1,5-Naphthalenedisulfonic acid, 2-[2-[8-[[4-chloro-6-[[4-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]amino]-1,3,5-triazin-2-yl]amino]-1-hydroxy-3,6-disulfo-2-naphthalenyl]diazenyl]-, sodium salt (1:5)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test compound is neither mutagenic in bacterial nor in mammalian cells.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

September 2020 - January 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Type of assay:

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

Target gene:

The mutation assay method used in this study is based on the identification of L5178Y colonies which have become resistant to a toxic thymidine analogue trifluorothymidine (TFT).This analogue can be metabolised by the enzyme thymidine kinase (TK) into nucleosides, which are used in nucleic acid synthesis resulting in the death of TK-competent cells.TK-deficient cells, which are presumed to arise through mutations in the TK gene, cannot metabolise trifluorothymidine and thus survive and grow in its presence. In the L5178Y mouse lymphoma cells, the gene which codes for the TK enzyme is located on chromosome 11. Cells which are heterozygous at the TK locus (TK+/−) may undergo a single step forward mutation to the TK−/− genotype in which little or no TK activity remains. The cells used, L5178Y TK+/−, are derived from one of the two clones originated from a thymic tumour induced in a DBA/2 mouse by methylcholanthrene. The use of the TK mutation system in L5178Y mouse lymphoma cells has been well characterised and validated (D. Clive et al., 1979) and is accepted by most of the regulatory authorities. The mouse lymphoma assay often produces a bimodal size distribution of TFT resistant colonies designated as small or large. It has been evaluated that point mutations and deletions within the active allele (intragenic event) produce large colonies. Small colonies result in part from lesions that affect not only the active TK allele but also a flanking gene whose expression modulates the growth rate of cells.

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI medium supplemented with Horse serum.
- Properly maintained: yes; Permanent stocks of the L5178Y TK+/− cells are stored in liquid nitrogen, and subcultures are prepared from the frozen stocks for experimental use.
- Periodically checked for Mycoplasma contamination: yes
- The generation time and mutation rates (spontaneous and induced) have been checked in this laboratory.
- Prior to use, cells were cleansed of pre-existing mutants.

Metabolic activation:

with and without

Metabolic activation system:

S9 tissue fraction: Species: Rat; Strain: Sprague Dawley; Tissue: Liver Inducing Agents: Phenobarbital – 5,6-Benzoflavone Producer: MOLTOX, Molecular Toxicology, Inc. Batch Number: 4263

Test concentrations with justification for top dose:

The test item was found to be soluble in RPMI 1640 minimal medium at 20.0 mg/mL. On the basis of this result, a concentration of 2000 µg/mL was selected as the top dose level for the preliminary cytotoxicity assay.

Based on the results obtained in the preliminary trial, the assy for mutation at the TK locus was performed using the following dose levels:
Main Assay 1 (+/-S9, 3 hour treatment): 2000, 1000, 500, 250 and 125 ug/mL.

Negative results were obtained in Main Assay II, thus a third experiment (Main Assay III) in the absence of S9 metabolism was performed, using a longer treatment time (24 hours) and the dose levels described in the following table:
Main Assay 2 (-S9, 24 hour treatment): 2000, 1600, 1280, 1020, 512, 256 and 128

Vehicle / solvent:

Test item solutions were prepared using complete medium (RPMI 1640 Minimal medium).

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

methylmethanesulfonate

Details on test system and experimental conditions:

Cytotoxicity assay
A preliminary cytotoxicity test was performed in order to select appropriate dose levels for the mutation assays. In this test a wide range of dose levels of the test item was used and the survival of the cells was subsequently determined. Treatments were performed in the absence and presence of S9 metabolic activation for 3 hours and for 24 hours only in the absence of S9 metabolic activation. A single culture was used at each test point. After washing in Phosphate Buffered Saline (PBS), cells were resuspended in 20mL of complete medium (10%). Cell concentrations were adjusted to 8 cells/mL using complete medium (20%) and, for each dose level, 0.2mL was plated into 96 microtitre wells. The plates were incubated at 37°C in a 5% CO2 atmosphere (100% nominal relative humidity) for 8 days. Wells containing viable clones were identified by eye using background illumination and then counted.

Mutation assay
The mutation assay was performed including vehicle and positive controls, in the absence and presence of S9 metabolising system.
Preparation of test cell cultures was performed as described in section 4.4. Duplicate cultures were prepared at each test point, with the exception of the positive controls which were prepared in a single culture. In the first experiment, the cells were exposed to the test item for a short treatment time (3 hours). Since negative results were obtained, a second experiment in the absence of S9 metabolism was performed, using a longer treatment time (24 hours).
After washing in Phosphate Buffered Saline (PBS), cells were resuspended in fresh complete medium (10%) and cell densities were determined. The number of cells was adjusted to give 2×105 cells/mL. The cultures were incubated at 37°C in a 5% CO2 atmosphere (100% nominal relative humidity) to allow for expression of the mutant phenotype.

Evaluation criteria:

For a test item to be considered mutagenic in this assay, it is required that:
1. The induced mutant frequency (IMF) is higher than the global evaluation factor (GEF) suggested for the microwell method (126×10^−6) at one or more doses.
2. There is a significant dose-relationship as indicated by the linear trend analysis.
Results which only partially satisfy the above criteria will be dealt with on a case-by-case basis. Similarly, positive responses seen only at high levels of cytotoxicity will require careful interpretation when assessing their biological significance. Any increase in mutant frequency should lie outside the historical control range to have biological relevance.

Statistics:

Statistical analysis was performed according to UKEMS guidelines (Robinson W.D., 1990).

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Survival after treatment
In the first experiment, using the 3 hour treatment time in the absence of S9 metabolism, moderate toxicity reducing RTG to 44% of the concurrent negative control, was observed at the highest concentration tested. In the presence of S9 metabolic activation the highest concentration (2000 μg/mL) yielded slight toxicity (RTG=69%), while no remarkable toxic effects were observed over the remaining dose levels.
A more pronounced toxic effect was noticed in the second experiment, using a long treatment time in the absence of S9 metabolic activation. Dose related toxic effects were observed from 512 μg/mL onwards reducing RTG to 9% and 1% at the two highest dose levels, 1600 and 2000 μg/mL, respectively. The next lower concentration (1280 μg/mL) yelded 20% RTG.
At low survival levels, the mutation data are prone to a variety of artefacts (selection effects, sampling error, founder effects). Mechanisms other than direct genotoxicity per se can lead to positive results that are related to cytotoxicity and not genotoxicity (e.g. events associated with apoptosis, endonuclease release fromlysosomes, etc.). For this reason it is generally recommended that such data are treated with caution or excluded from consideration.
Accordingly, we have excluded from the statistical analyses, mutation data obtained using the long treatment time at 1600 and 2000 µg/mL.

Mutation results
No statistically significant or biologically relevant increase in mutant frequency values was observed in any experiment, at any concentration tested, in the absence or presence of S9 metabolism, using the short or long treatment time. On the basis of these results and in accordance with the criteria for outcome of the study, the test item was not considered to induce mutation in L5178Y mouse lymphoma cells.

see Final Report

Conclusions:

It is concluded that the test substance does not induce mutation at the TK locus of L5178Y mouse lymphoma cells in vitro in the absence or presence of S9 metabolic activation, under the reported experimental conditions.

Executive summary:

The test item was examined for mutagenic activity by assaying for the induction of 5 trifluorothymidine resistant mutants in mouse lymphoma L5178Y cells after in vitro treatment, in the absence and presence of S9 metabolic activation, using a fluctuation method. The test item was found to be soluble in RPMI 1640 minimal medium at 20.0 mg/mL. On the basis of this result, a concentration of 2000 µg/mL was selected as the top dose level for the preliminary cytotoxicity assay. Both in the absence and presence of S9 metabolic activation, the test item was assayed at a maximum dose level of 2000 µg/mL and at a wide range of lower dose levels: 1000, 500, 250, 125, 62.5, 31.3, 15.6 and 7.81 µg/mL.

No precipitation of the test item was noted upon addition of the test item to the cultures or by the end of any treatment period. Using the 3 hour treatment time in the absence of S9 metabolic activation, slight or mild toxicity was observed at the three highest dose levels reducing relative survival (RS) up to 53% of the concurrent negative control value. In the presence of S9 metabolism mild toxicity was noticed only at the highest dose level. No remarkable toxicity was observed over the remaining concentrations tested. Using the 24 hour treatment time in the absence of S9 metabolic activation, dose ralated toxicity was observed reducing RS to 37% of the concurrent negative control value at 1000 µg/mL. At the next higher concentration (2000 µg/mL) a severe toxic effect was noticed (RS=1%).

Based on the results obtained in the preliminary trial, two independent assays for mutation at the TK locus were performed using the following dose levels:

 

Main Assay 1: (+/- S9, 3 hour treatment): 2000, 1000, 500, 250 and 125 µg/mL

Main Assay 2: (-S9, 24 hour treatment): 2000, 1600, 1280, 1020, 512, 256 and 128 µg/mL

 

Negative and positive control treatments were included in each mutation experiment in the absence and presence of S9 metabolism. The cloning efficiency at the time of mutant selection and the suspension growth for the solvent control cultures were within the acceptable range in all treatment series. The mutant frequencies in the solvent control cultures fell within the normal range. Marked increases were obtained with the positive control treatments indicating the correct functioning of the assay system. The study was then accepted as valid.

Using the 3 hour treatment in the absence of S9 metabolism, moderate toxicity reducing the Relative Total Growth (RTG) to 44% was observed at 2000 µg/mL, while, in the presence of S9 metabolic activation, this concentration yielded 69% RTG. Using the 24 treatment in the absence of S9 metabolism, dose related toxic effects were observed from 512 μg/mL onwards reducing RTG below 10% at the two highest dose levels. No relevant increases in mutant frequency were observed at any concentration tested, in any experiment.

It is concluded that the test substance does not induce mutation at the TK locus of L5178Y mouse lymphoma cells in vitro in the absence or presence of S9 metabolic activation, under the reported experimental conditions.

Reference 2

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:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

84/449/EWG, B.13, B.14 (Standard Platten-Test und Preincubations-Test)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

bacteria, other: TA1535, TA1537, TA1538, TA98, TA100, E.coli WP2uvrA

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix from Rats and Hamster liver

Test concentrations with justification for top dose:

Concentration range in the first test (with and without metabolic activation): 4, 20, 100, 500, 2500 and 5000 µg/plate
Concentration range in the second test (with and without metabolic activation): 4, 20, 100, 500, 2500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: none

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

Species / strain:

S. typhimurium TA 98

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:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

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:

valid

Positive controls validity:

valid

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:

valid

Positive controls validity:

valid

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:

valid

Positive controls validity:

valid

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:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1538

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:

valid

Positive controls validity:

valid

Conclusions:

The test compound did not cause a significant increase in the number of revertant colonies with any of the tester strains either in the absence or in the presence of S9 Mix. No dose dependent effect was obtained.

Executive summary:

Reactive Red 239 was tested for mutagenicity with the strains TA100, TA1535, TA1537, TA1538, TA98 of salmonella typhimurium and Escherichia coli WP2uvrA.

The mutagenicity studies were conducted in the absence and in the presence of a metabolizing system derived from rat liver homogenate. A dose range of 6 different dosees from 4 µg/plate to 5000 µg/plate was used.

Control plates without mutagen showed that the number of spontaneous revertant colonies was similar to that described in the literature. All the positiv control compounds gave the expected increase in the number of revertant colonies.

Toxicity: the test compound proved to be not toxic o f the bacterial strains. On the basis of the preliminary test results the top dose level did no exceed 5000 µg/plate.

Mutagenicity: In the absence of the metabolic activation system the test compound did not show a dose dependent increase in the number of revertants in any of the bacterial strains. Also in the presence of a metabolic activation system, treatment of the cells with Reactive Red 239 did no result in relevant increases in the number of revertant colonies.

Sumarizing, it can be stated that Reactive Red 239 is not mutagenic in these bacterial test systems niether with or without exogenous metabolic activation at the dose levels investigated.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1989

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix from rat and hamster liver

Test concentrations with justification for top dose:

Concentration range in the first test (with and without metabolic activation): 4, 20, 100, 500, 2500, 10000 µg/plate
Concentration range in the second test (with and without metabolic activation): 4, 20, 100, 500, 2500, 10000 µg/plate

Vehicle / solvent:

none

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

congo red

other:

Details on test system and experimental conditions:

According to the modification proposed by Prival using 30 minutes preincubation in the presence of 30% hamster S-9 (Syrien golden hamster)

Species / strain:

S. typhimurium TA 98

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:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

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:

valid

Positive controls validity:

valid

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:

valid

Positive controls validity:

valid

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:

valid

Positive controls validity:

valid

Conclusions:

The test compound did not cause a significant increase in the number of revertant colonies with any of the tester strains either in the absence or in the presence of S9 Mix. No dose dependent effect was obtained.

Executive summary:

Reactive Red 239 was tested for mutagenicity with the strains TA 100, TA 1535, TA 1537 and TA 98 of Salmonella typhimurium. The mutagenicity studies were conducted in the standard plate test (Ames Test) and in a modified preincubation test (Prival Test) in the absence and in the presence of a metabolizing system derived from rat or hamster liver homogenate, respectively. A dose range of 6 different doses from 4 to 5000 µg/plate was used.

Control plates without mutagen showed that the number of spontaneous revertant colonies was similar to that described in the literature. All the positive control compounds gave the expected increase in the number of revertant colonies.

The test compound proved to be not toxic to the bacterial strains. 5000 µg/plate was chosen as top dose level for the mutagenicity study.

In the standard plate test, Reactive Red 239 did not show a dose dependent increase in the number of revertants in any of the bacterial strains in the absence or presence of a metabolic activation system.

In the presence of hamster liver S-9 using the preincubation method according to Prival, Reactive Red 239 did not induce a significant increase in the number of revertant colonies, with any of the tester strains.

Summarizing, it can be stated that Reactive Red 239 is not mutagenic in the standard plate test (Ames Test) and in the preincubation method according to Prival.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

The test substance did not cause any adverse effects in the mouse micronucleus test.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

84/449/EWG, B.12

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

NMRI

Sex:

male/female

Route of administration:

oral: gavage

Vehicle:

deionised water

Duration of treatment / exposure:

animals were killed after 24, 48 or 72 hours after administration of the test compound

Frequency of treatment:

The test compound was given in two equal parts within two hours

Remarks:

Doses / Concentrations:
6250 mg/kg
Basis:
nominal in water

No. of animals per sex per dose:

Male: 6250 mg/kg; No. of animals: 5; Sacrifice time: 24 hours
Male: 6250 mg/kg; No. of animals: 5; Sacrifice time: 48 hours
Male: 6250 mg/kg; No. of animals: 5; Sacrifice time: 72 hours
Female: 6250 mg/kg; No. of animals: 5; Sacrifice times: 24 hours
Female: 6250 mg/kg; No. of animals: 5; Sacrifice times: 48 hours
Female: 6250 mg/kg; No. of animals: 5; Sacrifice times: 72 hours

Positive control(s):

endoxane, 50 mg/kg BW

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The incidence of micronucleated polychromatic erythrocytes in the dose groups of Reaktiv-Rot F-52167 FW was within the normal range of the negative control groups. No statistically significant increase of micronucleated polychromatic erythrocytes has been observed.
Due to the colour of the test compound urine, faeces and skin were red coloured.

Conclusions:

Interpretation of results (migrated information): negative
not mutagenic

Executive summary:

Reactive Red 239 was tested in the micronucleus test. The test compound was administered orally by gavage to male and female mice. Doses levels of 0 and 6250 mg per kg bodyweight were tested. The 6250 mg per kg bodyweight dose level was chosen since a preliminary study had shown it to be the maximal applicable dose. The test compound was given in two equal parts within two hours and according to the test procedure the animals were killed 24, 48, 72 hours after administration of the test compound.

Endoxan was used as positive control substance and was administered orally at a dose of 50 mg per kg bodyweight.

The incidence of micronucleated polychromatic erythrocytes of the animals treated with Reactive Red 239 was within the normal range of the negative control. The number of normochromatic erythrocytes containing micronuclei was not increased. The ratio of polychromatic/normochromatic erythrocytes in both male and female animals remained unaffected by the treatment with Reactive Red 239 and was statistically not different form the control values.

Endoxan induced a marked statistically significant increase in the number of polychromatic cells with micronuclei in both males and females, indicating the sensitivity of the system. The ratio of polychromatic erythrocytes to normocytes was not changed to a significant extend.

The results indicated that, under the conditions of the present study, Reactive Red 239 is not cytogenic in the micronucleus test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Reactive Red 239 was tested for mutagenicity with the strains TA100, TA1535, TA1537, TA1538, TA98 of salmonella typhimurium and Escherichia coli WP2uvrA.

The mutagenicity studies were conducted in the absence and in the presence of a metabolizing system derived from rat liver homogenate. A dose range of 6 different dosees from 4 µg/plate to 5000 µg/plate was used.

Control plates without mutagen showed that the number of spontaneous revertant colonies was similar to that described in the literature. All the positiv control compounds gave the expected increase in the number of revertant colonies. The test compound proved to be not toxic o f the bacterial strains. On the basis of the preliminary test results the top dose level did no exceed 5000 µg/plate.

Reactive Red 239 did no result in relevant increases in the number of revertant colonies in the absence or presence of a metabolic activation system.

Hence, Reactive Red 239 is not mutagenic in these bacterial test systems either with or without exogenous metabolic activation at the dose levels investigated.

Reactive Red 239 was tested for mutagenicity with the strains TA 100, TA 1535, TA 1537 and TA 98 of Salmonella typhimurium. The mutagenicity studies were conducted in the standard plate test (Ames Test) and in a modified preincubation test (Prival Test) in the absence and in the presence of a metabolizing system derived from rat or hamster liver homogenate, respectively. A dose range of 6 different doses from 4 to 5000 µg/plate was used.

Control plates without mutagen showed that the number of spontaneous revertant colonies was similar to that described in the literature. All the positive control compounds gave the expected increase in the number of revertant colonies.

The test compound proved to be not toxic to the bacterial strains. 5000 µg/plate was chosen as top dose level for the mutagenicity study.

In the standard plate test, Reactive Red 239 did not show a dose dependent increase in the number of revertants in any of the bacterial strains in the absence or presence of a metabolic activation system.

In the presence of hamster liver S-9 using the preincubation method according to Prival, Reactive Red 239 did not induce a significant increase in the number of revertant colonies, with any of the tester strains.

Summarizing, it can be stated that Reactive Red 239 is not mutagenic in the standard plate test (Ames Test) and in the preincubation method according to Prival.

The test item was examined for mutagenic activity by assaying for the induction of 5 trifluorothymidine resistant mutants in mouse lymphoma L5178Y cells after in vitro treatment, in the absence and presence of S9 metabolic activation, using a fluctuation method. The test item was found to be soluble in RPMI 1640 minimal medium at 20.0 mg/mL. On the basis of this result, a concentration of 2000 µg/mL was selected as the top dose level for the preliminary cytotoxicity assay. Both in the absence and presence of S9 metabolic activation, the test item was assayed at a maximum dose level of 2000 µg/mL and at a wide range of lower dose levels: 1000, 500, 250, 125, 62.5, 31.3, 15.6 and 7.81 µg/mL.

No precipitation of the test item was noted upon addition of the test item to the cultures or by the end of any treatment period. Using the 3 hour treatment time in the absence of S9 metabolic activation, slight or mild toxicity was observed at the three highest dose levels reducing relative survival (RS) up to 53% of the concurrent negative control value. In the presence of S9 metabolism mild toxicity was noticed only at the highest dose level. No remarkable toxicity was observed over the remaining concentrations tested. Using the 24 hour treatment time in the absence of S9 metabolic activation, dose ralated toxicity was observed reducing RS to 37% of the concurrent negative control value at 1000 µg/mL. At the next higher concentration (2000 µg/mL) a severe toxic effect was noticed (RS=1%).

Based on the results obtained in the preliminary trial, two independent assays for mutation at the TK locus were performed using the following dose levels:

Main Assay 1: (+/- S9, 3 hour treatment): 2000, 1000, 500, 250 and 125 µg/mL

Main Assay 2: (-S9, 24 hour treatment): 2000, 1600, 1280, 1020, 512, 256 and 128 µg/mL

Negative and positive control treatments were included in each mutation experiment in the absence and presence of S9 metabolism. The cloning efficiency at the time of mutant selection and the suspension growth for the solvent control cultures were within the acceptable range in all treatment series. The mutant frequencies in the solvent control cultures fell within the normal range. Marked increases were obtained with the positive control treatments indicating the correct functioning of the assay system. The study was then accepted as valid.

Using the 3 hour treatment in the absence of S9 metabolism, moderate toxicity reducing the Relative Total Growth (RTG) to 44% was observed at 2000 µg/mL, while, in the presence of S9 metabolic activation, this concentration yielded 69% RTG. Using the 24 treatment in the absence of S9 metabolism, dose related toxic effects were observed from 512 μg/mL onwards reducing RTG below 10% at the two highest dose levels. No relevant increases in mutant frequency were observed at any concentration tested, in any experiment.

It is concluded that the test substance does not induce mutation at the TK locus of L5178Y mouse lymphoma cells in vitro in the absence or presence of S9 metabolic activation, under the reported experimental conditions.

 

Reactive Red 239 was tested in the micronucleus test. The test compound was administered orally by gavage to male and female mice. Doses levels of 0 and 6250 mg per kg bodyweight were tested. The 6250 mg per kg bodyweight dose level was chosen since a preliminary study had shown it to be the maximal applicable dose. The test compound was given in two equal parts within two hours and according to the test procedure the animals were killed 24, 48, 72 hours after administration of the test compound.

Endoxan was used as positive control substance and was administered orally at a dose of 50 mg per kg bodyweight.

The incidence of micronucleated polychromatic erythrocytes of the animals treated with Reactive Red 239 was within the normal range of the negative control. The number of normochromatic erythrocytes containing micronuclei was not increased. The ratio of polychromatic/normochromatic erythrocytes in both male and female animals remained unaffected by the treatment with Reactive Red 239 and was statistically not different form the control values.

Endoxan induced a marked statistically significant increase in the number of polychromatic cells with micronuclei in both males and females, indicating the sensitivity of the system. The ratio of polychromatic erythrocytes to normocytes was not changed to a significant extend.

The results indicated that, under the conditions of the present study, Reactive Red 239 is not cytogenic in the micronucleus test.