Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Bacterial Reverse Mutation Test (B.13/14, GLP), with and without metabolic activation, S.typhimurium TA98, TA100, TA 1535 and TA1537, E.coli WP2 uvrA:

-Mutagenic for tester strains S. typhimurium TA 98 and TA 100 with metabolic activation,

-Non-mutagenic for all the used tester strains without metabolic activation as well as for S. typhimurium TA 1537, TA 1535 TA and E.Coli with metabolic activation.

Read Across to Analogue Substance:

- Micronucleus Test in cultured CHO Cells in vitro (OECD 487, GLP), with and without metabolic activation, Chinese hamster ovary (CHO-K1) cells: no indications of genotoxic properties.

- Mouse Lymphoma Assay forward Mutation assay in vitro (B.17, GLP), with and without metabolic activation, L5178Y (TK+/-) mouse lymphoma cells:

no indications of genotoxic properties

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
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 see attached Read Across supporting document in Section 13 which includes the following:
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
3. ANALOGUE APPROACH JUSTIFICATION
4. DATA MATRIX
Reason / purpose:
read-across source
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Results for Read across source substance are reported. Read across target substance expected to give the same results.
Conclusions:
Based on read across to an analogue substance, Solvent Red 19T is predicted to give negative results both with and without metabolic activation in a gene mutation assay in cultured mammalian cells.
Executive summary:

A read across approach was used to predict the genotoxic properties of Solvent Red 19T ( 1-({2-methyl-4-[(2-methylphenyl)diazenyl]phenyl}diazenyl)-N-tridecylnaphthalen-2-amine).

The analogue source substance was Solvent Red 19E, which has the same structure, but where the tridecyl group has been replaced with a 2 -ethyl hexyl group. This material was

was assessed for genotoxicity in cultured mammalian cells (L5178Y TK +/ ) both in the presence and absence of metabolic activation according to OECD Test Guideline 476 and EC No.

440/2008 B.17: Mutagenicity in vitro mammalian cell gene test. The concentration range was 15.63 to 250 μg/mL. The mutation frequencies with the test material were within the same range as the negative control and hnce no mutagenicity was observed. Based on the similarity in structure and manufacturing process, Solvent Red 19T would be expected to have the same properties.

Endpoint:
in vitro cytogenicity / micronucleus study
Remarks:
Type of genotoxicity: chromosome aberration
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 see attached Read Across supporting document in Section 13 which includes the following:
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
3. ANALOGUE APPROACH JUSTIFICATION
4. DATA MATRIX
Reason / purpose:
read-across source
Key result
Species / strain:
Chinese hamster Ovary (CHO)
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
Remarks on result:
other: Result for Read Across Source substance, expected to be the same for Read Across Target substance
Conclusions:
Based on read across to an analogue substance, Solvent Red 19T is predicted to show no indications of genotoxic properties.
Executive summary:

A read across approach was used to predict the genotoxic properties of Solvent Red 19T ( 1-({2-methyl-4-[(2-methylphenyl)diazenyl]phenyl}diazenyl)-N-tridecylnaphthalen-2-amine).

In-vitromicronucleus test according to OECD 487 was carried out for an analogue subtance (Solvent Red 19E) with the same structure, but where the tridecyl group has been replaced with a 2 -ethyl hexyl group concluded that the substance did not have genotoxic properties. The analogue substance, did not result in a statistically significant increase of micronucleus frequencies.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26.07.2017 - 31.08.2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
OECD Guidelines for testing of Chemicals, Section 4, No. 471 “Bacterial Reverse Mutation Test”, adopted 21st July 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
Commission Regulation (EC) No. 440/2008, B.13/14. “Mutagenicity: Reverse Mutation Test Using Bacteria”, 30 May 2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
EPA Health Effects Test Guidelines, OPPTS 870.5100 “Bacterial Reverse Mutation Test” EPA 712-C-98-247, August 1998
EPA Health Effects Test Guidelines, OPPTS 870.5100 “Escherichia coli WP2 and WP2 uvrA Reverse Mutation Assays” EPA 712-C-96-247, June 1996 (Public Draft)
Deviations:
no
Principles of method if other than guideline:
M.J. PRIVAL and V.D. MITCHELL: Analysis of a method for testing azo dyes for mutagenicity in Salmonella typhimurium in the presence of flavine mononucleotide and hamster liver S9, Mutation Research 97: 103-116, 1982
BRUCE N. AMES, JOYCE MCCANN and EDITH YAMASAKI: Methods for Detecting Carcinogens and Mutagens with the Salmonella / Mammalian-Microsome Mutagenicity Test. Mutation Research, 31: 347-364, 1975
DOROTHY M. MARON and BRUCE N. AMES: Revised Method for the Salmonella Mutagenicity Test. Mutation Research, 113: 173-215, 1983
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
No further details specified in the study report.
Target gene:
histidine and tryptophan
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 Mix
Test concentrations with justification for top dose:
The examined test concentrations in the Initial Mutation Test in Salmonella typhimurium TA98, TA100, TA1535 and TA1537 strains with metabolic activation were 5000, 1581, 500, 158.1, 50, 15.81 and 5 μg/plate. The examined test concentrations in the Initial Mutation Test in Salmonella typhimurium TA98, TA100, TA1535 and TA1537 strains without metabolic activation were 500, 158.1, 50, 15.81, 5, 1.581 and 0.5 μg/plate.
The examined test concentrations in the Confirmatory Mutation Test in Salmonella typhimurium TA98, TA100, TA1535 and TA1537 strains with metabolic activation were 5000, 1581, 500, 158.1, 50, 15.81, 5 and 1.581 μg/plate. The examined test concentrations in the Confirmatory Mutation Test in Salmonella typhimurium TA98, TA100, TA1535 and TA1537 strains without metabolic activation were 500, 158.1, 50, 15.81, 5, 1.581, 0.5 and 0.1581 μg/plate.
The examined test concentrations in the Initial Mutation Test and in the Confirmatory Mutation Test in Escherichia coli WP2 uvrA strain with and without metabolic activation were 5000, 1581, 500, 158.1, 50, 15.81, 5 and 1.581 μg/plate.

Concentrations were selected on the basis of the Preliminary Solubility Test and Preliminary Range Finding Test (Informatory Toxicity Test). In the Initial Mutation Test and Confirmatory Mutation Test different concentrations were used.
Vehicle / solvent:
The solubility of the test item was examined using distilled water, dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF) and Acetone. At 100 mg/mL, insolubility was observed using distilled water and, partial dissolution was detected in DMSO.
At the same concentration, a purple red solution was observed in DMF and Acetone. Due to the better biocompatibility, DMF was selected as vehicle (solvent) for the study.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Distilled water; Dimethyl sulfoxide (DMSO); N,N-Dimethylformamide (DMF).
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
methylmethanesulfonate
other: 4-nitro-1,2-phenylenediamine (NPD); 2-aminoanthracene (2AA)
Details on test system and experimental conditions:
Genotypes
In addition to histidine or tryptophan mutation, each strain has additional mutations, which enhances its sensitivity to mutagens. The uvrB (uvrA) strains are defective in excision repair, making them more sensitive to the mutagenic and lethal effects of a wide variety of mutagens because they cannot repair DNA damage. The presence of 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 (WP2 uvrA) is also defective in DNA excision repair.

Storage
The strains are stored at -80 ± 10ºC in the Culture Collection of the Microbiological Laboratory of the CiToxLAB Hungary Ltd. Frozen permanent cultures of the tester strains were prepared from fresh, overnight cultures to which DMSO was added as a cryoprotective agent.

Confirmation of Phenotypes of Tester Strains
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. and Maron and Ames.
Established procedures (Standard Operating Procedures) for the preparations of each batch of frozen stock culture, raw data and reports of phenotype confirmation are stored in the Microbiological Laboratory of CiToxLAB Hungary Ltd.

Spontaneous Reversion of Tester Strains
Each test strain reverts spontaneously at a frequency that is characteristic of the strain.
Spontaneous reversion of the test strains to histidine (Salmonella typhimurium strains) or tryptophan (Escherichia coli WP2 uvrA strain) independence is measured routinely in mutagenicity experiments and expressed as the number of spontaneous revertants per plate.
Historical control values for spontaneous revertants (revertants/plate) for untreated control sample without metabolic activation were in the period of 2011-2016 were (as guide) as follows: Salmonella typhimurium TA98: 9-50, TA100: 54-210, TA1535: 1-46, TA1537: 1-24, Escherichia coli WP2 uvrA: 11-82.

Procedure for Growing Cultures
The frozen bacterial cultures were thawed at room temperature and 200 μL inoculum were used to inoculate each 50 mL of Nutrient Broth No. 2 (Section 5.4.2.) for the overnight cultures in the assay. The cultures were incubated for 10-14 hours at 37 °C in a Gyrotory water bath shaker.

Viability of the Testing Cultures
The viability of each testing culture was determined by plating 0.1 mL of the 10E+5, 10E+6, 10E+7 and 10E+8 dilutions prepared by sterile physiological saline on Nutrient Agar plates.
The viable cell number of the cultures was determined by manual counting after approximately 24-hour incubation at 37 °C.

METABOLIC ACTIVATION SYSTEM
Test bacteria were also exposed to the test item in the presence of an appropriate metabolic activation system, which was a cofactor-supplemented post-mitochondrial S9 fraction.
The post-mitochondrial fraction (S9 fraction) was prepared by the Microbiological Laboratory in the CiToxLAB Hungary Ltd according to Ames et al. and Maron and Ames. The documentation of the preparation of this post-mitochondrial fraction is stored in the reagent notebook in the Microbiological Laboratory which is archived yearly.
For azo-dyes and diazo-compounds the modified protocol proposed by Prival and Mitchell is referred to in the OECD guideline No. 471.
This modified protocol differs from the standard plate incorporation assay in five ways:
1. uninduced hamster liver S9 instead of induced rat liver S9 is used
2. the hamster liver S9 mix contains 30% hamster liver extract
3. flavine mononucleotide is added to the S9 mix
4. exogeneous glucose 6-phosphate dehydrogenase, NADH, and four times the standard amount of glucose 6-phosphate is added to the S9 mix
5. a 30 minutes pre-incubation step is used before addition of top agar.

These modifications are needed in order to test the mutagenic potential under conditions in which reduction of the compound to its constituent aromatic amines occurs. The hamster liver post-mitochondrial fraction (S9 fraction) was obtained from Trinova Biochem GmbH.

Hamster Liver S9 fraction
Hamster Liver Homogenate S9 Fraction
The S9 fraction of Syrian golden hamster liver was provided by Trinova Biochem GmbH (Rathenaustraße 2; D-35394 Gießen, Germany); Manufacturer: MOLTOX-Molecular Toxicology Inc. (Industrial Park Dr. 157; Boone, North Carolina, 28607 USA). Certificate of Analysis is obtained from the supplier, stored in the Microbiological Laboratory.

DESCRIPTION OF THE TEST PROCEDURE
The study included a Preliminary Solubility Test, a Preliminary Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test and a Confirmatory Mutation Test. In the Preliminary Concentration Range Finding Test as well as in the Initial Mutation Test, and in the Confirmatory Mutation Test, the pre-incubation method was used. At the request of the Sponsor all the tests were performed by using the Prival modification.

Concentration Range Finding Test (Informatory Toxicity Test)
Based on the solubility test, 100 mg/mL stock solution was prepared in DMF. Seven test concentrations were prepared by successive dilutions of the stock solution, spaced by factors of 2, 2.5 and approximately √10. The revertant colony numbers and the inhibition of the background lawn of auxotrophic cells of two of the tester strains (Salmonella typhimurium TA98 and TA100) were determined at concentrations of 5000, 2500, 1000, 316, 100, 31.6 and 10 μg/plate of the test item, in the absence and presence of metabolic activation. In the Preliminary Concentration Range Finding Test the plate incorporation method was used.

Test Item Concentrations in the Mutagenicity Tests (Initial Mutation Test and Confirmatory Mutation Test)
Based on the results of the preliminary tests, 100 mg/mL stock solution was prepared from the test item with DMF, which was diluted by serial dilutions to obtain lower doses. The maximum test concentration was 5000 μg test item/plate.
The examined test concentrations in the Initial Mutation Test in Salmonella typhimurium TA98, TA100, TA1535 and TA1537 strains with metabolic activation were 5000, 1581, 500, 158.1, 50, 15.81 and 5 μg/plate. The examined test concentrations in the Initial Mutation Test in Salmonella typhimurium TA98, TA100, TA1535 and TA1537 strains without metabolic activation were 500, 158.1, 50, 15.81, 5, 1.581 and 0.5 μg/plate.
The examined test concentrations in the Confirmatory Mutation Test in Salmonella typhimurium TA98, TA100, TA1535 and TA1537 strains with metabolic activation were 5000, 1581, 500, 158.1, 50, 15.81, 5 and 1.581 μg/plate. The examined test concentrations in the Initial Mutation Test in Salmonella typhimurium TA98, TA100, TA1535 and TA1537 strains without metabolic activation were 500, 158.1, 50, 15.81, 5, 1.581, 0.5 and 0.1581 μg/plate.
The examined test concentrations in the Initial Mutation Test and in the Confirmatory Mutation Test in Escherichia coli WP2 uvrA strain with and without metabolic activation were 5000, 1581, 500, 158.1, 50, 15.81, 5 and 1.581 μg/plate.

Control Groups Used in the Tests
Strain-specific positive and negative (solvent) controls, both with and without metabolic activation were included in each test. In addition, an untreated control was used demonstrating that the chosen vehicle induced no deleterious or mutagenic effects.

Test Method
A pre-incubation procedure was performed, as a preliminary range finding test, an initial and a confirmatory mutation test. Bacteria (cultured in Nutrient Broth No.2) were exposed to the test item both in the presence and absence of an appropriate metabolic activation system.
Before the overlaying, the test item formulation (or vehicle/solvent or reference control), the bacterial culture and the S9 mix or phosphate buffer was added into appropriate tubes to provide direct contact between bacteria and the test item (in its vehicle/solvent). The tubes (3 tubes per control and 3 tubes for each concentration level) were gently mixed and incubated for 30 min at 30 °C in a shaking incubator.
Molten top agar was prepared and kept at 45 °C. 2 mL of top agar was aliquoted into individual test tubes (3 tubes per control or concentration level). The equivalent number of minimal glucose agar plates was properly labelled. The test item and other components were prepared freshly and added to the overlay (45 °C).
The content of the tubes:
top agar 2000 μL
solvent or test item solution (or reference controls) 50 μL
overnight culture of test strain 100 μL
phosphate buffer (pH 7.4) or S9 mix 500 μL

This solution was mixed and poured on the surface of minimal agar plates. For activation studies, instead of phosphate buffer, 0.5 mL of the S9 mix was added to each overlay tube.
The entire test consisted of non-activated and activated test conditions, with the addition of untreated, negative and positive controls. After preparation, the plates were incubated at 37 °C for 48 ± 1 hours.
Rationale for test conditions:
In accordance with the test guidelines.
Evaluation criteria:
The colony numbers on the untreated / negative (solvent) / positive control and test item treated plates were determined by manual counting.

Criteria for Validity:
The study was considered valid if:
- the number of revertant colonies of the negative (vehicle/solvent) and positive controls are in the relevant historical control range, generated at the test facility, in all tester strains of the main tests (with or without S9-mix);
- at least five analysable concentrations are presented in all strains of the main tests;

Criteria for a Positive Response:
A test item was considered mutagenic if:
- a dose–related increase in the number of revertants occurred and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurred in at least one strain with or without metabolic activation.
An increase was considered biologically relevant if:
- the number of reversions at least two times higher than the reversion rate of the solvent control in Salmonella typhimurium TA98, TA100 and Escherichia coli WP2 uvrA bacterial strains
- the number of reversions at least three times higher than the reversion rate of the solvent control in Salmonella typhimurium TA1535 and TA1537 bacterial strains

Criteria for a Negative Response:
A test article was considered non-mutagenic if:
- the total number of revertants in tester strain Salmonella typhimurium TA98, TA100 or Escherichia coli WP2 uvrA is not greater than two times the concurrent vehicle control, and the total number of revertants in tester strain Salmonella typhimurium TA1535 or TA1537 is not greater than three times the concurrent vehicle control;
- the negative response should be reproducible in at least one follow up experiment.
Statistics:
According to the guidelines, statistical method may be used as an aid in evaluating the test results. However, statistical significance should not be the only determining factor for a positive response.
The mean number of revertants per plate, the standard deviation and the mutation factor values were calculated for each concentration level of the test item and for the controls using Microsoft ExcelTM software.
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
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 nor precipitates, but tested up to recommended limit concentrations
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:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
PRELIMINARY RANGE FINDING TEST (INFORMATORY TOXICITY TEST)
In the Preliminary Range Finding Test, the pre-incubation method (Prival modification) was used. The preliminary test was performed using Salmonella typhimurium TA98 and Salmonella typhimurium TA100 tester strains in the presence and absence of metabolic activation system (±S9 Mix) with appropriate untreated, negative (solvent) and positive controls. In the test each samples (including the controls) were tested in triplicate.
In the Range Finding Test the concentrations examined were: 5000, 2500, 1000, 316, 100, 31.6 and 10 μg/plate.
In the Preliminary Concentration Range Finding Test, a dose-related increase was observed in the number of revertant colonies in Salmonella typhimurium TA98 strain with metabolic activation. In Salmonella typhimurium TA100 strain with metabolic activation an increase was observed in the number of revertant colonies at concentration levels of 1000 and 316 μg/plate.
The calculated mutation factor values were over the biologically relevant threshold values 2.
Slight precipitate was observed in the Preliminary Range Finding Test in Salmonella typhimurium TA98 strain with metabolic activation on the plates at 5000 μg/plate concentration.
Absent/reduced/slightly reduced background lawn was detected in the Preliminary Range Finding Test in all examined bacterial strains without metabolic activation on the plates at 5000, 2500, 1000 and 316 μg/plate concentrations and with metabolic activation on the plates at 5000 μg/plate concentration.
Based on the results of the Range Finding Test and the solubility findings, the maximum final concentration to be tested in the main experiments was 5000 μg/plate.

INITIAL AND CONFIRMATORY MUTATION TESTS
In the Initial Mutation Test and in the Confirmatory Mutation Test, the pre-incubation method (Prival modification) was used. The Initial Mutation Test and Confirmatory Mutation Test were carried out using Salmonella typhimurium strains (TA98, TA100, TA1535 and TA1537) and Escherichia coli WP2 uvrA strain. Each test was performed in the presence and absence of metabolic activation system (±S9 mix) with appropriate untreated, negative (solvent) and positive controls. In the main test each sample (including the controls) were tested in triplicate.
Based on the results of the preliminary experiment, the examined test concentrations in the Initial Mutation Test in Salmonella typhimurium TA98, TA100, TA1535 and TA1537 strains with metabolic activation were 5000, 1581, 500, 158.1, 50, 15.81 and 5 μg/plate. The examined test concentrations in the Initial Mutation Test in Salmonella typhimurium TA98, TA100, TA1535 and TA1537 strains without metabolic activation were 500, 158.1, 50, 15.81, 5, 1.581 and 0.5 μg/plate.
The examined test concentrations in the Confirmatory Mutation Test in Salmonella typhimurium TA98, TA100, TA1535 and TA1537 strains with metabolic activation were 5000, 1581, 500, 158.1, 50, 15.81, 5 and 1.581 μg/plate. The examined test concentrations in the Confirmatory Mutation Test in Salmonella typhimurium TA98, TA100, TA1535 and TA1537 strains without metabolic activation were 500, 158.1, 50, 15.81, 5, 1.581, 0.5 and 0.1581 μg/plate.
The examined test concentrations in the Initial Mutation Test and in the Confirmatory
Mutation Test in Escherichia coli WP2 uvrA strain with and without metabolic activation were 5000, 1581, 500, 158.1, 50, 15.81, 5 and 1.581 μg/plate.
Slight precipitate was observed in all examined bacterial strains with metabolic activation in the Initial Mutation Test on the plates at 5000 μg/plate concentration, in the Confirmatory Mutation Test with metabolic activation on the plates at 5000 μg/plate concentration, without metabolic activation on the plates at 500 μg/plate concentration and for Escherichia coli WP2 uvrA strain on the plates at 5000 and 1581 μg/plate concentrations.
Reduced/slightly reduced background lawn was detected in the Initial Mutation Test in all examined bacterial strains with metabolic activation on the plates at 5000 μg/plate concentration, in Salmonella typhimurium strains without metabolic activation on the plates at 500 μg/plate concentration, for Salmonella typhimurium TA98 strain on the plates at 158.1 μg/plate concentration and in Escherichia coli WP2 uvrA strain without metabolic activation on the plates at 5000 and 1581 μg/plate concentrations.
Absent/reduced/slightly reduced background lawn was detected in the Confirmatory Mutation Test in all examined bacterial strains with metabolic activation on the plates at 5000 μg/plate concentration, in Salmonella typhimurium strains without metabolic activation on the plates at 500 μg/plate concentration, for Salmonella typhimurium TA98, TA1537 strains on the plates at 158.1 μg/plate concentration and in Escherichia coli WP2 uvrA strain on the plates at 5000 and 1581 μg/plate concentrations.
Taking into account the results at all concentrations for each bacterial strain, the results for Salmonella typhimurium TA98, TA100 strains without metabolic activation, Salmonella typhimurium TA1535, TA1537 and Escherichia coli WP2 uvrA strains with and without metabolic activation were negative. The results for Salmonella typhimurium TA98 and TA100 strains with metabolic activation using the pre-incubation method (Prival modification) were dose-related and reproducibly positive.

VALIDITY OF THE TESTS
Untreated, negative (vehicle/solvent) and positive controls were run concurrently. The mean values of revertant colony numbers of untreated, negative (solvent) and positive control plates were within the historical control range in all strains. At least five analysable concentrations were presented in all strains with and without metabolic activation.
The reference mutagens showed a distinct increase of induced revertant colonies in each strain with and without metabolic activation. The viability of the bacterial cells was checked by a plating experiment in each test. The study was considered to be valid.

Summary Table of the range Finding Test

Concentration (μg/plate)

Mean values of revertants / Mutation factor (MF)

Salmonella typhimuriumtester strains

TA 98

TA 100

-S9

+S9

-S9

+S9

Untreated control

Mean

24.7

34.3

110.0

86.0

MF

1.03

1.11

1.10

0.87

Distilled water control

Mean

-

-

107.0

-

MF

-

-

1.07

-

DMSO control

Mean

26.0

35.7

-

89.7

MF

1.08

1.15

-

0.91

DMF control

Mean

24.0

31.0

99.7

98.7

MF

1.00

1.00

1.00

1.00

5000

Mean

0.0

11.0

0.0

38.3

MF

0.00

0.35

0.00

0.39

2500

Mean

0.0

299.3

0.0

171.7

MF

0.00

9.66

0.00

1.74

1000

Mean

0.0

253.0

0.0

273.0

MF

0.00

8.16

0.00

2.77

316

Mean

6.0

210.7

60.7

217.3

MF

0.25

6.80

0.61

2.20

100

Mean

21.7

135.3

96.7

190.0

MF

0.90

4.37

0.97

1.93

31.6

Mean

23.7

70.0

97.0

121.7

MF

0.99

2.26

0.97

1.23

10

Mean

24.3

29.0

99.0

85.7

MF

1.01

0.94

0.99

0.87

NPD (4μg)

Mean

416.0

-

-

-

MF

16.00

-

-

-

2AA (2μg)

Mean

-

2410.0

-

2372.0

MF

-

67.57

-

26.45

SAZ (2μg)

Mean

-

-

1246.0

-

MF

-

-

11.64

-

 

Summary Table of the Initial Mutation Test (Pre-Incubation Method)

Concentration (μg/plate)

Mean values of revertants / Mutation factor (MF)

Salmonella typhimuriumtester strains

Escherichia coli

TA 98

TA 100

TA 1535

TA 1537

WP2uvrA

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Untreated control

Mean

21.7

39.3

100.0

81.3

13.7

11.7

12.0

17.0

43.7

42.3

MF

1.02

1.23

1.09

1.00

1.05

0.92

0.77

1.02

0.96

0.98

Distilled water control

Mean

-

-

93.0

-

14.0

-

-

-

45.3

-

MF

-

-

1.01

-

1.08

-

-

-

1.00

-

DMSO control

Mean

23.3

21.7

-

85.0

-

12.0

14.7

17.3

-

45.0

MF

1.09

0.68

-

1.05

-

0.95

0.94

1.04

-

1.04

DMF control

Mean

21.3

31.7

91.7

81.3

13.0

12.7

15.7

16.7

45.3

453.3

MF

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

5000

Mean

--

5.0

--

15.0

--

30.0

--

7.0

0.0

30.7

MF

--

0.16

--

0.18

--

2.37

--

0.42

0.00

0.71

1581

Mean

--

264.0

--

142.3

--

18.7

--

22.7

16.3

54.3

MF

--

8.34

--

1.75

--

1.47

--

1.36

0.36

1.25

500

Mean

8.3

184.7

31.0

160.3

12.3

15.3

1.0

22.7

35.0

45.7

MF

0.39

5.83

0.34

1.97

0.95

1.21

0.06

1.36

0.77

1.05

158.1

Mean

13.3

139.0

77.0

170.0

10.3

12.7

10.3

19.7

42.3

44.7

MF

0.63

4.39

0.84

2.09

0.79

1.00

0.66

1.18

0.93

1.03

50

Mean

17.0

95.3

85.3

115.3

10.3

12.2

13.7

17.7

44.7

45.7

MF

0.80

3.01

0.93

1.42

0.79

0.97

0.87

1.06

0.99

1.05

15.81

Mean

19.3

46.7

80.7

78.3

12.0

11.0

15.7

17.3

47.0

41.3

MF

0.91

1.47

0.88

0.96

0.92

0.87

1.00

1.04

1.04

0.95

5

Mean

23.3

42.0

92.3

85.0

13.0

15.0

12.7

16.7

39.0

38.7

MF

1.09

1.33

1.01

1.05

1.00

1.18

0.81

1.00

0.86

0.89

1.581

Mean

21.3

--

87.3

--

14.0

--

16.7

--

35.0

37.3

MF

1.00

--

0.95

--

1.08

--

1.06

--

0.77

0.86

0.5

Mean

18.7

--

98.7

--

12.0

--

11.7

--

--

--

MF

0.88

--

1.08

--

0.92

--

0.84

--

--

--

NPD (4μg)

Mean

416.0

-

-

-

-

-

-

-

-

-

MF

17.83

-

-

-

-

-

-

-

-

-

2AA (2μg)

Mean

-

2405.3

-

2400.0

-

208.0

-

218.7

-

-

MF

-

111.02

-

28.24

-

17.33

-

12.62

-

-

2AA (50μg)

Mean

-

-

-

-

-

-

-

-

-

280.3

MF

-

-

-

-

-

-

-

-

-

6.23

SAZ (2μg)

Mean

-

-

1194.7

-

1288.0

-

-

-

-

-

MF

-

-

12.85

-

92.00

-

-

-

-

-

9AA (50μg)

Mean

-

-

-

-

-

-

406.7

-

-

-

MF

-

-

-

-

-

-

27.73

-

-

-

MMS (2μL)

Mean

-

-

-

-

-

-

-

-

1072.0

-

MF

-

-

-

-

-

-

-

-

23.65

-

 

Summary Table of the Confirmatory Mutation Test (Pre-Incubation Method)

Concentration (μg/plate)

Mean values of revertants / Mutation factor (MF)

Salmonella typhimuriumtester strains

Escherichia coli

TA 98

TA 100

TA 1535

TA 1537

WP2uvrA

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Untreated control

Mean

23.7

28.36

73.7

78.3

12.0

13.3

12.2

14.7

44.0

43.0

MF

1.04

0.93

0.95

1.06

1.00

1.21

1.16

1.10

1.02

0.84

Distilled water control

Mean

-

-

73.7

-

13.0

-

-

-

42.7

-

MF

-

-

0.95

-

1.08

-

-

-

0.99

-

DMSO control

Mean

23.7

30.0

-

77.7

-

12.7

12.0

11.3

-

45.7

MF

1.04

0.99

-

1.05

-

1.15

1.13

0.85

-

0.90

DMF control

Mean

22.7

30.3

77.3

73.7

12.0

11.0

10.7

13.3

45.0

51.0

MF

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

5000

Mean

--

0.0

--

0.0

--

5.0

--

2.0

0.0

31.7

MF

--

0.00

--

0.00

--

0.45

--

0.15

0.00

0.62

1581

Mean

--

171.3

--

242.7

--

16.7

--

20.0

2.7

52.3

MF

--

5.65

--

3.29

--

1.52

--

1.50

0.06

1.03

500

Mean

10.0

160.0

37.3

207.3

6.3

15.0

1.0

13.3

29.7

45.7

MF

0.44

5.27

0.48

2.81

0.53

1.36

0.09

1.00

0.69

0.90

158.1

Mean

13.3

110.0

85.3

191.0

7.0

12.7

5.7

12.7

53.3

55.7

MF

0.59

3.63

1.10

2.59

0.58

1.15

0.53

0.95

1.24

1.09

50

Mean

19.3

70.3

93.7

146.0

10.0

11.0

11.7

11.0

53.7

49.0

MF

0.85

2.32

1.21

1.98

0.83

1.00

1.09

0.83

1.25

0.96

15.81

Mean

19.3

47.3

94.3

103.0

9.3

11.7

10.0

10.3

51.7

56.7

MF

0.85

1.56

1.22

1.40

0.79

1.06

0.94

0.78

1.20

1.11

5

Mean

21.3

35.3

97.7

93.3

11.0

14.0

11.0

13.7

53.0

51.3

MF

0.94

1.16

1.26

1.27

0.92

1.27

10.3

10.3

1.23

1.01

1.581

Mean

17.0

26.3

90.7

87.7

10.3

9.3

9.7

13.3

50.3

48.3

MF

0.75

0.87

1.17

1.19

0.86

0.85

0.91

1.00

1.17

0.95

0.5

Mean

18.0

--

100.3

--

11.7

--

11.0

--

--

--

MF

0.79

--

1.30

--

0.97

--

1.03

--

--

--

0.1581

Mean

20.0

--

95.7

--

12.7

--

12.0

--

--

--

MF

0.88

--

1.24

--

1.06

--

1.13

--

--

--

NPD (4μg)

Mean

408.0

-

-

-

-

-

-

-

-

-

MF

17.24

-

-

-

-

-

-

-

-

-

2AA (2μg)

Mean

-

2384.0

-

2398.7

-

205.3

-

200.0

-

-

MF

-

79.47

-

30.88

-

16.21

-

17.65

-

-

2AA (50μg)

Mean

-

-

-

-

-

-

-

-

-

262.7

MF

-

-

-

-

-

-

-

-

-

5.75

SAZ (2μg)

Mean

-

-

1205.3

-

1172.0

-

-

-

-

-

MF

-

-

16.36

-

90.15

-

-

-

-

-

9AA (50μg)

Mean

-

-

-

-

-

-

389.7

-

-

-

MF

-

-

-

-

-

-

32.47

-

-

-

MMS (2μL)

Mean

-

-

-

-

-

-

-

-

979.3

-

MF

-

-

-

-

-

-

-

-

22.95

-

 

Conclusions:
The test item Solvent Red 19T was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay.
The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537), and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a metabolic activation system, which was a cofactor-supplemented post-mitochondrial S9 fraction prepared from the livers of uninduced hamsters.
The study included a Preliminary Solubility Test, a Preliminary Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test and a Confirmatory Mutation Test. At the request of the Sponsor all the tests were performed by using a modified pre-incubation method (Prival modification).
The reported data of this mutagenicity assay show that under the experimental conditions applied the test item induced gene mutations by base pair changes and frameshifts in the genome of the strains used.
In conclusion, the test item Solvent Red 19T (Batch Number: TE 2139) was shown to be mutagenic in this study. The test item had mutagenic activity in Salmonella typhimurium TA98 and TA100 bacterial strains with metabolic activation. No mutagenic activity was observed in Salmonella typhimurium TA98, TA100 strains without metabolic activation, Salmonella typhimurium TA1535, TA1537 and Escherichia coli WP2 uvrA bacterial strains with and without metabolic activation under the test conditions used in this study.
Executive summary:

The test item Solvent Red 19T was tested for potential mutagenic activity using the Bacterial

Reverse Mutation Assay.

 

The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537) and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9 fraction) prepared from the livers of uninduced hamsters.

 

The study included a Preliminary Solubility Test, a Preliminary Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Pre-Incubation Method, Prival modification) and a Confirmatory Mutation Test (Pre-Incubation Method, Prival modification).

 

Based on the results of the Solubility Test, the test item was dissolved in N,N-dimethylformamide (DMF). The examined test concentrations in the Initial Mutation Test in Salmonella typhimurium TA98, TA100, TA1535 and TA1537 strains with metabolic activation were 5000, 1581, 500, 158.1, 50, 15.81 and 5 μg/plate. The examined test concentrations in the Initial Mutation Test in Salmonella typhimurium TA98, TA100, TA1535 and TA1537 strains without metabolic activation were 500, 158.1, 50, 15.81, 5, 1.581 and 0.5 μg/plate.

The examined test concentrations in the Confirmatory Mutation Test in Salmonella typhimurium TA98, TA100, TA1535 and TA1537 strains with metabolic activation were 5000, 1581, 500, 158.1, 50, 15.81, 5 and 1.581 μg/plate. The examined test concentrations in the Confirmatory Mutation Test in Salmonella typhimurium TA98, TA100, TA1535 and TA1537 strains without metabolic activation were 500, 158.1, 50, 15.81, 5, 1.581, 0.5 and 0.1581 μg/plate.

The examined test concentrations in the Initial Mutation Test and in the Confirmatory Mutation Test in Escherichia coli WP2 uvrA strain with and without metabolic activation were 5000, 1581, 500, 158.1, 50, 15.81, 5 and 1.581 μg/plate.

 

In the Preliminary Concentration Range Finding Test, in the Initial Mutation Test and Confirmatory Mutation Test in the case of Salmonella typhimurium TA98 and TA100 strains with metabolic activation, a dose-related, reproducible positive effect was obtained using the pre-incubation method (Prival modification).

 

Slight precipitate was observed in the Preliminary Range Finding Test in Salmonella typhimurium TA98 strain with metabolic activation on the plates at 5000 μg/plate concentration. This effect was detected in all examined bacterial strains with metabolic activation in the Initial Mutation Test on the plates at 5000 μg/plate concentration, in the Confirmatory Mutation Test with metabolic activation on the plates at 5000 μg/plate concentration, without metabolic activation on the plates at 500 μg/plate concentration and for Escherichia coli WP2 uvrA strain on the plates at 5000 and 1581 μg/plate concentrations.

 

Absent/reduced/slightly reduced background lawn was detected in the Preliminary Range Finding Test in all examined bacterial strains without metabolic activation on the plates at 5000, 2500, 1000 and 316 μg/plate concentrations and with metabolic activation on the plates at 5000 μg/plate concentration.

This effect was observed in the Initial Mutation Test in all examined bacterial strains with metabolic activation on the plates at 5000 μg/plate concentration, in Salmonella typhimurium strains without metabolic activation on the plates at 500 μg/plate concentration, for Salmonella typhimurium TA98 strain on the plates at 158.1 μg/plate concentration and in Escherichia coli WP2 uvrA strain on the plates at 5000 and 1581 μg/plate concentrations.

The same effect was detected in the Confirmatory Mutation Test in all examined bacterial strains with metabolic activation on the plates at 5000 μg/plate concentration, in Salmonella typhimurium strains without metabolic activation on the plates at 500 μg/plate concentration, for Salmonella typhimurium TA98, TA1537 strains on the plates at 158.1 μg/plate concentration and in Escherichia coli WP2 uvrA strain on the plates at 5000 and 1581 μg/plate concentrations.

 

The mean values of revertant colonies of the solvent control plates were within the historical control data range, the reference mutagens showed the expected increase in the number of revertant colonies, the viability of the bacterial cells was checked by a plating experiment in each test. The tests were considered to be valid.

 

The reported data of this mutagenicity assay show that under the experimental conditions applied the test item induced gene mutations by base pair changes and frameshifts in the genome of the strains used.

 

In conclusion, the test item Solvent Red 19T (Batch Number: TE 2139) was shown to be mutagenic in this study. The test item had mutagenic activity in Salmonella typhimurium TA98 and TA100 bacterial strains with metabolic activation. No mutagenic activity was observed in Salmonella typhimurium TA98, TA100 strains without metabolic activation, Salmonella typhimurium TA1535, TA1537 and Escherichia coli WP2 uvrA bacterial strains with and without metabolic activation 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

The test material was examined in the Ames test (Bacterial Reverse Mutation Assay ) which gave a postive result. The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537), and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a metabolic activation system, which was a cofactor-supplemented post-mitochondrial S9 fraction prepared from the livers of uninduced hamsters. The study included a Preliminary Solubility Test, a Preliminary Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test and a Confirmatory Mutation Test. At the request of the Sponsor all the tests were performed by using a modified pre-incubation method (Prival modification). The test item had mutagenic activity in Salmonella typhimurium TA98 and TA100 bacterial strains with metabolic activation. No mutagenic activity was observed in Salmonella typhimurium TA98, TA100 strains without metabolic activation, Salmonella typhimurium TA1535, TA1537 and Escherichia coli WP2 uvrA bacterial strains with and without metabolic activation under the test conditions used in this study.

A read across approach was then used to 2 further genotoxic endpoints (chromosomal aberration assay, mammalian cell mutation assay). An analogue substance (Solvent Red 19E) showed no genotoxic potential in either of these tests. Based on the results of these studies and according to the mutagenicity testing strategy (Guidance on information requirements and chemical safety assessment, Chapter R.7a: Endpoint specific guidance) there is no need to perform further testing. The substance is considered as not genotoxic.

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.