumulate the metaphase cells.
(2) At the end of culture, the cells were washed with PBS (-).
(3) The cells were treated with 0.25% trypsin-EDTA at 37°C for 5 minutes and dissociated from the plate. MEM culture medium was added to the dissociated cell.
(4) The cell suspension was collected in a centrifuge tube and centrifuged (1000 rpm for 5 minutes; the same conditions were applied to the following centrifugation steps) to collect the cells.
(5) After the resulting supernatant was removed, the cells were subjected to hypotonic treatment with 4 mL of 0.075 mol/L potassium chloride solution at 37°C for 15 minutes.
(6) The cells and 0.5 mL of the ice-cold fixative mixture (ethanol/acetic acid mixture [3: 1, v/v]) were mixed. The mixture was centrifuged and the supernatant was removed.
(7) The cells and 4 mL of the ice-cold fixative mixture were mixed. The mixture was centrifuged and the supernatant was removed.
(8) The procedure in (7) was conducted again.
(9) The cells were suspended in a small amount of the ice-cold fixative mixture.
(10) The cell suspension was dropped onto two sites on a slide glass placed on wet cloth, and then was dried. Two slides were prepared from each plate.
(11) The cells were stained for 20 minutes with 3% Giemsa's solution. The slides were washed with water and dried.
(12) The slides were mounted with a cover glass and the mounting medium.

- Measurement of cell growth index (RPD, RCC)
(l) A portion of the cell suspensions harvested as described in section Preparation of specimens (3) was subjected to cell counting with a cell counter. This measurement of the cell number was conducted for all treatment conditions (the test substance, negative control substance, and positive control substance treatment groups).
(2) The RPD value was calculated. In addition, a Relative Cell Count (RCC, the mean of the negative control values was regarded as 100%) was calculated.

- Observation
Selection of concentrations for metaphase analysis
Main test 1
The concentrations at which the RPD was around 50% were 10 and 50 µg/mL in the -S9 mix and +S9 mix assays, respectively. In the 24-hour assay, the RPD was 31.1% at 10 µg/mL.
Based on the results of the measurement of RPD, the specimens of the following concentrations were used for the metaphase analysis.
+S9 mix assay: 30, 40, and 50 µg/mL
The metaphase analysis was not conducted in the -S9 mix and 24-hour assays because the accepted concentrations of the test substance treatment group for the metaphase analysis were less than three.

Main test 2
The concentrations at which the RPD was around 50% were 15 and 10 µg/mL in the -S9 mix and 24-hour assays, respectively.
Based on the results of the measurement of RPD, the specimens of the following concentrations were used for the metaphase analysis.
-S9 mix assay: 5, 10, and 15 µg/mL
24-hour assay: 2.5, 5, and 10 µg/mL

Preliminary observation
The specimens selected and those of the negative and positive control groups were preliminarily examined by Auto Metaphase Finder to confirm whether the test was performed appropriately. The followings were confirmed.
(1) In total of two slides prepared from each treatment plate, 150 or more metaphase cells were observed for each plate.
(2) The chromosomal aberrations were properly induced at the expected levels in the negative and positive control groups.

- Observation
Method for coding specimens
The specimens were rearranged randomly. The identification of the specimens was covered with labels and then observed.
Criteria for selection of observable metaphase cells
(1) Chromosomes should be well spread.
(2) Structural aberrant cell: Number of centromeres should be 25±2
Numerical aberrant cell: Number of centromeres should be 25±2 or 38 or more
Number of cells analyzed
150 cells per plate (300 cells per concentration)
Structural aberration
(1) Chromatid break
(2) Chromatid exchange
(3) Chromosome break
(4) Chromosome exchange (dicentric, ring etc.)
(5) Fragmentation
Gap
A "gap" was defined as an achromatic region in a single chromatid that was narrower than the width of the chromatid.
The number of gaps was listed in a separate column from the other aberrations. Gaps were not included in the structural aberrations.
Numerical aberration
(1) Polyploid cells with 38 or more centromeres
(2) Endoreduplicated cells

Evaluation criteria:

Criterion for acceptance of data
The data from the plate in which 150 metaphase cells (300 cells per concentration) could be observed were accepted.

Criteria for acceptance of the test
The test was accepted when the criteria described below were satisfied for each treatment condition.
(1) In the negative and positive controls and at three or more concentrations of the test substance treatment group, the criterion for acceptance of the data was satisfied.
(2) The incidences of both structural and numerical aberrant cells in the negative control group were within the historical negative control range (mean ± 2SD).
(3) The incidence of structural aberrant cells in the positive control group was within the historical positive control range (mean ± 3SD) and a significant difference from the negative control value was detected.

Judgment
Comparison with background data
When the incidences of both structural and numerical aberrant cells in the test substance treatment group were within the historical negative control range (mean ± 2SD) of the test facility, the test substance was judged as negative. When the incidence of structural or numerical aberrant cells in the test substance treatment group was more than the historical negative control range (mean ± 2SD) of the test facility, the statistical analysis described below was performed.

Statistics:

Statistical analysis was performed to detect the differences in the incidences of structural aberrant cells between the negative control and the positive control groups. Fisher's exact test was used for statistical analysis by the computer program package "Package software EXSUS, ver. 7.7.1 The level of significance was set at 5% (two-side).
Since the incidences of numerical aberrant cells at 30 µg/mL in the +S9 mix assay and structural aberrant cells at 10 µg/mL in the -S9 mix assay were more than the historical negative control range of the test facility, statistical analysis was performed to detect the differences in the incidences of aberrant cells between the negative control and the test substance treatment groups by Fisher's exact test. Since the statistically significant increase was not detected by Fisher's exact test, Cochran-Armitage test to detect the dose-response relationship was not performed.

Results and discussion

Test results

Key result

Species / strain:

Chinese hamster lung (CHL/IU)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Cell growth inhibition test
Precipitation of the test substance was observed in the freatment mixture at the beginning and the end of the treatnent at 50 µg/mL or more in the -S9 mix and +S9 mix assays and at 25 µg/mL or more in the 24-hour assay. Moreover, precipitation of the test substance was observed in the treatment rnixture at the beginning of the treatment at 25 µg/mL in the -S9 mix and +S9 mix assays. The IC50 were 27.8, 51.9, and 25.0 µg/mL in the -S9 mix, +S9 mix, and 24-hour assays, respectively.

Chromosomal aberration test
Main test 1
Precipitation of the test substance was observed in the treatment mixture at the beginning and the end of the freatment at 30 µg/mL or more in the -S9 mix and +S9 mix assays and at 25 µg/mL or more in the 24-hour assay. Moreover, precipitation of the test substance was observed in the treatment mixture at the beginning of the treatnent at 25 µg/mL in the -S9 mix assay. The concentrations at which the RPD was around 50% were 10 and 50 µg/mL in the -S9 mix and +S9 mix assays, respectively. In the 24-hour assay, the RPD was 31.1% at 10 µg/mL. None of the specimens prepared in the -S9 mix and 24-hour assays were used for the metaphase analysis because the accepted concentrations of the test substance treatment group for the metaphase analysis were less than three.
Based on the results of the RPD, the concentations for the metaphase analysis were selected at 30, 40, and 50 µg/mL in the +S9 mix assay. In the preliminary observation, all selected specimens were acceptable to perform metaphase analysis.
As a result of the metaphase analysis, the incidences of structural aberrant cells in the +S9 mix assay in the test substance treatment group were within the historical negative control range of the test facility, but the incidence of numerical aberrant cells was 0.7% at 30 µg/mL in the +S9 mix assay and outside the historical negative control range of the test facility.
However, no statistically significant increase of the incidence of numerical aberrant cells compared to the negative control value was detected by Fisher's exact test at 30 µg/mL in the +S9 mix assay.
In the negative control group, the incidences of both structural aberrant cells and numerical aberrant cells were within the historical negative control range under the treatment condition. In the positive control group, the incidence of structural aberrant cells was within the historical positive control range and a significant difference from the negative control value was detected under the treatment condition

Main test 2
Precipitation of the test substance was observed in the treatment mixture at the beginning and the end of the treatrnent at 25 µg/mL or more in the -S9 mix and 24-hour assays. Moreover, precipitation of the test substance was observed in the treatment mixture at the end of the treatment at 20 µg/mL in the 24-hour assay. The concentrations at which the RPD was around 50% were 15 and 10 µg/mL in the -S9 mix and 24-hour assays, respectively.
Based on the results of the RPD, the concentrations for the metaphase analysis were selected at 5, 10, and 15 µg/mL in the -S9 mix assay and at 2.5, 5, and 10 µg/mL in the 24-hour assay. In the preliminary observation, all selected specimens were acceptable to perform metaphase analysis.
As a result of the metaphase analysis, the incidences of structural and numerical aberrant cells in the 24-hour assay and the incidences of numerical aberrant cells in the -S9 mix assay in the test substance treatment group were within the historical negative control range of the test facility, but the incidence of structural aberrant cells was 3.7% at 10 µg/mL in the -S9 mix assay and outside the historical negative control range of the test facility.
However, no statistically significant increase of the incidence of structural aberrant cells compared to the negative control value was detected by Fisher's exact test at 10 µg/mL in the -S9 mix assay.
In the negative control group, the incidences of both structural aberrant cells and numerical aberrant cells were within the historical negative control range under the respective treatment conditions. In the positive control group, the incidence of structural aberrant cells was within the historical positive control range and a significant difference from the negative control value was detected under the respective treatment conditions.

Any other information on results incl. tables

Table 1 Results of Cell Growth Inhibition Test

Concentration (ug/mL)	Relative population doubling (%)
	Short-term treatment assay		Continuous treatment assay
	—S9 mix assay	+S9 mix assay	24-hour assay
Negative control (Saline)	100.0	100.0	100.0
3.13	92.7	97.7	94.7
6.25	87.9	98.0	92.8
12.5	79.6	94.7	76.3
25	59.9	84.0	58.2
50	12.1	53.3	0.0
100	0.0	9.7	0.0
200	0.0	0.0	0.0

Table 2 Results of the cell growth index in the chromosomal aberration test (main test 1)

Concentration (µg/mL)	Short-term treatment assay (-S9 mix assay)		Continuous treatment assay (24-hour assay)
	RPI) (%)	RCC (%)	RPI) (%)	RCC
Negative control (Saline)	100.0	100.0	100.0	100.0
10	41.8	67.4	31.1	58.4
15	16.8	56.7	0.0	41.2
20	0.0	46.7	0.0	32.9
25	0.0	40.8	0.0	24.2
30	0.0	33.8	0.0	20.6
35	0.0	27.0	0.0	16.8
40	0.0	25.6	0.0	13.7
45	0.0	24.1	0.0	11.4

Applicant's summary and conclusion

Conclusions:

The test item was not considered to have the potential to induce chromosomal aberration in CHL/IU cells under the conditions employed in this study.

Executive summary:

An in vitro chromosomal aberration study was conducted with CHL/IU cells derived from the lungs of female Chinese hamsters as indicator cells according to OECD 473.

The cell growth inhibition test was conducted at 3.13, 6.25, 12.5, 25, 50, 100, and 200 µg/mL in the absence of S9 mix (-S9 mix assay) and the presence of S9 mix (+S9 mix assay) in the short-term treatment assay and in the continuous treatment assay for 24 hours (24-hour assay).

As a result of the cell growth inhibition test, the 50% cell growth inhibition concentration (IC50) was 27.8, 51.9, and 25.0 µg/mL in the -S9 mix, +S9 mix, and 24-hour assays, respectively.

Based on the above results, the chromosomal aberration test (main test 1) was conducted at 10, 15, 20, 25, 30, 35, 40, and 45 µg/mL in the -S9 mix and 24-hour assays and at 10, 20, 30, 40, 50, 60, 70, and 80 µg/mL in the +S9 mix assay.

 

As a result, the incidence of numerical aberrant cells at 30 µg/mL in the +S9 mix assay was outside the historical negative control range of the test facility. However, no statistically significant increase of the incidence of numerical aberrant cells compared to the negative control value was detected by Fisher's exact test at 30 µg/mL in the +S9 mix assay. The incidences structural aberrant cells were within the historical negative control range of the test facility.

The chromosomal aberration test (main test 2) was conducted at 1.25, 2.5, 5, 7.5, 10, 15, 20, 25, 30, and 35 µg/mL in the -S9nff< assay and at 0.625, 1.25, 2.5, 5, 10, 15, 20, 25, 30, and 35 µg/mL in the 24-hour assay.

Based on the results of the cell growth index and the preliminary observation, the concentrations for the metaphase analysis were selected at 5, 10, and 15 µg/mL in the -S9 mix assay and at 2.5, 5, and 10 µg/mL in the 24-hour assay.

As a result, the incidence of structural aberrant cells at 10 µg/mL in the -S9 mix assay was outside the historical negative control range of the test facility. However, no statistically significant increase of the incidence of structural aberrant cells compared to the negative control value was detected by Fisher's exact test at 10 µg/mL in the -S9 mix assay. The incidences of structural and numerical aberrant cells in the 24-hour assay and the incidences of numerical aberrant cells in the -S9 mix assay were within the historical negative control range of the test facility.

In conclusion, the test item was not considered to have the potential to induce chromosomal aberration in CHL/IU cells under the conditions employed in this study.