Hydrogen 9-amino-7-phenyl-5-(phenylamino)-4,10-disulphonatobenzo[a]phenazinium, disodium salt

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

No mutagenic effects were detected in a bacterial reverse mutation assay (Ames) in five strains after incubation in presence and absence of metabolic activation. No increase of forward mutations was detected in mammalian cells (HPRT in CHO cells).

No chromosomal aberrations were detected in vitro in human peripheral lymphocytes.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: well performed GLP and OECD guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/ß-Naphthoflavone induced rat liver S9 Mix

Test concentrations with justification for top dose:

Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment II: 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Vehicle / solvent:

deionised water - The solvent was chosen because of its solubility properties.

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Without metabolic activation: sodium azide (TA 1535, TA 100), 4-nitro-o-phenylene-diamine (TA 1537, TA 98), methyl methane sulfonate (WP2 uvrA) / With metabolic activation: 2-aminoanthracene (TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA)

Details on test system and experimental conditions:

METHOD OF APPLICATION: plate incorporation test (experiment I) and the pre-incubation test (experiment II)

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.

A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.

An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.

A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

a statistical analysis of the data is not mandatory

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: Minor toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), were observed at 5000 µg/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

Non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay

Executive summary:

This study was performed to investigate the potential of the test item to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment II: 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments.

Minor toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), were observed at 5000 µg/plate without metabolic activation in strains TA 1537, TA 98 (experiment I), and TA 1535, TA 98, and TA 100 (experiment II), and with metabolic activation in strains TA 98 and TA 100 (experiment I), and in strains TA 1537 and TA 98 (experiment II).

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 May 2017 to 05 August 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

In accordance with the OECD guidelines for testing of chemicals, No. 473, “In Vitro Mammalian Chromosome Aberration Test” adopted on 29 July 2016.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: In vitro Mammalian Chromosomal Aberration Test

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Human peripheral blood lymphocytes
- Suitability of cells:Human peripheral lymphocytes from the blood of healthy, young, non-smoking male donors (24 and 31 years of age) with no known recent exposure to genotoxic chemicals or radiation were used from individual donors
- Sex, age and number of blood donors if applicable: Male, 24 and 31 years of age. number of donors 2.
- Whether whole blood or separated lymphocytes were used if applicable: whole blood
- Modal number of chromosomes: 2n=46

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: RPMI media supplemented with 10% FBS and antibiotics (1% penicillin-streptomycin) was used, with 37±1ºC with 5±1% CO2
- Properly maintained: yes

Cytokinesis block (if used):

Not Applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 homogenate was thawed immediately before use and mixed with the co-factor solution containing 4 mM NADP, 5 mM Glucose-6-phosphate, 8 mM MgCl2 and 33 mM KCl in Phosphate Buffer Saline (PBS)

Test concentrations with justification for top dose:

Based on the results of solubility, precipitation and pH tests, an initial cytotoxicity test was conducted for the selection of test concentrations for the chromosomal aberration test. The concentrations selected for initial cytotoxicity test were 0.01562, 0.03125, 0.0625 and 0.125 mg/mL.

Based on the results of initial cytotoxicity test, the concentrations selected for the chromosome aberration test are 0.0156, 0.03125, 0.0625 and 0.125 mg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: dimethyl sulfoxide (DMSO) was selected as vehicle and found soluble with DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

- OTHER:
DURATION
- Exposure duration: 3 to 6 hours (with and without S9), 20 to 24 hours (without S9)
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 2 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 2 hours

SPINDLE INHIBITOR (cytogenetic assays):: Colchicine

STAIN (for cytogenetic assays): 5% Giemsa

NUMBER OF REPLICATIONS: Two

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: few drops of the cells suspension was aspirated and dropped onto the chilled slides pre labeled . The slides were air dried. Three slides were prepared for each treatment. Slides were stained using 5% Giemsa stain for 20 minutes.

NUMBER OF CELLS EVALUATED: 500 cells were scored per each replicate for determination of mitotic index.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 300 metaphase

Rationale for test conditions:

The primary cell cultures of lymphocytes from the human whole blood are selected on the basis of growth ability in culture, stability of the karyotype. This provides the opportunity to test using the same test system which the in vitro test is predictive of in vivo genotoxic events.

Evaluation criteria:

Cytotoxicity was determined by calculating percentage reduction in mitotic index (%).
The cells were evaluated for structural aberrations in 150 metaphase plates for each replicate and the metaphases with aberrations were recorded in raw data and presented in study report.
Gaps were recorded separately and reported but generally not included in the total aberration frequency
a test chemical is considered to be clearly positive if, in any of the experimental conditions examined:
a) At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative/vehicle control.
b) The increase is dose-related when evaluated with an appropriate trend test.
When all of these criteria are met, the test chemical is then considered able to induce chromosomal aberrations in cultured mammalian cells in this test system.
• Providing that all acceptability criteria are fulfilled, a test chemical is considered clearly negative if, in all experimental conditions examined:
a) None of the test concentrations exhibits a statistically significant increase compared with the concurrent negative/vehicle control
b) There is no concentration-related increase when evaluated with an appropriate trend test

Statistics:

Data (Percentage of cells with aberrations) was analyzed using SPSS Software version 22 for differences among solvent/vehicle control, positive control and test item groups using ANOVA following Dunnett’s test at a 95% level of confidence (P<0.05)

Key result

Species / strain:

lymphocytes:

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no mutagenic potential (based on QSAR/QSPR prediction)

TABLE 1.           SUMMARY OF PERCENTAGE MITOTIC INDEX FOR INITIAL CYTOTOXICITY TEST

Set No.		Treatment	Concentration (mg/mL)		Mitotic Index				Mean Mitotic Index	Mean Percentage Mitotic Index		Percentage Reduction in Mitotic Index
					R1		R2
Set 1 (+S9)                  (3-6 hours)		Vehicle control	-		0.0456		0.0496		0.0476	4.76		-
		Test item [Acid Violet 50]	0.01562		0.0407		0.0439		0.0423	4.23		11.13
			0.03125		0.0404		0.0400		0.0402	4.02		15.55
			0.0625		0.0345		0.0324		0.0335	3.35		29.62
			0.125		0.0288		0.0323		0.0305	3.05		35.92
Set 2 (-S9)                     (3-6 hours)	Vehicle control			-		0.0441	0.0485	0.0463			4.63		-
	Test item [Acid Violet 50]			0.01562		0.0402	0.0399	0.0400			4.00		13.61
				0.03125		0.0431	0.0323	0.0377			3.77		18.57
				0.0625		0.0307	0.0323	0.0315			3.15		31.97
				0.125		0.0305	0.0289	0.0297			2.97		35.85
Set 3 (-S9)                   (20-24 hours)		Vehicle control	-		0.0409		0.0417		0.0413	4.13		-
		Test item [Acid Violet 50]	0.01562		0.0380		0.0340		0.0360	3.60		12.83
			0.03125		0.0320		0.0344		0.0332	3.32		19.61
			0.0625		0.0302		0.0267		0.0285	2.85		30.99
			0.125		0.0272		0.0248		0.0260	2.60		37.05

+S9: With metabolic activation; -S9: Without metabolic activation  

 

TABLE 2.     SUMMARY OFCHROMOSOMAL ABERRATIONSAND MITOTIC INDEX

                                                                                             

	Treatment	Concentration (mg/mL)	Mean % MI	Mean % Reduction in MI	Mean of Total Aberrations with Gaps	Mean of Total Aberrations without Gaps	Mean of Total Aberrant cells without Gaps	Mean of Percentage Aberrated Cells
Set 1 (+S9) (3-6 hours)	Vehicle control	-	5.04	NA	1	1	1.0	0.70
	Positive Control (Cyclophosphamide)	10 µg/mL	4.26	15.45	17.5	17.0	15.5	10.35*
	Test item  [Acid Violet 50]	0.01562	4.15	17.65	1	1.0	1.0	0.7
		0.03125	3.95	21.59	1.5	1.0	1.0	0.7
		0.0625	3.75	25.70	2.0	1.5	1.5	0.7
		0.125	3.54	29.77	2.5	2.0	2.0	1.30

MI: Mitotic Index; *: Statistically significant;+S9: With metabolic activation

 

 

 

 

TABLE 2 (Contd..,). SUMMARY OFCHROMOSOMAL ABERRATIONSAND MITOTIC INDEX

                                                                                        

Set No.	Treatment	Concentration (mg/mL)	Mean % MI	Mean % Reduction in MI	Mean of Total Aberrations with Gaps	Mean of Total Aberrations without Gaps	Mean of Total Aberrant cells without Gaps	Mean of Percentage Aberrated Cells
Set 2 (-S9) (3-6 hours)	Vehicle control	-	4.77	NA	2.0	1.0	1.0	0.7
	Positive Control (Mitomycin-C)	0.05 µg/mL	4.13	13.36	17.0	16.5	15.5	10.35*
	Test item  [Acid Violet 50]	0.01562	4.37	8.40	2.0	2.0	2.0	1.3
		0.03125	3.76	21.01	2.0	1.5	1.5	1.0
		0.0625	3.55	25.52	1.5	1.0	1.5	1.0
		0.125	3.47	27.11	2.5	2.0	2.0	1.3

MI: Mitotic Index; *: Statistically significant;-S9: Without metabolic activation

 

 

 

TABLE 2 (Contd..,). SUMMARY OFCHROMOSOMAL ABERRATIONSAND MITOTIC INDEX

Set No.	Treatment	Concentration (mg/mL)	Mean % MI	Mean % Reduction in MI	Mean of Total Aberrations with Gaps	Mean of Total Aberrations without Gaps	Mean of Total Aberrant cells without Gaps	Mean of Percentage Aberrated Cells
Set 3 (-S9) (20-24 hours)	Vehicle control	-	4.84	NA	1.5	1.5	1.5	1.0
	Positive Control (Mitomycin-C)	0.05 µg/mL	4.16	14.16	20.5	19.5	17.5	11.65*
	Test item  [Acid Violet 50]	0.01562	4.06	16.08	1.0	1.0	1.0	0.7
		0.03125	3.78	21.91	2.0	1.0	1.0	0.7
		0.0625	3.36	30.60	2.0	2.0	2.0	1.30
		0.125	3.28	32.30	3.0	2.5	2.5	1.65

MI: Mitotic Index; *: Statistically significant;-S9: Without metabolic activation

 

 

Conclusions:

Based on the results obtained, the test item, Acid Violet 50 is considered as non-clastogenic at and up to the concentration of 0.125 mg/mL, both in the presence and absence of metabolic activation under the test conditions.

Executive summary:

The test item was evaluated for chromosomal aberration in human lymphocytes, as per the OECD guideline for the testing of chemicals,No. 473 “In vitro Mammalian Chromosome AberrationTest” adopted on 29 July 2016. Test item was found soluble in dimethyl sulfoxide (DMSO) at 100 mg/mL. Precipitation test was conducted at concentrations of 0.0625, 0.125, 0.25, 0.5 and 1.0 mg/mL of test item. Precipitation was seen at all the concentrations except 0.0625 mg/mL. After 24 hours of incubation, no change in pH was observed at any of the concentrations tested at and up to 1 mg/mL. As there was a slight to moderate precipitation at 0.125 mg/mL, 0.125 mg/mL was selected as highest concentration for testing in the initial cytotoxicity test. The other concentrations selected were 0.01562, 0.03125 and 0.0625 mg/mL. In the initial cytotoxicity test, the test item resulted in not greater than 37.05% reduction of Mitotic Index (MI) at the concentrations of 0.01562, 0.03125, 0.0625 and 0.125 mg/mL both at short term (3 to 6 hours) and long term treatment (20 to 24 hours). Hence, the above indicated concentrations were selected for the chromosomal aberration test.In the chromosomal aberration test, the cells were treated withthe test itemat the concentrations of 0.125, 0.0625, 0.03125, and 0.01562 mg/mLconcentrations using DMSO as vehicle. The treatment was carried out in duplicates for short term (3 to 6 hours) both in the presence and absence of metabolic activation and long term (20 to 24 hours) in the absence of metabolic activation. Concentrations of 10 µg/mL of Cyclophosphamide Monohydrate (+S9) and 0.05µg/mLof Mitomycin-C (-S9 both for short term and long term) were used as positive controls. Cells were arrested at metaphase using 0.3 µg/mL of colchicine. The cells were harvested between 2 to 3 hours of cell cycle arrest and slides were prepared and stained using 5% Giemsa stain.

The results indicated no statistically significant increase in the number of aberrant cells when compared with vehicle control at any of the concentrations tested. The mean percentage reduction in mitotic index observed at 0.125 mg/mL, the highest concentration tested was 31.06 in the presence of metabolic activation and 27.11 and 32.30 in the absence of metabolic activation for short and long term treatments, respectively.

The respective positive controls tested induced 10.35% to 11.65% of mean aberrant cells which was statistically significant. The mean percentage reduction in mitotic index was in the range of 13.36 to 17.01 when compared with the respective vehicle controls.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 April 2017 to 31 August 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

In vitro Mammalian Cell Gene Mutation Tests using the Hprt and xprt genes” adopted on 29th July 2016

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: In vitro Mammalian Cell Gene Mutation Tests using the Hprt and xprt genes

Target gene:

Hprt and xprt genes

Species / strain / cell type:

other: CHO-AA8

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: ATCC
- Suitability of cells:The CHO AA8 cells are one of the recommended test systems by regulatory agencies for conducting in vitro mammalian gene mutation test
- Methods for maintenance in cell culture if applicable:Cells were maintained in Alpha Minimal Essential Medium (MEM) containing 10% FBS with antibiotics (1% Penicillin and Streptomycin) and incubated at 37±1°C and 5±1% CO2.


MEDIA USED
- Type and identity of media including CO2 concentration if applicable:Alpha Minimal Essential Medium (MEM) containing 10% FBS with antibiotics (1% Penicillin and Streptomycin) and incubated at 37±1°C and 5±1% CO2.
- Properly maintained: [yes]
- Periodically checked for Mycoplasma contamination: [yes]
- Periodically checked for karyotype stability: [yes]
- Periodically 'cleansed' against high spontaneous background: [yes]

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

Not applicable

Metabolic activation:

with and without

Metabolic activation system:

One mL of S9 homogenate was thawed immediately before use and mixed with 9 mL of co-factor solution

Test concentrations with justification for top dose:

Since the test item slight precipitated at 0.125 mg/mL, same was considered as the highest concentration in the initial cytotoxicity test.
Up to the concentration of 0.125 mg/mL the Relative Survival was more than 20%. At higher concentrations the value of RS is < 10% was obtained. Therefore 0.125 mg/mL was selected as the highest concentration for testing in the Gene mutation test.
Four concentrations i.e. 0.0156, 0.0312, 0.0625 and 0.125 mg/mL were selected for gene mutation test, based on initial cytotoxicity test.

Vehicle / solvent:

- Vehicle used: DMSO
- Justification for choice of vehicle:The test item was soluble in DMSO at 100 mg/mL

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

benzo(a)pyrene

Details on test system and experimental conditions:

- Cell density at seeding (if applicable):Approximately 20 Lakh cells per culture flask were seeded using culture medium with 10% FBS with antibiotics (1% Penicillin and Streptomycin).

DURATION
- Exposure duration: 3 to 6 hours
- Expression time (cells in growth medium):9 days of expression period
- Selection time (if incubation with a selection agent): 7 to 9 days

SELECTION AGENT (mutation assays):6 Thioguanine

NUMBER OF REPLICATIONS:Tetraplates

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth, relative survival

Rationale for test conditions:

Not applicable

Evaluation criteria:

There are several criteria for determining a positive result, such as a concentration-related increase or a reproducible increase in mutant frequency. Biological relevance of the results should be considered first. Statistical methods may be used as an aid in evaluating the test results. Statistical significance should not be the only determining factor for a positive response.
A test item, for which the results do not meet the above criteria, is considered non-mutagenic in this system

Statistics:

Data of mutant frequencies were analyzed for differences among vehicle control, treatment and positive control groups using SPSS Software version 22 at a 95% level (p<0.05) of significance.

Key result

Species / strain:

other: CHO-K1, CHO AA8 Cells

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:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No Change
- Water solubility: No
- Precipitation:Yes

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:No
- Negative vehicle) historical control data:No

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: RICC, Cloning efficiency, Relative survival

Remarks on result:

other: non-mutagenic

Not applicable

Conclusions:

Based on the results obtained, the test item, Acid Violet 50 is considered as non-mutagenic at and up to the concentration of 0.125 mg/mL both in the presence and absence of metabolic activation under the laboratory conditions tested.

Executive summary:

The test itemAcid Violet 50,was evaluated for gene mutation test in CHO AA8 cells, as per the OECD guideline for the testing of chemicals,No. 476 “In vitroMammalian Cell Gene Mutation Tests using the hprt and xprt genes” adopted on 29^thJuly 2016.

The test item was soluble in DMSO at 100 mg/mL. The test item did not precipitate at and up to 0.0625 mg/mL in culture medium. Slight precipitation was observed at the concentration of 0.125 mg/mL and precipitation was observed at up to the concentration at 1 mg/mL. The pH tested at concentrations up to 1 mg/mL was comparable to the vehicle control. Based on these results, 0.125 mg/mL was chosen as the highest concentration for the initial cytotoxicity test.

The results of the initial cytotoxicity test indicated that the test item was not cytotoxic to CHO AA8 cells as the Relative Survival of the treated CHO AA8 cells at the tested concentrations up to 0.125 mg/mL was more than 20%, when compared with the respective vehicle control, both in the presence and absence of metabolic activation.

The Gene mutation test was conducted at the concentrations of 0.0156, 0.0312, 0.0625 and 0.125 mg/mL using DMSO as a vehicle in four plates/group in the presence and absence of metabolic activation (3 to 6 hours).

Positive controlswere used for the gene mutation test.

Cytotoxicity was assessed by determining the Adjusted Cloning Efficiency and Relative Survival in the test. 

There was no statistically significant increase in the number of mutant colonies at any of the concentrations tested when compared with vehicle control.

Positive controls resulted in mutant frequencies,which were statistically significant when compared with the vehicle control.

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

No classification

No indication of genotoxic effects were detected in bacterial and mammalian cell. No clastogenic effects were observed in an in vitro chromosomal aberration study in human peripheral lymphocytes.