1,3-Benzenediamine, 4-methyl-, reaction products with 4-nitrobenzenamine, p-phenylenediamine and sodium sulfide (Na2(Sx))

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Three genetic toxicity studies were performed according to OECD guidelines 471 (with the test substance), 473 (with the read across substance CAS 90268-98-7) and 476 (with the read across substance CAS 90268-98-7) to determine the genetic toxicity potential of the test item. No genetic toxicity was observed in all three studies.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

Please refer to the attached read-across justification in section 13.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

29th July, 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

14 February 2017

Deviations:

no

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 examined

Positive controls validity:

valid

Remarks on result:

other: In conclusion, the test item did not induce structural chromosome aberrations in Chinese Hamster lung V79 cells, when tested up to cytotoxic concentrations in the absence and presence of metabolic activation.

Remarks:

test item is considered as being non-clastogenic in this system

Conclusions:

The read across-item did not induce structural chromosome aberrations in Chinese Hamster lung V79 cells, when tested up to cytotoxic concentrations in the absence and presence of metabolic activation.

Executive summary:

The read across-substance suspended in DMSO was tested in a chromosome aberration assay in V79 cells in two independent experiments. For the cytogenetic experiments the following concentrations were selected on the basis of a pre-test on cytotoxicity (without and with metabolic activation using rodent S9 mix) in accordance with the current OECD Guideline 473.

Experiment A with 3/20 h treatment/sampling time

without: 15.6,1 31.3, 62.5, 125 and 1802g/mL test item

with S9 mix: 15.6,131.3, 62.5, 125 and 180g/mL test item

Experiment B with 20/20 h treatment/sampling time

without S9 mix: 3.9,17.8, 15.6, 31.3and 452g/mL test item

Experiment B with 20/28 h treatment/sampling time

without S9 mix: 3.9,17.8, 15.6, 31.3and 452g/mL test item

Experiment B with 3/28 h treatment/sampling time

with S9 mix: 15.6,131.3, 62.5, 125 and 180g/mL test item

Following treatment and recovery the cells were exposed to the spindle inhibitor colchicine (0.2 µg/mL) 2.5 hours prior to harvesting. Harvested cells were treated with fixative for ca. 10 minutes before being placed on slides and stained. In each experimental group duplicate cultures were evaluated for cytogenetic damage (150 metaphases per culture). Clear cytotoxicity of about 50 % was observed after test item treatment in all experimental parts. No relevant increases in cells carrying structural chromosomal aberrations were observed, neither in the absence nor in the presence of metabolic activation. In experiment A in the absence and presence of metabolic activation and in experiment B in the presence of metabolic activation, some values were slightly above the 95% control limits of the historical control data. However, no statistical significant differences were observed after test item treatment when compared to the concurrent solvent as well as the historical control groups. In addition, no dose-response relationships were observed and therefore, the findings were not considered as being biologically relevant. There were no biologically relevant increases in the rate of polyploid or endoreduplicated metaphases in either experiment in the presence or absence of metabolic activation. The number of aberrations found in the solvent controls was in the range of the historical laboratory control data. The concurrent positive controls ethyl methanesulphonate (0.4 and 1.0 L/mL) and cyclophosphamide (5 g/mL) caused the expected biologically relevant increases of cells with structural chromosome aberrations as compared to solvent controls and were compatible with the historical positive control data. Thus, the test item is considered as being non-clastogenic in this system.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

Please refer to the attached read-across justification in section 13.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Remarks:

Sub-line (K1)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

evidence of toxicity was seen at the highest tested concentration with the test item in presence and absence of metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

The read across substance was not mutagenic in this in vitro mammalian cell gene mutation test performed with Chinese hamster ovary cells.

Executive summary:

The read across-substance suspended in DMSO was tested in a Mammalian Gene Mutation Test in CHO-K1 cells. The following concentrations were selected on the basis of a pre-test on cytotoxicity without and with metabolic activation using S9 mix of phenobarbital and β-naphthoflavone induced rat liver:

Mutation Assay 5-hour treatment period without S9-mix: 62.5, 125, 250, 350 and 450 µg/mL

Mutation Assay 5-hour treatment period with S9-mix: 125, 250, 500, 750 and 1000 µg/mL

In the performed mutation assay the concentration levels were chosen mainly based on the cytotoxicity. Phenotypic expression was evaluated up to 8 days following exposure. In the absence and presence of metabolic activation clear cytotoxicity (survival approximately 16%) of the test item was observed at the highest concentration applied (450 µg/mL in the absence and 1000 µg/mL in the presence of S9 mix). In both experimental parts, there were no statistically significant increases in mutation frequency when compared to the concurrent solvent control and the laboratory historical control data at any concentration tested in the absence and presence of metabolic activation. In the absence of S9 mix, in the cultures treated with 350 and 450 µg/mL the mutation frequency exceeded the 95% confidence interval of the historical control data (1 of 4 and 4 of 4 cultures, respectively). In the presence of S9 mix, in the cultures treated with 500 and 750 µg/mL the mutation frequency exceeded the 95% confidence interval of the historical control data (1 of 4 and 2 of 4 cultures, respectively). These findings were not considered to be biologically relevant since no dose-response relationships were noted, all values were within the normal range of mutation frequency and no statistical difference to the concurrent controls and the historical control range were observed. The mutation frequency found in the solvent controls was in the range of historical laboratory control data. The concurrent positive controls ethyl methanesulfonate (1.0 µL/mL) and 7, 12-dimethyl benzanthracene (20 µg/mL) caused the expected biologically relevant increases of cells with mutation frequency as compared to solvent controls and were compatible with the historical positive control data. It is concluded that the test item was not mutagenic in this in vitro mammalian cell gene mutation test performed with in Chinese hamster ovary cells.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 March - 12 June, 2017

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:

21st July, 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

May 30, 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

August, 1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ICH Guidance S2(R1): Guidance on Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use, June 2012

Version / remarks:

June 2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Date of production: 16 June 2015
Expiration date: 16 June 2020

Target gene:

In addition to histidine and tryptophan mutation, each strain has additional mutations which enhance its sensitivity to mutagens. The uvrB (uvrA) strains are defective in excision repair. It causes the strains to be more sensitive to the mutagenic and lethal effects of a wide variety of mutagens because they cannot repair DNA damages. The rfa mutation increases the permeability of the bacterial lipopolysaccharide wall for larger molecules. The plasmid pKM101 (TA98, TA100) carries the muc+ gene which participates in the error-prone "SOS" DNA repair pathway induced by DNA damage. This plasmid also carries an ampicillin resistance transfer factor (R-factor) which is used to identify its presence in the cell. The Escherichia coli strain used in this test (WP2uvrA) is also defective in DNA excision repair.

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:

post mitochondrial supernatant (S9)

Test concentrations with justification for top dose:

5000; 1600; 500; 160; 50 and 16 µg a.i./plate corresonding to 9090, 2909, 909, 291, 91 and 29 µg test item/plate

Vehicle / solvent:

dimethyl sulfoxide (DMSO)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

methylmethanesulfonate

other: 4-Nitro-1,2-phenylenediamine, 2-aminoanthracene, Dimethyl sulfoxide

Details on test system and experimental conditions:

Tester strains: Salmonella typhimurium TA98, TA100, TA1535, TA1537 and Escherichia coli WP2 uvrA were obtained from:
Supplier: Trinova Biochem GmbH; Rathenau Str. 2; D-35394 Giessen, Germany;
Manufacturer: MOLTOX INC., P.O. BOX 1189; BOONE, NC 28607 USA.
Frozen stock cultures were prepared from the disc cultures.
Storage of Tester Strains

The strains are stored at -80 ± 10ºC in the Laboratory of TOXI-COOP ZRT. in the form of lyophilized discs and in frozen permanent copies. Frozen permanent cultures of the tester strains are prepared from fresh, overnight cultures to which DMSO (8 % (v/v)) is added as a cryoprotective agent.

The phenotypes of the tester strains used in the bacterial reverse mutation assays with regard to membrane permeability (rfa), UV sensitivity (uvrA and uvrB), ampicillin resistance (amp), as well as spontaneous mutation frequencies are checked regularly according to Ames et al.
Established procedures (Standard Operating Procedures) for the preparations of each batch of frozen stock culture and raw data and reports of phenotype confirmation are stored in the Laboratory of TOXI-COOP ZRT.

Each tester strain reverts spontaneously at a frequency that is characteristic for the strain. Spontaneous reversions of the test strains to histidine or tryptophan prototrophs are measured routinely in mutagenicity experiments and expressed as the number of spontaneous revertants per plate. The historical control values for spontaneous revertants (revertants/plate: 2008-2016).

The frozen bacterial cultures were thawed at room temperature and 200 µL inoculum was used to inoculate each 50 mL of Nutrient Broth No. 2 for the overnight cultures in the assay. The cultures were incubated for approximately 10-14 hours in a 37oC Benchtop Incubator Shaker.

The viability of each testing culture was determined by plating 0.1 mL of the 10-5, 10-6, 10-7 and 10-8 dilutions of cultures on nutrient agar plates. The viable cell number of the cultures was determined by manual colony counting.

The Minimal Glucose Agar (MGA) Plates

Ready-to-use minimal glucose agar (MGA) plates were used in the study. The origin of the ready-to use MGA plates:
Supplier: VWR International;
Manufacturer: Merck Life Science GmbH, Germany.
Certificates of Analysis1) were obtained from the supplier.
Typical composition (g/1000 mL) of MGA plates:
Glucose 20.0 g
Magnesium sulfate 0.2 g
Citric acid 2.0 g
di-Potassium hydrogenphosphate 10.0 g
Sodium ammonium hydrogenphosphate 3.5 g
Agar agar 13.0 g
1) Batch No.: 141538; Expiry date: 21 March 2017; (used in the Informatory Toxicity Tests)
143118; Expiry date: 09 July 2017; (used in the Initial and Confirmatory Mutation Tests)

Nutrient Broth No. 2

Nutrient broth No. 2. 25.0 g
Ultrapure water ad 1000.0 mL
Sterilization for 20 minutes was performed at 121˚C in an autoclave.

Nutrient Agar

Nutrient Agar 20.0 g
Ultrapure water ad 1000.0 mL
Sterilization for 20 minutes was performed at 121˚C in an autoclave.

Top Agar for Salmonella typhimurium Strains

Agar solution:
Agar Bacteriological 4.0 g
NaCl 5.0 g
Ultrapure water ad 1000.0 mL
Sterilization for 20 minutes was performed at 121˚C in an autoclave.

Histidine – Biotin solution (0.5 mM):
D-Biotin 122.2 mg
L-Histidine•HCl H2O 104.8 mg
Ultrapure water ad 1000.0 mL
Sterilization was performed by filtration through a 0.22 µm membrane filter.

Complete Top Agar for Salmonella typhimurium strains:
Histidine – Biotin solution (0.5 mM) 100.0 mL
Agar solution 900.0 mL

Top Agar for Escherichia coli Strain

Tryptophan solution (2 mg/mL):
L-Tryptophan 2000.0 mg
Ultrapure water ad 1000.0 mL
Sterilization was performed by filtration through a 0.22 µm membrane filter.

Complete Top Agar for Escherichia coli strain:
Nutrient Broth by 5.4.2 50.0 mL
Tryptophan solution (2 mg/mL) 2.5 mL
Agar solution by 5.4.4 947.5 mL

Metabolic Activation System

The test bacteria were also exposed to the test item in the presence of an appropriate metabolic activation system, which is a cofactor-supplemented post-mitochondrial fraction (S9).

Rat Liver S9 Fraction

The S9 fraction of phenobarbital (PB) and β-naphthoflavone (BNF)-induced rat liver was provided by Trinova Biochem GmbH (Rathenau Str. 2; D-35394 Giessen, Germany; Manufacturer: MOLTOX INC., P.O. BOX 1189; BOONE, NC 28607 USA).

The Quality Control & Production Certificate of each lot of S9 was obtained from the supplier. The original Quality Control & Production Certificates of rat liver S9 are stored in the Laboratory of TOXI-COOP ZRT. The copies of the quality control certificates of the used S9 lots1) are given in Appendix VIII. The following lots of the S9 were applied:
1) Lot Number: 3593; Expiry date: February 25, 2018; Protein content: 36.4 mg/mL
(used in the Informatory Toxicity Tests);
Lot Number: 3634; Expiry date: May 12, 2018; Protein content: 40.3 mg/mL
(used in the Confirmatory Mutation Test);
Lot Number: 3650; Expiry date: June 16, 2018; Protein content: 36.5 mg/mL
(used in the first Informatory Toxicity Test);
Lot Number: 3662; Expiry date: July 07, 2018; Protein content: 40.5 mg/mL
(used in the Informatory Toxicity Tests and in the Initial Mutation Test);
Lot Number: 3727; Expiry date: December 01, 2018; Protein content: 33.7 mg/mL
(used in the Confirmatory Mutation Test).

The S9 Mix (with Rat Liver S9)

Salt solution for S9 mix Final concentration in S9 mix
NADP Na 7.66 g 4 mM
D-glucose-6 phosphate Na 3.53 g 5 mM
MgCl2 1.90 g 8 mM
KCl 6.15 g 33 mM
Ultrapure water ad 1000 mL
Sterilized by filtration through a 0.22 µm membrane filter.

The complete S9 mix was freshly prepared containing components as follows:
Ice cold 0.2 M sodium phosphate-buffer, pH 7.4 500 mL
Rat liver homogenate (S9) 100 mL
Salt solution for S9 mix 400 mL
The S9 mix (containing 10 % S9) was kept in an ice bath before it was added to the culture medium.

Sodium Phosphate Buffer (0.2 M, pH 7.4)

Solution A:
Na2HPO4 x 12H2O 71.63 g
Ultrapure water ad 1000 mL
Solution B:
NaH2PO4 x H2O 27.6 g
Ultrapure water ad 1000 mL

Solution A 880 mL
Solution B 120 mL*
* The components were mixed in the above ratio; thereafter the pH was checked and corrected. The correction was performed with admixture of the solution A or B.
After the pH setting the sterilization was performed by filtration through a 0.22 µm membrane filter.

Rationale for test conditions:

Based on the solubility tests, stock suspensions with a concentration of 50 mg/mL were prepared in ultrapure water and dimethyl sulfoxide (DMSO), respectively and diluted accordingly.
In the informatory toxicity tests any correction factor, based on the active component of the test item (55 %) was not taken into consideration; therefore the 50 mg/mL stock suspension concentration corresponded to 27.5 mg active component/mL.
The revertant colony numbers and the inhibition of the background lawn of auxotrophic cells of two of the tester strains (Salmonella typhimurium TA98, TA100) were determined in both tests.
In the informatory toxicity tests the revertant colony numbers of solvent control plates with and without S9 mix were in line with the corresponding historical control data ranges. The positive control treatments showed the expected biological relevant increases in induced revertant colonies in both tester strains.

The test item concentrations investigated in the initial and confirmatory mutation tests are as follows:
5000, 1600, 500, 160, 50 and 16 µg/plate.

Evaluation criteria:

The colony numbers on the untreated, solvent control, positive control and the test item treated plates were determined visually by manual counting, and the mean values, standard deviations and the mutation rates were calculated.

A test item is considered mutagenic if:
- a dose–related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic
activation.

An increase is considered biologically relevant if:
- in strain Salmonella typhimurium TA100 the number of reversions is at least twice as high as the reversion rate of the solvent control,
- in strain Salmonella typhimurium TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA the number of reversions is at least three times higher than the reversion rate of the solvent control.

According to the guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.
Criteria for a negative response:
A test item is considered non-mutagenic if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.

Statistics:

The mean values and appropriate standard deviations and mutation rates were calculated by EXCEL software.

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

Untreated negative 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

Untreated negative 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

Untreated negative 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

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Validity of the Performed Experiments

The tester strains used in this study demonstrated the specific phenotype characteristics, were in line with the corresponding historical control data ranges (and showed the adequate strain culture titer.
Each batch of the S9 fraction used in this test had the appropriate biological activity was active in the applied system
Each of the investigated reference mutagens showed the expected increase (at least a 3-fold increase) in induced revertant colonies over the mean value of the respective vehicle control in all main experimental phases and the number of revertants fell in the corresponding historical control ranges in most cases, thereby meeting the criteria for the positive control in the main experimental phases, in both tester strains.
The spontaneous revertant colony numbers of the dimethyl sulfoxide (DMSO) solvent control plates showed characteristic mean numbers agreed with the actual historical control data ranges in all strains in both main experimental phases.
Six concentration levels were investigated in the main mutation experiments (initial and confirmatory mutation tests).
In the performed main experimental phases there were at least five analyzable concentrations and a minimum of three non-toxic and non-precipitated dose levels at each tester strain.
All criteria for the validity of the performed experiments have therefore been met.

In the performed initial and confirmatory mutation test multiple test items were tested with reference values from the common parallel controls.
In the initial and confirmatory mutation tests the revertant colony numbers of the dimethyl sulfoxide (DMSO) solvent control plates with and without S9 mix were in line with the corresponding historical control data ranges.
The reference mutagen treatments (positive controls) showed the expected, biological relevant increases in induced revertant colonies, but in the confirmatory mutation test, in the case of S. typhimurium TA100 and TA1535 the revertant colony numbers of sodium azide (SAZ), in the case of E. coli WP2 uvrA the revertant colony numbers of methyl methanesulfonate (MMS) were above the corresponding historical control data ranges; however these were considered as acceptable without any effect on the final conclusion of the study.

The revertant colony numbers of the untreated and ultrapure water control plates in different experimental phases were slightly higher or lower than the DMSO control plates. The higher or lower revertant counts of these controls remained in the corresponding historical control data ranges.
In summary, the actual values of untreated, solvent and positive controls were in line with the criteria for validity of the assay.

Initial and Confirmatory Mutation Tests (Plate Incorporation Test and Pre-Incubation Test)

No substantial increases were observed in revertant colony numbers of any of the five test strains following treatment with Brown 3 at any concentration level, either in the presence or absence of metabolic activation (S9 mix) in the performed experiments.
In the performed experiments, sporadically increased revertant colony numbers were observed. These increases did not show a dose-response relationship, were of minor intensity, and all of the increases remained far below the biologically relevant thresholds for being positive. The obtained increases were therefore considered as biologically not relevant, being in the range of the biological variability of the applied test system.
The highest revertant colony number increase was observed in the initial mutation test (plate incorporation test) in S. typhimurium TA98 strain, at 1600 μg/plate, in the presence of metabolic activation (+S9). This value however remained in the range of the corresponding solvent historical control data and additional clear concentration related increase in revertant colony counts was not noticed. The mutation rate was 1.89, which was far below the genotoxicological threshold for being positive.
In the confirmatory mutation test an unequivocal inhibitory effect of the test item on bacterial growth was observed in the case of S. typhimurium TA1535 and TA1537 strains, in the absence of exogenous metabolic activation ( S9 mix). The cytotoxicity was indicated by absent or decreased revertant colony counts (some of them below the corresponding historical control data ranges) and affected background lawn development: reduced or slightly reduced background lawn. Cytotoxicity was noticed in S. typhimurium TA1535 at the concentration levels of 5000 and 1600 µg/plate ( S9 mix) and in S. typhimurium TA1537 in the concentration range of 5000-500 µg/plate ( S9 mix).
In general, 500 µg/plate was considered as lowest concentration showing cytotoxicity.

When evaluated by naked eye, non-interfering test item precipitate was noticed after about 48 hours incubation on the plates in the examined strains at the highest examined concentration of 5000 µg/plate in absence and in the presence of S9 following the plate incorporation and pre-incubation procedures.

Summary Table of the Results of the First Concentration Range Finding Test

Concentration Range Finding Test (Informatory Toxicity Test)
Concentrations (mg/plate)	Salmonella typhimuriumtester strains
	TA 98				TA 100
	-S9		+S9		-S9		+S9
Mean values of revertants per plate and Mutation rate (MR)	Mean	MR	Mean	MR	Mean	MR	Mean	MR
Untreated Control	21.0	1.17	26.3	1.39	100.0	1.15	121.7	1.09
DMSO Control	12.3	1.00	24.0	1.00	–	–	104.0	1.00
Ultrapure Water Control	18.0	1.00	19.0	1.00	87.0	1.00	111.7	1.00
2750	23.3	1.30	21.7	1.14	101.3	1.16	101.7	0.91
880	22.0	1.22	24.0	1.26	107.3	1.23	101.7	0.91
275	18.3	1.02	24.0	1.26	96.7	1.11	101.0	0.90
88	22.0	1.22	23.3	1.23	90.7	1.04	102.0	0.91
27.5	19.7	1.09	24.0	1.26	98.7	1.13	122.3	1.10
8.8	24.3	1.35	25.3	1.33	94.0	1.08	116.7	1.04
2.75	24.0	1.33	24.3	1.28	109.3	1.26	117.0	1.05
NPD (4mg)	210.7	17.08	–	–	–	–	–	–
SAZ (2mg)	–	–	–	–	1061.3	12.20	–	–
2AA (2mg)	–	–	2573.3	107.22	–	–	2322.7	22.33

MR:Mutation Rate

NPD:4-Nitro-1,2-phenylenediamine

SAZ:Sodium azide

2AA:2-aminoanthracene

Summary Table of the Results of the Second Concentration Range Finding Test

Concentration Range Finding Test (Informatory Toxicity Test)
Concentrations (mg/plate)	Salmonella typhimuriumtester strains
	TA 98				TA 100
	-S9		+S9		-S9		+S9
Mean values of revertants per plate and Mutation rate (MR)	Mean	MR	Mean	MR	Mean	MR	Mean	MR
Untreated Control	18.0	1.04	28.3	1.01	100.7	1.13	112.0	1.11
DMSO Control	17.3	1.00	28.0	1.00	89.3	1.00	101.0	1.00
Ultrapure Water Control	–	–	–	–	90.3	1.00	–	–
2750	23.7	1.37	23.7	0.85	99.3	1.11	93.7	0.93
275	17.7	1.02	25.3	0.90	115.0	1.29	118.7	1.17
27.5	25.0	1.44	22.3	0.80	106.7	1.19	121.3	1.20
2.75	22.7	1.31	29.3	1.05	120.7	1.35	128.7	1.27
NPD (4mg)	280.0	16.15	–	–	–	–	–	–
SAZ (2mg)	–	–	–	–	1130.7	12.52	–	–
2AA (2mg)	–	–	1586.7	56.67	–	–	2349.3	23.26

MR:Mutation Rate

NPD:4-Nitro-1,2-phenylenediamine

SAZ:Sodium azide

2AA:2-aminoanthracene

Summary Table of the Results of the Initial Mutation Test

Initial Mutation Test (Plate Incorporation Test)
Concentrations (mg/plate)	Salmonella typhimuriumtester strains																Escherichiacoli
	TA 98				TA 100				TA 1535				TA 1537				WP2uvrA
	-S9		+S9		-S9		+S9		-S9		+S9		-S9		+S9		-S9		+S9
Mean values of revertants per plate Mutation rate (MR)	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR
Untreated Control	24.0	1.57	26.7	1.25	96.3	1.11	101.3	1.07	12.7	1.09	11.0	0.89	7.0	1.00	8.0	0.96	22.7	1.03	32.3	0.87
DMSO Control	15.3	1.00	21.3	1.00	87.0	1.00	95.0	1.00	11.7	1.00	12.3	1.00	7.0	1.00	8.3	1.00	22.0	1.00	37.0	1.00
Ultrapure Water Control	–	–	–	–	85.3	1.00	–	–	9.7	1.00	–	–	–	–	–	–	25.0	1.00	–	–
5000	17.7	1.15	34.7	1.63	95.3	1.10	91.0	0.96	12.3	1.06	11.3	0.92	5.7	0.81	6.3	0.76	22.7	1.03	25.0	0.68
1600	18.0	1.17	40.3	1.89	93.3	1.07	110.7	1.16	9.3	0.80	13.3	1.08	8.7	1.24	6.0	0.72	19.7	0.89	31.3	0.85
500	21.7	1.41	31.0	1.45	100.7	1.16	91.0	0.96	12.0	1.03	12.7	1.03	7.3	1.05	8.3	1.00	31.0	1.41	32.7	0.88
160	23.0	1.50	31.3	1.47	86.0	0.99	86.3	0.91	11.0	0.94	12.3	1.00	10.3	1.48	8.0	0.96	27.3	1.24	36.0	0.97
50	19.0	1.24	26.7	1.25	94.0	1.08	90.3	0.95	10.0	0.86	12.3	1.00	9.7	1.38	8.7	1.04	32.7	1.48	32.7	0.88
16	22.0	1.43	33.7	1.58	84.3	0.97	92.0	0.97	13.7	1.17	14.3	1.16	7.0	1.00	6.0	0.72	28.7	1.30	45.3	1.23
NPD (4mg)	240.3	15.67	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–
SAZ (2mg)	–	–	–	–	1154.7	13.53	–	–	962.7	99.59	–	–	–	–	–	–	–	–	–	–
9AA (50mg)	–	–	–	–	–	–	–	–	–	–	–	–	318.0	45.43	–	–	–	–	–	–
MMS (2mL)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	644.0	25.76	–	–
2AA (2mg)	–	–	1325.3	62.13	–	–	1429.3	15.05	–	–	146.7	11.89	–	–	95.7	11.48	–	–	–	–
2AA (50mg)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	204.0	5.51

MR:Mutation Rate;          NPD:4-Nitro-1,2-phenylenediamine;SAZ: Sodium azide;9AA:9-Aminoacridine;MMS:Methyl methanesulfonate;2AA: 2-aminoanthracene

Summary Table of the Results of the Confirmatory Mutation Test

Confirmatory Mutation Test (Pre-Incubation Test)
Concentrations (mg/plate)	Salmonella typhimuriumtester strains																Escherichia coli
	TA 98				TA 100				TA 1535				TA 1537				WP2uvrA
	-S9		+S9		-S9		+S9		-S9		+S9		-S9		+S9		-S9		+S9
Mean values of revertants per plate Mutation rate (MR)	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR
Untreated Control	16.3	0.94	26.3	1.18	105.7	1.06	121.3	1.18	16.7	1.39	13.0	1.11	4.7	1.08	6.3	0.83	23.0	0.92	28.7	0.99
DMSO Control	17.3	1.00	22.3	1.00	99.7	1.00	103.0	1.00	12.0	1.00	11.7	1.00	4.3	1.00	7.7	1.00	25.0	1.00	29.0	1.00
Ultrapure Water Control	–	–	–	–	96.0	1.00	–	–	15.0	1.00	–	–	–	–	–	–	21.3	1.00	–	–
5000	14.3	0.83	20.7	0.93	80.3	0.81	112.0	1.09	2.3	0.19	8.0	0.69	0.0	0.00	7.3	0.96	20.0	0.80	24.7	0.85
1600	12.0	0.69	34.3	1.54	80.3	0.81	112.0	1.09	5.3	0.44	9.3	0.80	0.7	0.15	8.3	1.09	20.0	0.80	30.7	1.06
500	11.7	0.67	24.7	1.10	107.3	1.08	109.7	1.06	11.3	0.94	10.0	0.86	2.3	0.54	6.7	0.87	25.3	1.01	37.3	1.29
160	27.3	1.58	24.3	1.09	98.0	0.98	100.3	0.97	12.0	1.00	10.7	0.91	6.0	1.38	7.0	0.91	28.0	1.12	29.3	1.01
50	22.7	1.31	23.3	1.04	103.7	1.04	113.0	1.10	11.0	0.92	12.3	1.06	4.7	1.08	8.0	1.04	26.0	1.04	36.0	1.24
16	22.7	1.31	30.3	1.36	95.3	0.96	101.7	0.99	10.7	0.89	11.0	0.94	5.7	1.31	8.3	1.09	26.7	1.07	31.7	1.09
NPD (4mg)	220.7	12.73	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–
SAZ (2mg)	–	–	–	–	1978.7	20.61	–	–	2125.3	141.69	–	–	–	–	–	–	–	–	–	–
9AA (50mg)	–	–	–	–	–	–	–	–	–	–	–	–	253.0	58.38	–	–	–	–	–	–
MMS (2mL)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	1352.0	63.38	–	–
2AA (2mg)	–	–	1757.3	78.69	–	–	1125.3	10.93	–	–	210.7	18.06	–	–	136.3	17.78	–	–	–	–
2AA (50mg)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	205.3	7.08

MR:Mutation Rate;          NPD:4-Nitro-1,2-phenylenediamine;SAZ: Sodium azide;9AA:9-Aminoacridine;MMS:Methyl methanesulfonate;2AA: 2-aminoanthracene

Historical Control Values for Revertants/Plate (for the Period of 2008-2016)

			Bacterial strains
Historical control data of untreated control	‑S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	21.0	105.0	10.5	8.1	25.4
		SD	3.7	25.7	1.4	2.3	5.2
		Minimum	9	66	3	2	11
		Maximum	39	155	23	19	45
		n	226	236	216	214	215
	+S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	27.5	117.1	11.8	9.0	33.9
		SD	4.3	18.1	1.4	1.9	5.2
		Minimum	12	75	4	2	17
		Maximum	46	166	23	20	56
		n	226	236	216	214	215
			Bacterial strains
Historical control data of DMSO control	‑S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	20.4	100.1	10.3	7.9	24.7
		SD	3.6	24.8	1.3	2.4	4.6
		Minimum	10	64	3	2	11
		Maximum	38	147	23	20	45
		n	226	236	216	214	215
	+S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	26.5	113.8	11.8	8.8	33.7
		SD	4.1	18.3	1.5	1.9	5.0
		Minimum	15	71	3	3	16
		Maximum	47	162	25	20	57
		n	226	236	216	214	215
			Bacterial strains
Historical control data of Water control	‑S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	21.9	104.7	10.5	7.6	26.1
		SD	3.7	25.9	1.5	2.2	5.5
		Minimum	12	68	3	2	12
		Maximum	35	154	24	16	48
		n	89	236	216	89	215
	+S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	27.4	117.3	11.4	8.7	34.9
		SD	4.0	18.5	1.3	2.2	4.9
		Minimum	15	83	4	3	18
		Maximum	43	167	22	16	57
		n	89	152	149	89	148

Abbreviations:   TA98, TA100, TA1535, TA1537: Salmonella typhimuriumTA98, TA100, TA1535,

                               TA1537;E. coli:Escherichia coliWP2uvrA

                                               SD: Standard deviation;    DMSO: Dimethyl sulfoxide;n: number of studies

Historical Control Values for Revertants/Plate (for the Period of 2008-2016)(continued)

			Bacterial strains
Historical control data of positive controls	‑S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	260.1	977.2	847.3	478.6	724.5
		SD	31.8	150.6	126.3	104.5	65.0
		Minimum	123	521	359	110	320
		Maximum	664	1970	1855	1601	1313
		n	226	236	216	214	215
	+S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	1222.7	1436.4	164.1	147.0	257.7
		SD	274.9	318.3	33.1	20.1	72.5
		Minimum	386	583	85	69	140
		Maximum	2676	2988	498	399	477
		n	226	236	216	214	215

Abbreviations:   TA98, TA100, TA1535, TA1537: Salmonella typhimuriumTA98, TA100, TA1535,

                               TA1537;E. coli:Escherichia coliWP2uvrA

                               SD: Standard deviation; DMSO: Dimethyl sulfoxide;         n: number of studies

.

Conclusions:

The test item did not induce gene mutations in the bacterial strains tested.

Executive summary:

The test item was tested with regard to a potential mutagenic activity using the Bacterial Reverse Mutation Assay. The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimuriumTA98, TA100, TA1535 and TA1537), and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coliWP2uvrA) in the presence and absence of a post mitochondrial supernatant (S9) prepared from livers of Phenobarbital/b-naphthoflavone-induced rats. Based on the results of the preliminary concentration range finding tests (informatory toxicity tests) the following concentrations of the test item (based on the 55 % active ingredient (a.i.) content of the test item) were prepared and investigated in the initial and confirmatory mutation tests: 5000;1600; 500; 160; 50 and 16 µg a.i./plate. DMSO was used as solvent. In the confirmatory mutation test an inhibitory effect of the test item was observed in the S. typhimurium TA1535 and TA1537 strains in the absence of exogenous metabolic activation. The inhibitory effect was indicated by absent or decreased revertant colony counts (some of them below the corresponding historical control data ranges) and affected background lawn development: reduced or slightly reduced background lawn. In general, 500 µg/plate (noticed in S. Typhimurium TA1537) was considered as lowest concentration showing cytotoxicity. The reference mutagen treatments (positive controls) showed the expected, biological relevant increases (more than 3-fold increase)in induced revertant colonies and the number of revertants mostly fell in the corresponding historical control ranges, thereby meeting the criteria for the positive control in all experimental phases, in all tester strains. No biologically relevant increases were observed in revertant colony numbers of any of the five test strains following treatment with the test item at any concentration level, either in the presence or absence of metabolic activation (S9 mix) in the performed experiments. In conclusion, the test item has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Ames test:

The test item was tested with regard to a potential mutagenic activity using the Bacterial Reverse Mutation Assay. The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimuriumTA98, TA100, TA1535 and TA1537), and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coliWP2uvrA) in the presence and absence of a post mitochondrial supernatant (S9) prepared from livers of Phenobarbital/b-naphthoflavone-induced rats. Based on the results of the preliminary concentration range finding tests (informatory toxicity tests) the following concentrations of the test item (based on the 55 % active ingredient content (a.i., reflecting the dye content) of the test item) were prepared and investigated in the initial and confirmatory mutation tests: 5000;1600; 500; 160; 50 and 16 µg a.i./plate. DMSO was used as solvent. In the confirmatory mutation test an inhibitory effect of the test item was observed in the S. typhimurium TA1535 and TA1537 strains in the absence of exogenous metabolic activation. The inhibitory effect was indicated by absent or decreased revertant colony counts (some of them below the corresponding historical control data ranges) and affected background lawn development: reduced or slightly reduced background lawn. In general, 500 µg a.i./plate (noticed in S. Typhimurium TA1537) was considered as lowest concentration showing cytotoxicity. The reference mutagen treatments (positive controls) showed the expected, biological relevant increases (more than 3-fold increase)in induced revertant colonies and the number of revertants mostly fell in the corresponding historical control ranges, thereby meeting the criteria for the positive control in all experimental phases, in all tester strains. No biologically relevant increases were observed in revertant colony numbers of any of the five test strains following treatment with the test item at any concentration level, either in the presence or absence of metabolic activation (S9 mix) in the performed experiments. In conclusion, the test item has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.

 

Chromosome Aberration test (read across from CAS 90268-98-7):

The read across-substance suspended in DMSO was tested in a chromosome aberration assay in V79 cells in two independent experiments. For the cytogenetic experiments the following concentrations were selected on the basis of a pre-test on cytotoxicity (without and with metabolic activation using rodent S9 mix) in accordance with the current OECD Guideline 473: Experiment A with 3/20 h treatment/sampling time: with and without rodent S9 mix: 15.6,1 31.3, 62.5, 125 and 180 µg/mL test item; Experiment B with 20/20 h and 20/28 h treatment/sampling time: without S9 mix: 3.9,17.8, 15.6, 31.3 and 45 µg/mL test item; Experiment B with 3/28 h treatment/sampling time: with S9 mix: 15.6,131.3, 62.5, 125 and 180 µg/mL test item. Following treatment and recovery the cells were exposed to the spindle inhibitor colchicine (0.2 µg/mL) 2.5 hours prior to harvesting. Harvested cells were treated with fixative for ca. 10 minutes before being placed on slides and stained. In each experimental group duplicate cultures were evaluated for cytogenetic damage (150 metaphases per culture). Clear cytotoxicity of about 50 % was observed after test item treatment in all experimental parts. No relevant increases in cells carrying structural chromosomal aberrations were observed, neither in the absence nor in the presence of metabolic activation. In experiment A in the absence and presence of metabolic activation and in experiment B in the presence of metabolic activation, some values were slightly above the 95 % control limits of the historical control data. However, no statistical significant differences were observed after test item treatment when compared to the concurrent solvent as well as the historical control groups. In addition, no dose-response relationships were observed and therefore, the findings were not considered as being biologically relevant. There were no biologically relevant increases in the rate of polyploid or endoreduplicated metaphases in either experiment in the presence or absence of metabolic activation. The number of aberrations found in the solvent controls was in the range of the historical laboratory control data. The concurrent positive controls ethyl methanesulphonate (0.4 and 1.0 µL/mL) and cyclophosphamide (5 µg/mL) caused the expected biologically relevant increases of cells with structural chromosome aberrations as compared to solvent controls and were compatible with the historical positive control data. Thus, the read across substance is considered as being non-clastogenic in this system.

 

In vitro mammalian gene mutation test (read across from CAS 90268-98-7):

The read across-substance suspended in DMSO was tested in a Mammalian Gene Mutation Test in CHO-K1 cells. The following concentrations were selected on the basis of a pre-test on cytotoxicity without and with metabolic activation using S9 mix of phenobarbital and β-naphthoflavone induced rat liver: Mutation Assay 5-hour treatment period without S9-mix: 62.5, 125, 250, 350 and 450 µg/mL; Mutation Assay 5-hour treatment period with S9-mix: 125, 250, 500, 750 and 1000 µg/mL. In the performed mutation assay the concentration levels were chosen mainly based on the cytotoxicity. Phenotypic expression was evaluated up to 8 days following exposure. In the absence and presence of metabolic activation clear cytotoxicity (survival approximately 16 %) of the test item was observed at the highest concentration applied (450 µg/mL in the absence and 1000 µg/mL in the presence of S9 mix). In both experimental parts, there were no statistically significant increases in mutation frequency when compared to the concurrent solvent control and the laboratory historical control data at any concentration tested in the absence and presence of metabolic activation. In the absence of S9 mix, in the cultures treated with 350 and 450 µg/mL the mutation frequency exceeded the 95% confidence interval of the historical control data (1 of 4 and 4 of 4 cultures, respectively). In the presence of S9 mix, in the cultures treated with 500 and 750 µg/mL the mutation frequency exceeded the 95% confidence interval of the historical control data (1 of 4 and 2 of 4 cultures, respectively). These findings were not considered to be biologically relevant since no dose-response relationships were noted, all values were within the normal range of mutation frequency and no statistical difference to the concurrent controls and the historical control range were observed. The mutation frequency found in the solvent controls was in the range of historical laboratory control data. The concurrent positive controls ethyl methanesulfonate (1.0 µL/mL) and 7, 12-dimethyl benzanthracene (20 µg/mL) caused the expected biologically relevant increases of cells with mutation frequency as compared to solvent controls and were compatible with the historical positive control data. It is concluded that the test item was not mutagenic in this in vitro mammalian cell gene mutation test performed with in Chinese hamster ovary cells.

 

Conclusion: Three genetic toxicity studies were performed according to OECD guidelines 471, 473 (with the read across substance CAS 90268-98-7) and 476 (with the read across substance CAS 90268-98-7) to determine the genetic toxicity potential of the test item. No genetic toxicity was observed in all three studies.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. As a result the substance is not considered to be classified for genetic toxicity under Regulation (EC) No 1272/2008, as amended for the twelfth time in Regulation (EU) No 2019/521