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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In Vitro Mammalian Chromosome Aberration Test:

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

Ames study:

Mutagenicity of the test substance was assessed in the bacterial reverse mutation test using five strains, Salmonella typhimurium TA 100, TA 1535, TA98 and TA1537, and Escherichia coli WP2uvrA based on OECD 471.

It was concluded that the test item was not mutagenic under the conditions employed.

In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene:

The study was conducted according to a OECD 490 that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line.The test item did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the Global Evaluation Factor (GEF) of 126 E-6, consequently it is considered to be non-mutagenic in the mouse lymphoma assay.

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:

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

2014

Deviations:

no

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

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.

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.

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.

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.

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

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.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

This study was conducted between 16 June 2017 and 08 August 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

29 July 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

EC No. 440/2008 of 30 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Specific details on test material used for the study:

Information as provided by the Sponsor.
Identification: THEMIS (EC 500-429-8)
Purity: 100% (UVCB)
Description: Pale brown powder
Receipt date: 05 December 2016
Batch number: 161129
Expiry date: 28 November 2017
Storage Conditions: Room temperature in the dark

Target gene:

thymidine kinase, TK +/-, locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

Cell Line
The L5178Y TK+/- 3.7.2c mouse lymphoma cell line was obtained from Dr. J. Cole of the MRC Cell Mutation Unit at the University of Sussex, Brighton, UK. The cells were originally obtained from Dr. D. Clive of Burroughs Wellcome (USA) in October 1978 and were frozen in liquid nitrogen at that time.
Cell Culture
The stocks of cells are stored in liquid nitrogen at approximately -196°C. Cells were routinely cultured in RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/mL), Streptomycin (100 µg/mL), Sodium pyruvate (1 mM), Amphotericin B (2.5 µg/mL) and 10% donor horse serum (giving R10 media) at approximately 37 °C with 5% CO2 in air. The cells have a generation time of approximately 12 hours and were subcultured accordingly. RPMI 1640 with 20% donor horse serum (R20), 10% donor horse serum (R10), and without serum (R0), are used during the course of the study. Master stocks of cells were tested and found to be free of mycoplasma.

Metabolic activation:

with and without

Metabolic activation system:

Lot No. PB/βNF S9 30/06/17 and 31/03/17 was used in this study, and was pre-prepared inhouse following standard procedures. Prior to use, each batch of S9 is tested for its capability to activate known mutagens in the Ames test.
The S9-mix was prepared prior to the dosing of the test cultures and contained the S9 fraction (20% (v/v)), MgCl2 (8mM), KCl (33mM), sodium orthophosphate buffer pH 7.4 (100mM), glucose-6-phosphate (5mM) and NADP (5mM). The final concentration of S9, when dosed at a 10% volume of S9-mix into culture media, was 2%.

Test concentrations with justification for top dose:

Following solubility checks performed in-house, the test item was accurately weighed and formulated in DMSO prior to serial dilutions being prepared. The test item is a UVCB with a purity value of 100%; therefore the maximum recommended dose level was initially set at 5000 µg/mL. However the maximum achievable dose level was 2500 µg/mL due to formulation problems at 5000 µg/mL.

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Solvent (RO media) (Invitrogen batch 1858698 Expiry 30.04.18) exposure groups were used as the vehicle controls

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

Preliminary Toxicity Test
A preliminary toxicity test was performed on cell cultures at 5E+05 cells/mL, using a 4-hour exposure period both with and without metabolic activation (S9), and at 1.5E+05 cells/mL using a 24-hour exposure period without S9. The dose range was set at 0.63 to 160 µg/mL in all three exposure groups. The dose levels were selected to avoid excessive precipitate. Following the exposure period the cells were washed twice with R10, resuspended in R20 medium, counted using a Coulter counter and then serially diluted to 2 E+05 cells/mL.
The cultures were incubated at 37°C with 5% CO2 in air and sub-cultured after 24 hours by counting and diluting to 2E+05 cells/mL. After a further 24 hours the cultures were counted and then discarded. The cell counts were then used to calculate Suspension Growth (SG) values. The SG values were then adjusted to account for immediate post treatment toxicity, and a comparison of each treatment SG value to the concurrent vehicle control performed to give a percentage Relative Suspension Growth (%RSG) value.
Results from the preliminary toxicity test were used to set the test item dose levels for the mutagenicity experiments. Maximum dose levels were selected using the following criteria:
i) Maximum recommended dose level, 5000 µg/mL or 10 mM, whichever is the lowest concentration.
ii) The presence of precipitate regardless of where test item-induced toxicity was observed.
iii) Test item-induced toxicity, where the maximum dose level used should produce 10 to 20% survival (the maximum level of toxicity required). This optimum upper level of toxicity was confirmed by an IWGT meeting in New Orleans, USA (Moore et al., 2002).

Mutagenicity Test
Several days before starting the experiment, an exponentially growing stock culture of cells was set up so as to provide an excess of cells on the morning of the experiment. The cells were counted and processed to give 1E+06 cells/mL in 10 mL aliquots in R10 medium in sterile plastic universals for the 4-hour exposure groups in both the absence and presence of metabolic activation, and 0.3 E+06 cells/mL in 10 mL cultures were established in 25 cm2 tissue culture flasks for the 24-hour exposure group in the absence of metabolic activation. The exposures were performed in duplicate (A + B), both with and without metabolic activation (2% S9 final concentration) at 8 dose levels of the test item (0.06 to 8 µg/mL for all three exposure groups), vehicle and positive controls. 2 mL of S9-mix if required, 2 mL of the exposure dilutions, (0.2 mL or 0.15 mL for the positive controls), and sufficient R0 medium to bring the total volume to 20 mL (R10 was used for the 24 hour exposure group) were added to each universal.
The exposure vessels were incubated at 37°C for 4 or 24 hours with continuous shaking using an orbital shaker within an incubated hood.

Assessments
Measurement of Survival, Viability and Mutant Frequency
At the end of the treatment period, for each experiment, the cells were washed twice using R10 medium then resuspended in R20 medium at a cell density of 2 E+05 cells/mL. The cultures were incubated at 37°C with 5% CO2 in air and subcultured every 24 hours for the expression period of two days, by counting and diluting to 2E+05 cells/mL, unless the mean cell count was less than 3 E+05 cells/mL in which case all the cells were maintained.
On Day 2 of the experiment, the cells were counted, diluted to 1E+04 cells/mL and plated for mutant frequency (2000 cells/well) in selective medium containing 4 µg/mL 5-trifluorothymidine (TFT) in 96-well microtitre plates. Cells were also diluted to 10 cells/mL and plated (2 cells/well) for viability (%V) in non-selective medium.
The daily cell counts were used to obtain a Relative Suspension Growth (%RSG) value that gives an indication of post treatment toxicity during the expression period as a comparison to the vehicle control, and when combined with the Viability (%V) data a Relative Total Growth (RTG) value.

Plate Scoring
Microtitre plates were scored using a magnifying mirror box after ten to twelve days incubation at 37°C with 5% CO2 in air. The number of positive wells (wells with colonies) was recorded together with the total number of scorable wells (normally 96 per plate). The numbers of small and large colonies seen in the TFT mutation plates were also recorded as the additional information may contribute to an understanding of the mechanism of action of the test item (Cole et al, 1990). Colonies are scored manually by eye using qualitative judgment. Large colonies are defined as those that cover approximately ¼ to ¾ of the surface of the well and are generally no more than one or two cells thick. In general, all colonies less than 25% of the average area of the large colonies are scored as small colonies. Small colonies are normally observed to be more than two cells thick. To assist the scoring of the TFT mutant colonies 0.025 mL of thiazolyl blue tetrazolium bromide (MTT) solution, 2.5 mg/mL in phosphate buffered saline (PBS), was added to each well of the mutation plates. The plates were incubated for two hours. MTT is a vital stain that is taken up by viable cells and metabolised to give a brown/black color, thus aiding the visualization of the mutant colonies, particularly the small colonies.

Calculation of Percentage Relative Suspension Growth (%RSG)
The cell counts obtained immediately post treatment and over the 2-day expression period were used to calculate the Percentage Relative Suspension Growth.
4-Hour Suspension Growth (SG) = (24-hour cell count/2) x (48-hour cell count/2)
24-Hour Suspension Growth (SG) = (0-hour cell count/1.5) x (24-hour cell count/2) x (48 hour cell count/2)
Day 0 Factor = dose 0-hour cell count/vehicle control 0-hour cell count
%RSG = [(dose SG x dose Day 0 Factor)/vehicle control SG] x 100

Calculation of Day 2 Viability (%V)
Since the distribution of colony-forming units over the wells is described by the Poisson distribution, the day 2 viability (%V) was calculated using the zero term of the Poisson distribution [P(0)] method.

P(0) = number of negative wells/ total wells plated

%V = -ln P(0) x 100/ number of cells/well

Calculation of Relative Total Growth (RTG)
For each culture, the relative cloning efficiency, RCE, was calculated:

RCE = %V / Mean Solvent Control %V

Finally, for each culture RTG is calculated:

RTG = (RCE x RSG)/100%

Calculation of Mutation Frequency (MF)
MF per survivor = [(-ln P(0) selective medium)/cells per well in selective medium)]/surviving fraction in non-selective medium.
The experimental data was analysed using a dedicated computer program, Mutant 240C by York Electronic Research, which follows the statistical guidelines recommended by the UKEMS (Robinson W D et al., 1989). The statistical package used indicates the presence of statistically significant increases and linear-trend events.

Evaluation criteria:

Dose selection for the mutagenicity experiments was made using data from the preliminary toxicity test in an attempt to obtain the desired levels of toxicity. This optimum toxicity is approximately 20% survival (80% toxicity), but no less than 10% survival (90% toxicity). Relative Total Growth (RTG) values are the primary factor used to designate the level of toxicity achieved by the test item for any individual dose level. However, under certain circumstances, %RSG values may also be taken into account when designating the level of toxicity achieved. Dose levels that have RTG survival values less than 10% are excluded from the mutagenicity data analysis, as any response they give would be considered to have no biological or toxicological relevance.
An approach for defining positive and negative responses is recommended to assure that the increased MF is biologically relevant. In place of statistical analysis generally used for other tests, it relies on the use of a predefined induced mutant frequency (i.e. increase in MF above the concurrent control), designated the Global Evaluation Factor (GEF) of 126E-6, which is based on the analysis of the distribution of the vehicle control MF data from participating laboratories.
Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if, in any of the experimental conditions examined, the increase in MF above the concurrent background exceeds the GEF and the increase is concentration related (e.g., using a trend test). The test chemical is then considered able to induce mutation in this test system.
Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly negative if, in all experimental conditions examined there is no concentration related response or, if there is an increase in MF, it does not exceed the GEF. The test chemical is then considered unable to induce mutations in this test system.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Remarks:

At the TK+/- locus

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:

There was no evidence of toxicity following exposure to the test item in the all three exposure groups as indicated by the %RSG and RTG values. There was no evidence of reductions in viability (%V) in either of the three exposure groups, therefore indicating that residual toxicity had not occurred. The dose levels of 4 and 8 µg/mL in all three exposures were not plated out for 5-TFT resistance and viability due to excessive precipitate. The dose level of 2 µg/mL in the 24-hour exposure was plated out for 5-TFT resistance and viability but later discarded due to excessive precipitate. The precipitate observations did vary from the preliminary toxicity test, however at such low concentrations of the test item the precipitate was found difficult to identify. The OECD 490 guideline was met with only one precipitating dose level analysed in all three exposure groups. Acceptable levels of toxicity were seen with both positive control substances.
The vehicle controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. The positive controls produced marked increases in the mutant frequency per viable cell achieving the acceptability criterion, indicating that the test system was operating satisfactorily, and that the metabolic activation system was functional.
The test item did not induce any toxicologically significant increases in the mutant frequency E-6per viable cell in either of the three exposure groups. The GEF value of the test item dose levels were not exceeded in any of the three exposure groups. 

Preliminary Cytotoxicity Test

The dose range of the test item used in the preliminary toxicity test was 0.63 to 160 µg/mL. The results for the Relative Suspension Growth (%RSG) were as follows:

Dose (g/mL)	% RSG (-S9) 4-Hour Exposure	% RSG (+S9) 4-Hour Exposure	% RSG (-S9) 24-Hour Exposure
0	100	100	100
0.63	99p	80p	110p
1.25	102p	82p	78
2.5	90p	79p	85p
5	85p	77p	65p
10	86p	78p	46p
20	57p	44p	0p
40	0p	0p	0p
80	0p	0p	0p
160	0p	0p	0p

p= precipitate of test item at the end of exposure

In the all three exposure groups there was evidence of marked reductions in the relative suspension growth (%RSG) of cells treated with the test item when compared to the concurrent vehicle controls. A precipitate of the test item was observed at and above 0.63 µg/mL in all three exposure groups. In the subsequent mutagenicity experiments the maximum dose level was limited by precipitate of the test item.

Mutagenicity Test

Table 1            Summary of Results

Main Experiment

Treatment (µg/ml)	4-hours-S-9			Treatment (µg/ml)	4-hours+S-9
	%RSG	RTG	MF§		%RSG	RTG	MF§
0	100	1.00	180.09	0	100	1.00	172.59
0.06	96	0.93	187.01	0.06	103	1.11	148.41
0.13	100	0.84	183.71	0.13	111	1.03	168.14
0.25	104	0.93	197.72	0.25	98	0.99	152.39
0.5	94	0.88	182.17	0.5	103	1.19	151.25
1	93	0.89	150.57	1	96	1.03	172.23
2	94	1.04	139.48	2	94	0.91	170.25
4       Ø	85			4      Ø	96
8       Ø	90			8      Ø	95
MF threshold for a positive response 306.09				MF threshold for a positive response 298.59
EMS				CP
400	70	0.50	1489.81	1.5	94	0.58	1414.68

                     

Treatment (µg/mL)	24 -hours S9
	%RSG	RTG	MF§
0	100	1.00	163.62
0.06	103	0.84	184.53
0.13	101	0.89	161.64
0.25	98	1.00	135.92
0.5	97	1.03	145.16
1	99	0.89	166.75
2 X	106	0.83	162.05
4 Ø	109
8 Ø	87
MF Threshold for a positive response 289.62
EMS 150	37	0.49	1484.85

%RSG = Relative Suspension Growth

RTG = Relative Total Growth

MF§ =  5-TFT resistant mutants/10⁶viable cells 2 days after treatment

X = Excluded due to excessive precipitate

Ø = Not plated for viability or 5-TFT resistance

                                                       

Conclusions:

The test item did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the Global Evaluation Factor (GEF) of 126E-6, consequently it is considered to be non-mutagenic in this assay.

Executive summary:

The study was conducted according to OECD490 assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. 

One main Mutagenicity Test was performed. In this main test, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at 8 dose levels in duplicate, together with vehicle (R0 media), and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9), and a 24-hour exposure group in the absence of metabolic activation.

The dose range of test item used in the main test was selected following the results of a preliminary toxicity test. At the end of exposure precipitate of the test item was observed at and above 2 µg/mL in both 4-hour exposure groups. At the end of exposure in the 24-hour exposure group a precipitate of the test item was observed at and above 1 µg/mL. The dose levels plated for viability and expression of mutant colonies were 0.06, 0.13, 0.25, 0.5, 1, 2 µg/mL for 4-hour with/without S9 and 24-hour without S9.

The test item did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the Global Evaluation Factor (GEF) of 126 E-6, consequently it is considered to be non-mutagenic in this assay.  

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

This study was conducted between 11 March 2015 and 10 May 2016

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

Qualifier:

according to guideline

Guideline:

other: Testing Methods Concerning New Chernical Substances

Version / remarks:

Notification 0331 No.7, PFSB, MHLW, Japan; No.5 of March 29, 2011, MIB, METI, Japan; No.110331009, EPB, MOE, Japan; dated March 31, 2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Notice on Standards Issued by Ministry of Labor Based on Provisions of Article 57-3, Paragraph 1 of Industrial Safety and Health Act

Version / remarks:

Notice No.77, MOL, Japan, dated September 1, 1988

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Partial Revision of Nofice on Standards Issued by Ministry of Labor Based on Provisions of Article 57-3, Paragraph 1 of Industrial Safety and Health Act

Version / remarks:

Notice No.67, MOL, Japan, dated June 2, 1997

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Name of new chemical (IUPAC nomenclature) 4,4'-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with [(dimethylamino)methyl]phenol and piperazine
Other name THEMIS
Purity of new chemical tested 100.0 %
Lot No. of new chemical tested 151007
CAS No. 159034-96-5
Molecular weight (Industrial products) Mn: 3,000 - 30,000; Mw: 11,000 - 110,000; (Test substance) Mn: 3,000, Mw: 11,000
Melting point 120 - 140”C
Appearance at ambient temperature Powder, pale pink
Stability Swelled by water, Soften in 100”C or more
Solubility in several solvents Water Swelled (suspended at 50 mg/mL), Stable
DMSO Partially dissolved (not dissolved or suspended at 50 mg/mL)
Acetone Practically insoluble

Target gene:

Stains Amino acid requirement (1) UV sensitivity (2) Membrane mutation Drug resistance (4)
TA100 his- (base-pair change) ∆ uvrB rfa + (pKM101)
TA1535 his- (base-pair change) ∆ uvrB rfa -
TA98 his- (frarne-shift) ∆ uvrB rfa + (pKM101)
TA1537 his- (frame-shift) ∆ uvrB rfa -
WP2uvrA trp- (base-pair change) ∆ uvrA Wild Type -

(1) his-, histidine requirement; and trp-, fryptophan requirement
(2) ∆ uvrA and ∆ uvrB represent deletion of uvrA or uvrB gene (coding for DNA excision repair system), and the sfrains with each mutation have UV sensitivity.
(3) rfa denotes the deep rough character of cell wall, and the stains with this mutation have crystal violet sensitivity.
(4) + (pKM101) is the ampicillin resistance factor (R-factor), and the strains with this plasmid have ampicillin resistance.

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 Kikkoman Biochemifa Company
- method of preparation of S9
The S9 was prepared from the livers of 7-week old male Sprague-Dawley rats treated with phenobarbital and 5,6-benzoflavone.
- Protein content: 23.36 mg/mL
- Storage conditions: Below -80°C [Sanyo Ultralow freezer, MDF-192]
- S9 mix preparation:
One milliliter of S9 was added to 9 mL of the cofactor mix to give the proper ratio for the S9 mix. The S9 mix was freshly prepared just prior to use in each test and was kept in an ice bath until use.

Test concentrations with justification for top dose:

Preliminary test: 1.22, 4.88, 19.5, 78.1, 313, 1250, 5000 µg/plate with and without S9 mix
Main test I and main test II: 313, 625, 1250, 2500, 5000 µg/plate with and without S9 mix

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Sterilized distilled water (DW)

- Justification for choice of solvent/vehicle:
The test substance is swelled in water, partially dissolved in dimethyl sulfoxide (abbr. DMSO), practically insoluble in acetone by preliminary information from the sponsor.
In the solvent selection test, the test substance was suspended at 50 mg/mL in DW.
The test substance was not dissolved or suspended at 50 mg/mL in DMSO.
Increases in temperature, discoloration and foaming were not observed when the test substance was mixed with DW. According to these results, DW was selected as the solvent (negative control) for the test substance in this study.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

other: 2-(2-Furyl)-3-(5-nitro-2-furyl) acrylamide (AF-2), 9-Aminoacridine hydrochloride monohydrate (9-AA), 2-Amino Anthracene (2-AA)

Details on test system and experimental conditions:

BACTERIAL SUSPENSIONS
- Culture condition
Temperature: 37°C (kept at 40°C until culture)
Time: 8 hours
Method: Reciprocal shaking (shaking frequency: 90 times/minute)
Vessel: L-type test tube (volume: 22 mL)
Mediurn: Liquid growth medium (10 mL)
Stain and inoculate volume: A frozen stock suspension of the tester stain was thawed and 0.02 mL was inoculated.

- Measurement of the number of cells in bacterial suspension
After pre-culture, bacterial density of each strain was measured by a digital photometer (TAITEC Co., Ltd. Mini photo 518R), and then the bacterial suspension for counting the number of cells was prepared by the stepwise dilution method. The suspension was overlaid to a minimal glucose agar plate and incubated at 37 °C for 48 hours. The number of cells was calculated by counting the number of colonies on the plate.

MEDIA
- Preparation of liquid growth medium
Oxoid Nuffient Broth No.2 (Oxoid Ltd., Lot No. 1554986) 2.5 g was dissolved in 100 mL of purified water. The medium was autoclaved for 15 minutes at 121°C and stored in a cold storage.
- Preparation of top agar
Preparation of soft agar
Bacto-agar (Becton, Dickinson and Company, Lot No.5012909) 0.6 g and sodium chloride (Wako Pure Chemical Industries, Ltd.) 0.5 g were added to 100 mL of purified water. This mixture was autoclaved for 15 minutes at 121 °C and then was stored at 45°C until use.
Preparation of top agar
Each of the following each amino acid solution was added to the soft agar at a volume ratio of 1 to 10 just before use. The top agar was kept at 45°C until use.
Salmonella typhimurium: 0.5 mmol/L D-biotin and 0.5 mmol/L L-histidine solution mixture
Escherichia coli: 0.5 mmol/L L-tryptophan solution


PREPARATION OF THE TEST SUBSTANCE AND POSITIVE CONTROL SOLUTIONS
- Preparation of the test substance solutions
Preparation
(1) The test substance was weighed and suspended in DW with vortex mixing and ultrasonic wave treatment to make a 50 mg/mL suspension.
(2) A portion of this suspension was diluted stepwise with DW to make the test substance solutions of each concentration.
(3) All procedures, including those for weighing the test substance, dilution, dispensation and treatment of the solutions, were performed under a yellow light at room temperature.
Storage period and temperature
The storage period and temperature after preparation of the test substance solutions to the time of use were as follows:
Preliminary test: 2 hours and 5 minutes at room temperature (23°C) Main test I: 2 hours and 3 minutes at room temperature (22°C)
Main test Il: 1 hour and 41 minutes at room temperature (22°C)

- Preparation of positive control solutions
Preparation
Positive control solutions were prepared by the following method;
(1) NaN3 was dissolved in sterilized distilled water (Wako Pure Chemical Industies, Ltd.) and AF-2, 9-AA and 2-AA were dissolved in DMSO (Dojindo Laboratories).
(2) The above preparations were diluted with each solvent for preparing stock solutions.
Usage of the positive control solutions
The positive control solutions were prepared and stored as stock solutions, the stock solutions of positive control were thawed before use and used for the study.
2-AA stock solution was diluted more with DMSO at tested concentration. The remainders of thawed solutions were not reused.
Storage of the positive control solutions Divided into freezing tubes (1.0 mL) and frozen
Storage conditions
Below -20°C [Pharmaceutical refrigerator with freezer; MPR-414FS] (AF-2, NaN3 and 9-AA)
Below -80°C [Sanyo Ultralow freezer; MDF-192] (2-AA)

REVERSE MUTATION TEST
- Selection of the test method
This study was performed using the pre-incubation method with. and without S9 mix.
- Pre-incubation method
(1) For each treatment, 0.1 mL of the test substance solution, negative (solvent) control or positive control solution was added into a sterilized test tube with alunlinum cap.
(2) For assays without S9 mix, 0.5 mL of 0.1 mol/L sodium phosphate buffer (pH 7.4) was mixed and 0.1 mL of the bacterial suspension was added to this mixture.
(3) For assays with S9 mix, 0.5 mL of S9 mix was mixed and 0.1 mL of the bacterial suspension was added to this mixture.
(4) The mixture was incubated with gentle shaking (shaking frequency: 120 times/minute) for 20 minutes at 37 (pre-incubation).
(5) After pre-incubation, 2 mL of the molten top agar was added to this mixture and then poured onto a minimal glucose aga.r plate.
(6) After the overlaid agar had solidified, the plates were incubated for 48 hours at 37°C.

- Sterility test
In the preliminary test and two main tests, bacterial contamination was examined using one plate for each of the highest dose of the test substance suspension, S9 mix, and the 0.1 mol/L sodium phosphate buffer.
(1) 0.1 mL of the highest dose of the test substance suspension or 0.5 mL of the S9 mix or the 0.1 mol/L sodium phosphate buffer was mixed with 2 mL of the molten top agar.
(2) The mixture was poured onto a minimal glucose agar plate.
(3) After the overlaid agar had solidified, the plates were incubated for 48 hours at 37°C and then checked for bacterial contamination macroscopically.

- Observation
Precipitation: Precipitation was judged by observation of the plate surface macroscopically.
Microbial growth inhibition: Backgound lawn of the bacterial cells that have amino-acid requirement was observed by a stereoscopic microscope (Nikon, SMZ-10), and microbial growth inhibition was judged by the relationship between the test substance treated plates and the negative (solvent) control plate.

- Measurement of colony number
Revertant colonies were measured using an automatic colony counter (System Science Co., Ltd., CA-11D) or manual counting. Revertant colonies in positive control freatment groups, in TA100 of test substance treatment groups without precipitation, and in the test substance teatment groups in which many revertant colonies were observed were measured using an automatic colony counter and the others measured by manual counting. Correction of the measuring area was employed using instrumental analysis.

- Number of plate
Preliminary test: 1 plate/dose (2 plates/dose for the negative (solvent) and positive controls)
Main test I: 3 plates/dose
Main test II: 3 plates/dose

Expression of data
In the preliminary test, the mean of revertant colonies was calculated for the negative (solvent) control and the positive control.
In the main test, the mean and the standard deviation of revertant colonies were calculated for the negative (solvent) control, the positive control and the test substance treatment groups. The mean and the standard deviation were expressed by rounding to the first decimal place.

Evaluation criteria:

Acceptable criteria
The main tests were accepted as valid if all the following criteria were satisfied. The preliminary test was accepted as valid if the data by which doses were set in the main tests was provided.
(1) The negative (solvent) contol values (mean) and the positive confrol values (mean) are within the proper ranges calculated based on the historical data at our laboratory.
(2) The positive confrol values (mean) show clear positive responses in the respecfive tester strains, as evidenced by the number of revertant colonies being greater than 2-fold of the respective negative (solvent) control value (mean).
(3) There are more than 4 doses showing no microbial growth inhibition and more than 5 doses applicable to the evaluation.
(4) The result of the sterility test indicates that there is no bacterial contamination.
(5) There are no plates that became invalid for measurement due to contamination or other unexpected situations.

Judgment of test results
Test substance was judged to have mutagenicity (positive) when the test substance induced a dose-dependent increase in the number of the revertant colonies (mean) to a level equal to or greater than 2-fold of the negative (solvent) control value (mean value) in any one of the tester strains with or without S9 mix, and when the dose-dependent increase was reproducible. Other results were judged to be negative. Mutation activities (number of revertant colonies/mg) were not calculated because the test substance was judged to be negative.

Confirmation of the reproducibility
The reproducibility of the test result was confirmed in the two main tests.

Statistics:

No statistical analysis was performed with the test results in this study.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no 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:

no 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:

no 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:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

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:

Preliminary test
Number of revertant colonies: The number of revertant colonies treated with the test substance was less than twice that treated with the negative (solvent) control in all tester strains with or without S9 mix.
Microbial growth inhibition: Microbial growth inhibition was not observed in all tester strains with or without S9 mix.
Precipitation: Precipitation of the test substance was observed at the following dose levels.
Without S9 mix: 1250 µg/plate or more (all tester strains)
With S9 mix: 1250 µg/plate or more (all tester stains)

Main test I
Number of revertant colonies: The number of revertant colonies treated with the test substance was less than twice that treated with the negative (solvent) control in all tester strains with or without S9 mix.
Microbial growth inhibition: Microbial growth inhibition was not observed in all tester strains with or without S9 mix.
Precipitation: Precipitation of the test substance was observed at the following dose levels.
Without S9 mix: 1250 µg/plate or more (all tester stains)
With S9 mix: 1250 µg/plate or more (all tester stains)

Main test Il
Number of revertant colonies: The number of revertant colonies treated with the test substance was less than tmice that treated with the negative (solvent) control in all tester strains with or without S9 mix.
Microbial growth inhibition: Miicrobial growth inhibition was not observed in all tester strains with or without S9 rnix.
Precipitation: Precipitation of the test substance was observed at the following dose levels.
Without S9 mix: 1250 µg/plate or more (all tester strains)
With S9 mix: 1250 µg/plate or more (all tester sfrains)

Sterility test
Bacterial or fungous contamination was not observed in plates of the highest dose of the test substance suspension, S9 mix or the 0.1mol/L sodium phosphate buffer in the preliminary test and two main tests.

Conclusions:

From the results described above, it was concluded that the test item was not mutagenic under the conditions employed in this study.

Executive summary:

Mutagenicity of the test substance was assessed in the bacterial reverse mutation test using five strains, Salmonella typhimurium TA 100, TA 1535, TA98 and TA1537, and Escherichia coli WP2uvrA based on OECD 471. The test was conducted by the pre-incubation method with and without S9 mix.

A preliminary test was performed at 5000 µg/plate as the maximum dose using 7 doses with a common ratio of 4. As the results, the number of revertant colonies treated with the test substance was less than twice that treated with the negative (solvent) control in all tester strains with or without S9 mix.

Microbial growth inhibition of the test substance was not observed in all tester stains with or without S9 mix.

Precipitation of the test substance was observed at 1250 µg/plate or more in all tester strains with or without S9

Precipitation of the test substance was observed at 1250 µg/plate or more in all tester strains with or without S9 mix.

The main test II was performed at using the same doses as the main test I. As the results, the number of revertant colonies treated with the test substance was less than twice that treated with the negative (solvent) control in all tester strains with or without S9 mix. Microbial growth inhibition of the test substance was not observed in all tester strains with or without S9 mix.

Precipitation of the test substance was observed at 1250 µg/plate or more in all tester strains with or without S9 mix.

From the results described above, it was concluded that the test item was not mutagenic under the conditions employed in this study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

In Vitro Mammalian Chromosome Aberration Test: OECD 473, negative

Ames study: OECD 471, negative

In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene: OECD 490, negative

Therefore in accordance with Regulation (EC) No. 1272/2008 Table 3.5.1, the test substance should not be classified as germ cell mutagens.