Reaction product of salicylaldehyde and formaldehyde and phenol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genetic toxicity in vitro, Ames test. Noguchi (2005)

Under the conditions of this study, the test material was judged negative for the potential to induce gene mutation (mutagenicity).

Genetic toxicity in vitro, mammalian chromosome aberration test. Murata (2012)

Under the conditions of this study, the test material was considered negative for clastogenic potential.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 September 2005 to 29 September 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: Yakusyoku No.1121002 of the Pharmaceutical and Food Safety Bureau, Ministry of Health, Labour and Welfare

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: H15.11.13 seikyoku No.2 of the Manufacturing Industries Bureau, Ministry of Economy, Trade and Industry

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: kanpoki No.031121002 of the Environmental Policy Bureau, Ministry of the Environment

Version / remarks:

November 21, 2003

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Notification of Ministry of the Labour, Japan, No.77, September 1, 1988 and No.67 (revised), June 2, 1997.

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

S. typhimurium: Histidine locus
E. coli: Tryptophan locus

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Dr. T. Matsushima, Japan Bioassay Research Center, Japan Industrial Safety and Association, Hadano-City, Kanagawa.
- Strains were stored at -80 °C
- Prior to the master strains being used, characterisation checks were carried out to confirm the amino-acid requirement, presence of rfa, R factors, uvrB or uvrA mutation and the spontaneous reversion rate.
- In this assay, overnight sub-cultures of the appropriate coded stock cultures were prepared in nutrient broth and incubated at 37 C for 10 hours. Each culture was monitored spectrophotometrically for turbidity with titters determined by viable count analysis on nutrient agar plates.

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Dr. T. Matsushima, Japan Bioassay Research Center, Japan Industrial Safety and Association, Hadano-City, Kanagawa.
- Strains were stored at -80 °C
- Prior to the master strains being used, characterisation checks were carried out to confirm the amino-acid requirement, presence of rfa, R factors, uvrB or uvrA mutation and the spontaneous reversion rate.
- In this assay, overnight sub-cultures of the appropriate coded stock cultures were prepared in nutrient broth and incubated at 37 C for 10 hours. Each culture was monitored spectrophotometrically for turbidity with titters determined by viable count analysis on nutrient agar plates.

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Dose-Determination test: 4.88, 19.5, 78.1, 313, 1250 and 5000 µg/plate
Mutagenicity tests: 9.77, 19.5, 39.1, 78.1, 156 and 313 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test material was not dissolved in water at 50 mg/mL, but was dissolved in DMSO at 100 mg/mL in solubility checks performed in-house. DMSO was therefore selected as the vehicle of choice.
- The test material was dissolved in DMSO to make a stock solution of 100 mg/mL and further diluted to obtain desired concentrations.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

other: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (0.01 µg/ 50 µL/ plate for TA100 and WP2uvrA (-S9 mix), and 0.1 µg/ 50 µL/ plate for TA98 (-S9 mix))

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 minutes
- Measured aliquots (0.05 mL) of the test material formulation, vehicle or positive control were dispensed into sets of test tubes followed by either 0.5 mL of S9 mix or phosphate buffer, 0.1 mL of one of the bacterial cultures. The contents of each test tube were incubated at 37 °C for 20 min. and mixed with 2.0 mL of molten, trace histidine and biotin or tryptophan supplemented, top agar and equally distributed onto the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated, in duplicate, for each bacterial strain and for each concentration of test material both with and without S9 mix.
- Exposure duration: 48 hours at 37 °C

EVALUATION
- The frequency of revertant colonies was assessed using a colony analyser CA-11S (System Science Co., Ltd.).

OTHER
- Dose-Determination test: Six concentrations of the test material (4.88, 19.5, 78.1, 313, 1250 and 5000 µg/plate) were assayed in duplicate against each tester strain, using the pre-incubation method.

Evaluation criteria:

- The response was regarded positive if the maximum number of revertant colonies in the test material group increased ≥ 2 fold relative to the negative control group and dose-response and reproducibility were confirmed.

Key result

Species / strain:

S. typhimurium, other: TA 1535, TA 1537 TA 98 & TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

- The test material did not induce ≥ 2 fold increase in the number of revertant colonies relative to the negative control value for any strain with or without metabolic activation, and the reproducibility of the results was confirmed. From these results, the test material was judged negative for the potential to induce gene mutation (mutagenicity).
- Cytotoxicity were observed at and above 313 µg/plate with or without metabolic activation on dose-determination test, at and above 156 µg/plate without metabolic activation, at and above 313 µg/plate with metabolic activation on mutagenicity test 1 and 2. No precipitate of the test material was observed at any dose level. In the sterility test, no growth of bacteria was observed under any test conditions.
- All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9 mix and the sensitivity of the bacterial strains.

Table 1: Summary of Mutagenicity Test 1

± S9 Mix	Concentration (µg/plate)	Mean number of colonies/plate
		Base-pair Substitution Type			Frameshift Type
		TA100	TA1535	WP2uvrA	TA98	TA1537
-	Solvent 9.77 19.5 39.1 78.1 156 313	110 93 102 100 107 81* 65*	10 6 8 11 10 8* 0*	34 28 27 31 28 28* 0*	14 14 17 18 16 8* 0*	9 11 9 10 6 0* 0*
+	Solvent 9.77 19.5 39.1 78.1 156 313	114 128 115 115 118 108 102*	11 9 10 8 12 11 6*	31 32 26 33 29 37 39*	25 24 27 26 30 26 25*	10 11 12 11 14 16 9*
Positive Controls
-	Name	AF-2	SA	AF-2	AF-2	9AA
	Concentration (µg/plate)	0.01	0.5	0.01	0.1	80
	Mean no. colonies/plate	639	261	224	513	359
+	Name	2AA	2AA	2AA	2AA	2AA
	Concentration (µg/plate)	1	2	10	0.5	2
	Mean no. colonies/plate	1293	159	725	537	194

AF-2 = 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide

9AA = 9-aminoacridine

2AA = 2-aminoanthracene

SA = Sodium azide

When toxicity was observed, "*" was placed to the right of the number of the revertants.

Table 2: Summary of Mutagenicity Test 2

± S9 Mix	Concentration (µg/plate)	Mean number of colonies/plate
		Base-pair Substitution Type			Frameshift Type
		TA100	TA1535	WP2uvrA	TA98	TA1537
-	Solvent 9.77 19.5 39.1 78.1 156 313	122 123 96 109 113 74* 0*	9 7 8 10 6 6* 0*	26 35 32 28 23 31* 0*	12 15 15 12 11 10* 0*	9 7 7 7 8 0* 0*
+	Solvent 9.77 19.5 39.1 78.1 156 313	115 127 136 124 113 129 77*	9 10 8 10 8 8 6*	29 29 26 35 34 38 25*	24 22 28 28 32 24 20*	13 13 13 9 11 13 9*
Positive Controls
-	Name	AF-2	SA	AF-2	AF-2	9AA
	Concentration (µg/plate)	0.01	0.5	0.01	0.1	80
	Mean no. colonies/plate	627	314	229	520	331
+	Name	2AA	2AA	2AA	2AA	2AA
	Concentration (µg/plate)	1	2	10	0.5	2
	Mean no. colonies/plate	1317	210	719	576	211

AF-2 = 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide

9AA = 9-aminoacridine

2AA = 2-aminoanthracene

SA = Sodium azide

When toxicity was observed, "*" was placed to the right of the number of the revertants.

Conclusions:

Under the conditions of this study, the test material was judged negative for the potential to induce gene mutation (mutagenicity).

Executive summary:

The genetic toxicity of the test material was investigated in accordance with Japanese guidelines under GLP conditions. The mutagenic potential was assessed with a bacterial reverse mutation assay.

Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and Escherichia coli WP2uvrA were treated with the test material using the pre-incubation method at six dose levels, in duplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range for the dose-determination test was 4.88 to 5000 µg/plate. The experiment was repeated on a separate day using the dose range, 9.77 to 313 µg/plate, fresh cultures of the bacterial strains and fresh test substance formulations.

Cytotoxicity was observed at and above 313 µg/plate with or without metabolic activation on dose-determination test, at and above 156 µg/plate without metabolic activation, at and above 313 µg/plate with metabolic activation on mutagenicity test 1 and mutagenicity test 2. No precipitate of the test material was observed at any dose level.

No significant increases in the frequency of revertant colonies were recorded for any strain with or without metabolic activation.

Under the conditions of this study, the test material was judged negative for the potential to induce gene mutation (mutagenicity).

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 April 2012 to 07 May 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: Notification Yakusyoku 0331 No.7

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: H23.03.29 seikyoku No.5

Qualifier:

according to guideline

Guideline:

other: Kanpoki No.110331009

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Japan Health Sciences Foundation Health Sciences Research Resources Bank (HSRRB)
- Suitability of cells: The cell is suitable for chromosomal analysis due to relatively large chromosome size and a low number of chromosomes. There is a large amount of background data regarding chemical compounds.
- Freezing conditions: Cell suspension was frozen at a density of 1.5×10^6 cells/mL with 10 % (v/v) DMSO and was stored in liquid nitrogen.
- Number of passages if applicable: The cells were in their 5th passage at receipt. The cells within 30th passages were used for the test after thawing the 7th passage in the frozen state (frozen Lot No. 031212).
- Characteristic inspection: No morphological abnormality; chromosome number (modal number) of 25; cell doubling time of approximately 15 hours; and absence of mycoplasma contamination.

MEDIA USED
- Eagle’s MEM (SIGMA-ALDRICH Corporation) supplemented with 10% (v/v) bovine calf serum (SAFC Biosciences Inc., Lot No. 9D0732) was used. The bovine calf serum was inactivated in UBE Scientific Analysis Laboratory, Inc.
- The cells were cultured at 37 °C and 5 % CO2.
- Cells were pre-cultured at 2 x 10^4 cells per 5 mL culture medium per plate, for 3 days.

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Growth inhibition test (Short-term treatment process): 0.0781, 0.156, 0.313, 0.625, 1.25, 2.50 and 5.00 mg/mL
Growth inhibition test (Continuous treatment process): 0.0195, 0.0391, 0.0781, 0.156, 0.313, 0.625, 1.25, 2.50 and 5.00 mg/mL
Chromosome Aberration Test; cell growth rate (Short-term treatment process): 0.0781, 0.110, 0.156 and 0.221 mg/mL (-S9 mix) and 0.0391, 0.0552, 0.0781 and 0.110 mg/mL (+S9 mix)
Chromosome Aberration Test (Short-term treatment process): 0.0552, 0.0781, 0.110 and 0.156 mg/mL (-S9 mix) and 0.0138, 0.0195, 0.0276 and 0.0391 mg/mL (+S9 mix)
Chromosome Aberration Test (Continuous treatment process): 0.0276, 0.0391, 0.0552, 0.0781 and 0.110 mg/mL (-S9 mix)

- Dose selection (for Chromosome Aberration Test): As a result of the cell growth inhibition test, in both treatment groups, the top dose was considered as the dose level producing sufficient cytotoxicity, within doses of 0.156, 0.110, 0.0781… , resulting from 0.156 mg/mL with a common ratio of 2^1/2, and the following settings were used; top dose of 0.110 mg/mL at 5 levels in total with a common ratio of 2^1/2 (0.0276 - 0.110 mg/mL).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test material was insoluble in saline (at 50.0 mg/mL), and soluble in DMSO (at 500 mg/mL). Therefore, DMSO was selected as the solvent.
- The test material solution was prepared by sonication (for 4 minutes) and stirring at the time of use, and further diluted to obtain desired concentrations. Purity adjustment was not done at the preparation.

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:

EXPERIMENTAL PROCEDURE: Short-term treatment process
- Cell growth inhibition test (dose-finding test): The test was performed in the presence and absence of metabolic activation system. One culture plate was used per dose level. Cells were seeded at 2 x 10^4 cells per plate and pre-cultured for 3 days. A total of 0.03 mL of the test material solution was added and mixed in test tubes (corresponding to each dose level) containing a culture medium in advance. In the presence of metabolic activation system, 0.5 mL of S9 mix was also added and mixed in the tubes. The mixture was immediately replaced with the culture medium in the plate, and the cells were treated for 6 hours in a CO2 incubator. The solution volume per plate was 3 mL in total during treatment of the test material. After the end of the treatment period, the plates were checked for deposition of the test material. The plates were washed with PBS(-) (for test material removal), and then 5 mL of fresh culture medium was added and incubated for another 18 hours. After the end of incubation, cell growth conditions were examined under a microscope, and the cell growth rate was determined. The cell growth rate was calculated as a percentage of the negative control, which was set at 100%.
- Chromosome Aberration Test: Two culture plates were used per dose level. Pre-culture and treatment of the test material were conducted similarly to the cell growth inhibition test, and chromosome slides were prepared.

EXPERIMENTAL PROCEDURE: Continuous treatment process
- Cell growth inhibition test (dose-finding test): The test was performed in the 24 h and 48 h treatment. One culture plate was used per dose level. Cells were seeded at 2 x 10^4 cells per culture plate and pre-cultured for 3 days. A total of 0.05 mL of the test material solution was added and mixed in test tubes (corresponding to each dose level) containing a culture medium in advance. The mixture was immediately replaced with the culture medium in the plate, and the cells were treated for 24 or 48 hours in a CO2 incubator. The solution volume per plate was 5 mL in total during the treatment of the test material. After the end of the treatment period, the plates were checked for deposition of the test material and cell growth conditions, and then the cell growth rate was determined. The cell growth rate was calculated as a percentage of the negative control, which was set at 100 %.
- Chromosome Aberration Test: Two culture plates were used per dose level. Pre-culture and treatment of the test material were conducted similarly to the cell growth inhibition test, and chromosome slides were prepared.

CHROMOSOME PREPARATION
- In order to obtain metaphase cells, colcemid solution (GibcoBRL) was added to the culture plates at the final concentration of 0.2 g/mL, 2 hours prior to the end of incubation period. Next, cells were harvested with 0.25 % trypsin solution and centrifuged with each culture medium. Following removal of supernatants, hypotonic treatment was performed with 4 mL of 0.075 M potassium chloride solution in a thermostat water bath at 37 °C for 15 minutes. The cells were then semi-fixed with a few drops of the fixative solution (the mixture of alcohol and acetic acid). After centrifugation, this fixative solution was replaced with freshly prepared fixative solution. Replacement of fixative solutions was repeated several times in the same way and the cells were adequately fixed. Cell suspensions were prepared at an appropriate concentration, dropped on glass slides and air-dried. The chromosome slides were stained with 2 % Giemsa solution.

CELL COUNTING AND ANALYSIS
- Cells were harvested with 0.25 % trypsin solution, and some of the cells were stained by a trypan blue solution. Thereafter, viable cells were determined with a counting chamber.
- The chromosomal analysis was performed in a blind manner. Well-spread metaphase cells (in principle, 200 cells per dose) were examined under a microscope at magnification 600-1000x.
- Structural chromosome aberration: As the modal number of chromosomes in the CHL/IU cells was 25, cells with the chromosome number of 25 ± 2 were regarded as an object of the analysis. Structural aberrations were recorded according to the following categories, and a cell was counted as one aberrant cell if it had one or more aberrations of any type. The types of aberrations were also recorded. For example, if one cell had two break-type aberrations and three exchange-type aberrations, the number of aberrant cells would be recorded as one, and the number of aberrant types would be recorded as one for the break-type and one for the exchange-type. The structural aberration categories were: Chromatid break (ctb), Chromatid exchange (cte), Chromosome break (csb), Chromosome exchange (cse) and Fragmentation (Others).
- Numerical chromosome aberration: The number of cells was recorded when cells with chromosome doubling of haploid (triploid or hyperploid; called “poly”) or endoreduplication (called “end”) occurred.
- Gaps: Chromatid and chromosome gaps (called “g”) were recorded separately from the other aberrations and were not included in structural aberration. Achromatic regions smaller than the chromatid width in a coaxial line were considered as gaps, whereas achromatic regions larger than the chromatid width were considered as break-type aberration.

Evaluation criteria:

CRITERIA FOR JUDGEMENT
- The frequency (%) of structural aberrations and numerical aberrations was determined for dose levels according to the following criteria of Ishidate et al.:
< 5 % = Negative
≥ 5 and < 10 % = Equivocal
≥ 10 % = Positive
- The final judgment regarding the positive results was that the values of the treatment group showed both obvious increase compared with the negative control values and dose dependency. Additionally, where test series was judged “positive” for a single dose or “equivocal”, it was comprehensively judged “positive” if reproducibility of the result could be confirmed.

Statistics:

No statistical method was used for data analysis.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

SHORT-TERM TREATMENT PROCESS
- Cell growth inhibition test: In the presence and absence of a metabolic activation system, a cell growth inhibition test was performed with the dose range of 0.0781 to 5.00 mg/mL at 7 levels by a common ratio of 2. Deposition of the test material was observed at doses of ≥ 1.25 mg/mL at initiation of the treatment and doses of ≥ 2.50 mg/mL at completion of the treatment in the absence of metabolic activation system. In the presence of metabolic activation system, it was observed at doses of ≥ 1.25 mg/mL at initiation and completion of the treatment. More than 50% growth inhibition of the cell was observed at doses of ≥ 0.313 mg/mL in the absence of metabolic activation system, and at doses of ≥ 0.0781 mg/mL in the presence of metabolic activation system. Besides, the first round of chromosome aberration test in the presence and absence of metabolic activation system was discontinued because appropriate chromosome preparations for chromosomal analysis were not obtained in either group.
- Dose selection (for Chromosome Aberration Test): As a result of the cell growth inhibition test and first round of chromosome aberration test, the top dose was considered as the dose level producing sufficient cytotoxicity in the absence of metabolic activation system, and the following settings were used; top dose of 0.156 mg/mL at 4 levels in total with a common ratio of 2^1/2 (0.0552 - 0.156 mg/mL). In the presence of metabolic activation system, the top dose was similarly considered as the dose level producing sufficient cytotoxicity, with the following settings used; top dose of 0.0391 mg/mL at 4 levels in total with a common ratio of 2^1/2 (0.0138 - 0.0391 mg/mL).
- Chromosome Aberration Test: In the presence and absence of metabolic activation system, the second round of chromosome aberration test was performed at above dose setting. Deposition of the test material was not observed at any dose at initiation and completion of the treatment in either treatment group. More than 50% growth inhibition of the cell was observed at doses of ≥ 0.110 mg/mL in the absence of metabolic activation system and at dose of 0.0391 mg/mL in the presence of metabolic activation system. The frequency of chromosome aberrations was less than 5.0% for both structural aberrations and numerical aberrations at any dose in both groups.

CONTINUOUS TREATMENT PROCESS
- Cell growth inhibition test: In the 24 h and 48 h treatment, cell growth inhibition test was performed with the dose range of 0.0195 to 5.00 mg/mL at 9 levels by a common ratio of 2. Deposition of the test material was observed at doses of ≥ 1.25 mg/mL at initiation of the treatment and doses of ≥ 2.50 mg/mL at completion of the treatment in both treatment groups. More than 50% growth inhibition of the cell was observed at doses of ≥ 0.156 mg/mL in both groups.
- Dose selection (for Chromosome Aberration Test): As a result of the cell growth inhibition test, in both treatment groups, the top dose was considered as the dose level producing sufficient cytotoxicity, within doses of 0.156, 0.110, 0.0781… , resulting from 0.156 mg/mL with a common ratio of 2^1/2, and the following settings were used; top dose of 0.110 mg/mL at 5 levels in total with a common ratio of 2^1/2 (0.0276 - 0.110 mg/mL).
- Chromosome Aberration Test: In the 24 h and 48 h treatment, chromosome aberration test was performed at above dose setting. Deposition of the test material in the two treatment groups was not observed at any dose at initiation and completion of the treatment. More than 50% growth inhibition of the cell was observed at doses of ≥ 0.0781 mg/mL in the 24 h treatment, and at doses of ≥ 0.0552 mg/mL in the 48 h treatment. The frequency of chromosome aberrations was less than 5.0% for both structural aberrations and numerical aberrations at any dose in both groups.

Conclusions:

Under the conditions of this study, the test material was considered negative for clastogenic potential.

Executive summary:

The genetic toxicity of the test material was investigated in accordance with the Japanese guidelines: Notification Yakusyoku 0331 No.7 and H23.03.29 seikyoku No.5 and Kanpoki No.110331009 (March 31, 2011). The testing was performed under GLP conditions.

A chromosome aberration test using CHL/IU cells was performed with the test material. Testing was performed in the presence and absence of metabolic activation in the form of S9 mix. Following treatment with the test material viable cells were determined with a counting chamber. For the chromosomal analysis well-spread metaphase cells (in principle, 200 cells per dose) were examined under a microscope at magnification 600-1000x.

The frequency of chromosome aberrations was less than 5.0% for both structural aberrations and numerical aberrations at any dose by short-term treatment process in the presence and absence of metabolic activation system. For continuous treatment process in the 24 h and 48 h treatment, it was similarly less than 5.0% for both the aberrations at any dose. Accordingly, judging comprehensively, this test material was considered as “negative” for clastogenic potential.

The DMSO vehicle and appropriate positive controls (mitomycin C and benzo-a-pyrene) demonstrated that the test was properly conducted.

Under the conditions of this study, the test material was considered negative for clastogenic potential.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Genetic toxicity in vitro, Ames test. Noguchi (2005)

The genetic toxicity of the test material was investigated in accordance with Japanese guidelines under GLP conditions. The mutagenic potential was assessed with a bacterial reverse mutation assay. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and Escherichia coli WP2uvrA were treated with the test material using the pre-incubation method at six dose levels, in duplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range for the dose-determination test was 4.88 to 5000 µg/plate. The experiment was repeated on a separate day using the dose range, 9.77 to 313 µg/plate, fresh cultures of the bacterial strains and fresh test substance formulations.

Cytotoxicity was observed at and above 313 µg/plate with or without metabolic activation on dose-determination test, at and above 156 µg/plate without metabolic activation, at and above 313 µg/plate with metabolic activation on mutagenicity test 1 and mutagenicity test 2. No precipitate of the test material was observed at any dose level.

No significant increases in the frequency of revertant colonies were recorded for any strain with or without metabolic activation.

Under the conditions of this study, the test material was judged negative for the potential to induce gene mutation (mutagenicity).

Genetic toxicity in vitro, mammalian chromosome aberration test. Murata (2012)

The genetic toxicity of the test material was investigated In accordance with the Japanese guidelines: Notification Yakusyoku 0331 No.7 and H23.03.29 seikyoku No.5 and Kanpoki No.110331009 (March 31, 2011). The testing was performed under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

A chromosome aberration test using CHL/IU cells was performed with the test material. Testing was performed in the presence and absence of metabolic activation in the form of S9 mix. Following treatment with the test material viable cells were determined with a counting chamber. For the chromosomal analysis well-spread metaphase cells (in principle, 200 cells per dose) were examined under a microscope at magnification 600-1000x.

The frequency of chromosome aberrations was less than 5.0% for both structural aberrations and numerical aberrations at any dose by short-term treatment process in the presence and absence of metabolic activation system. For continuous treatment process in the 24 h and 48 h treatment, it was similarly less than 5.0% for both the aberrations at any dose. Accordingly, judging comprehensively, this test material was considered as “negative” for clastogenic potential.

The DMSO vehicle and appropriate positive controls (mitomycin C and benzo-a-pyrene) demonstrated that the test was properly conducted.

Under the conditions of this study, the test material was considered negative for clastogenic potential.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to genetic toxicity.