Tetrasodium 5-[4-chloro-6-(N-ethyl-anilino)-1,3,5-triazin-2-ylamino]-4-hydroxy-3-(1,5-disulfonatonaphthalen-2-ylazo)-naphthalene-2,7-disulfonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Reactive Red 245 was found to be not mutagenic in the Ames assay and also to be not clastogenic in the in vitro mammalian chromosomal abberation test. Additionally, using the principles of read across, the target substance Reactive Red 245 is also considered to be not mutagenic in mammalian cells as well basis of results on the source substance Reactive Red 238 which was non-mutagenic in the HPRT mutation assay. 

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

September 20, 1990 to November 13, 1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

other: Japan 1984, Toxicity Test Guideline, "Reversion Test with Bacteria"

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 6947/14
- Expiration date of the lot/batch: July, 1995

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: > 48 h in H20, DMSO, and DMF

Target gene:

histidine

Species / strain / cell type:

S. typhimurium, other: TA 1535, TA 1537, TA 1538, TA 98, TA 100

Details on mammalian cell type (if applicable):

The bacterial strains were obtained from Dr. Heinz Träger, Knoll AG, D-6700 Ludwigshafen, F.R.G.
Regular checking of the properties of the strains with regard to membrane permeability and ampicillin resistance as well as normal spontaneous mutation rates is performed in C C R according to Ames et al. (1). In this way it was ensured that the experimental conditions set down by Ames were fulfilled.

Metabolic activation:

with and without

Metabolic activation system:

aroclor 1254 induced rat liver S9 mix

Test concentrations with justification for top dose:

Experiment 1: 10, 100, 333.3, 1000, and 5000 µg/plate.
Experiment 2: 100, 333.3, 1000, 2500 and 5000 µg/plate.

Vehicle / solvent:

water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

all strains (TA 1535, TA 1537, TA 1538, TA 98, TA 100), with metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Strains: TA 1535, TA 100, without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene-diamine

Remarks:

Strains: TA 1537, TA 1538, TA 98, without metabolic activation

Details on test system and experimental conditions:

Method of application: in agar

Duration: After solidification the plates were incubated upside down for 72 h at 37 °C in the dark.

Determination of cytotoxicity: Toxic effects were evidenced by a reduction in the number of spontaneous revertants, a clearing of the bacterial background lawn, or by degree of survival of treated cultures.

Number of replications: Two independent experiments performed in triplicate.

Evaluation criteria:

A test article is considered as positive if either a significant dose-related increase in the number of revertants or a significant and reproducible increase for at least one test concentration is induced. A test article producing neither a significant dose-related increase in the number of revertants nor a significant and reproducible positive response at any one of the test points is considered non-mutagenic in this system. A significant response is described as follows: A test article is considered as mutagen if in strain TA 100 the number of reversions is at least twice as high and in strains TA 1535, TA 1537, TA 1538, and TA 98 it is at least three times higher as compared to the spontaneous reversion rate. Also, a dose-dependent increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test article regardless whether the highest dose induced the above described enhancement factors or not.

Statistics:

No appropriate statistical method is available.

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

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

with S9 mix both at the highest investigated dose.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

without S9 mix at the highest investigated dose.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

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

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
- To evaluate the toxicity of the test article, a pre-study was performed with strains TA 98 and TA 100 exposed to 1, 3.3, 10, 33.3, 100, 333.3, 1000 and 5000 µg/plate. The plates with the test article showed normal background growth up to 5000 µg/plate in strain TA 98 and TA 100.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Toxic effects, evidenced by a reduction in the number of revertants, occurred in strain TA 1537 without S9 mix and TA 1538 with S9 mix both at the highest investigated dose in experiment I. The plates incubated with the test article showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used.

Conclusions:

FAT 40406/A did not show any mutagenic activity in any Salmonella typhimurium strains.

Executive summary:

In a GLP-compliant Ames test, performed according to OECD guideline 471, 5 Salmonella typhimurium strains (TA 1535, TA 1537, TA 1538, TA 98, and TA 100) were used to the test the mutagenic potential of the test substance (10, 100, 333.3, 1000, 2500, 5000 µg per plate), both with and without metabolic activation. Cytotoxicity was observed in strain TA 1537 without S9 mix and TA 1538 with S9 mix at the highest concentration tested (5000 µg/plate) in experiment 1. Up to the highest investigated dose, neither a significant and reproducible increase of the number of revertants was found in any strain as compared to the solvent control nor a concentration-dependent enhancement of the revertant number exists. The presence of liver microsomal activation did not influence these findings. Therefore, it was concluded that FAT 40406/A did not show any mutagenic activity in any Salmonella typhimurium strains.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Chromosome abberation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

October 04, 1990 to June 21, 1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

other: Revised Chemical Substance Law (1987) according to the notification of December 9, 1986 by EA, Environmental Agency (no. 700); MHW, Ministry of Health and Welfare (No. 1039) and MITI, Ministry of International Trade and Industry (No. 1014), Japan.

GLP compliance:

yes

Remarks:

The OECD Principles of Good Laboratory Practice", Paris 1981

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 6947/14
- Expiration date of the lot/batch: July 1995

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: in water, DMSO, DMF for at least 48 h

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

aroclor 1254 induced rat liver S9 mix

Test concentrations with justification for top dose:

Evaluated test concentrations:
without S9 mix:
7 h: 0.30; 1, 2.50, 5 mg/ml
18 h: 0.03, 0.10, 0.30, 1, 2.50, 5 mg/ml
28 h: 0.30; 1.00; 2.50; 5.00 mg/ml
with S9 mix:
7 h: 0.30; 1.00; 2.50; 5.00 mg/ml
18 h: 0.03; 0.10; 0.30; 1.00; 2.50; 5.00 mg/ml
28 h: 0.30; 1.00; 2.50; 5.00 mg/ml

Vehicle / solvent:

culture medium without fetal calf serum

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation: Two days old logarithmically growing stock cultures more than 50 % confluent were trypsinised. The cells were seeded into Quadriperm dishes ( Heraeus, D-6450 Hanau, F.R.G. ) which contained microscopic slides (2 chambers per dish and test group). In each chamber 5E4 - 1E5 cells were seeded with regard to preparation time. The medium was MEM + 10 % FCS.
- Exposure: After 48 h (7 h, 28 h preparation interval) and 55 h (18 preparation interval) the medium was replaced with serum-free medium containing the test article, either without S9 mix or with 20 µL/mL S9 mix. After 4 h this medium was replaced with normal medium after rinsing twice.
- Fixation: 5, 15.5 and 25.5 h after the start of the treatment colcemid was added (0.2 µg/mL culture medium) to the cultures. 2 h (7 h interval) or 2.5 h later, (18 h and 28 h interval) the cells were treated on the slides in the chambers with hypotonic solution (0.4 % KCl) for 20 minutes at 37°C. After incubation in the hypotonic solution the cells were fixed with 3 + 1 absolute methanol + glacial acetic acid. Both slides per group were prepared. After fixation the cells were stained with Giemsa (Merck, D-6100 Darmstadt, F.R.G.).

NUMBER OF CELLS EVALUATED:
- At least 100 well spread metaphases per slide were scored for cytogenetic damage on coded slides. Only metaphases with characteristic chromosome number of 22 ± 1 were included in the analysis.

DETERMINATION OF CYTOTOXICITY
- To describe a cytotoxic effect the mitotic index (% cells in mitosis) was determined.

Evaluation criteria:

A test article is classified as mutagenic if it induces either a significant dose-related increase in the number of structural chromosomal aberrations or a significant positive response for at least one of the test points. A test article producing neither a significant dose-related increase in the number of structural chromosomal aberrations nor a significant and reproducible positive response at any one of the test points is considered non-mutagenic in this system.

Statistics:

chi-square test

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

5 mg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

- Additional information on genotoxicity: Both, in the absence and presence of S9 mix the test article did not increase the frequency of cells with aberrations at any fixation interval. The aberration rates of the cells after treatment with the test article (1.0 % - 4.0 %) were in or near to the range of the control values : 1.0 % - 3.0 %. At fixation interval 18 h, in the presence of S9 mix with increasing dose levels (0.3; 2.5; 5.0 mg/mL) of the test article increasing aberration rates were found (1.0; 1.5; 4.0 % ) . However, the aberration rates are within our historical control range (0.0 - 4.0 % ) . Therefore, this result is not regarded as biologically relevant.

- Additional information on cytotoxicity: The concentration range of the test article applied had been determined in a pre-experiment using the plating efficiency assay as indicator for toxicity response. Treatment with the highest concentration (5.0 mg/mL) reduced distinctly the plating efficiency of the V79 cells. Also the mitotic index was reduced after treatment with the highest scorable concentrations at each fixation interval in the presence and absence of S9 mix except at interval 18 h without S9 mix.

At fixation interval 18 h, in the presence of S9 mix with increasing dose levels (0.3; 2.5; 5.0 mg/ml) of the test article increasing aberration rates were found (1.0; 1.5; 4.0 % ). However, the aberration rates are within our historical control range (0.0 - 4.0 %). Therefore, this result is not regarded as biologically relevant. EMS (0.72 mg/ml) and CPA (1.40 µg/ml) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

Conclusions:

FAT 40406/A is considered to be not mutagenic in this chromosomal aberration test.

Executive summary:

In a GLP-compliant chromosome aberration test, tested according to OECD guideline 473, FAT 40406/A was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro. Preparation of chromosomes was done 7 h (high dose), 18 h (low, medium and high dose), and 28 h (high dose) after start of treatment with the test article. The treatment interval was 4 h. In each experimental group two parallel cultures were used. Per culture 100 metaphases were scored for structural chromosomal aberrations.

The following dose levels were evaluated:

without S9 mix:                            with S9 mix:

7 h: 1.0 mg/ml                            7 h: 1.0 mg/ml

18 h: 0.3; 2.5; 5.0 mg/ml          18 h: 0.3; 2.5; 5.0 mg/ml

28 h: 1.0 mg/ml                          28 h: 1.0 mg/ml

The concentration range of the test article applied had been determined in a pre-experiment using the plating efficiency assay as indicator for toxicity response. Treatment with the highest concentration (5.0 mg/ml) reduced distinctly the plating efficiency of the V79 cells. Also the mitotic index was reduced after treatment with the highest scorable concentrations at each fixation interval in the presence and absence of S9 mix except at interval 18 h without S9 mix. Higher concentrations of the test article than 5.0 mg/ml were not dissolved in the culture medium. There was no relevant increase in cells with structural aberrations after treatment with the test article at any fixation interval either without or with metabolic activation by S9 mix. Appropriate reference mutagens were used as positive controls and showed distinct increases in cells with structural chromosome aberrations. In conclusion, it can be stated that in the study described and under the experimental conditions reported, the test article did not induce structural chromosome aberrations as determined by the chromosomal aberration test in the V79 Chinese hamster cell line. Therefore, FAT 40406/A is considered to be non-mutagenic in this chromosomal aberration test.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

HPRT

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-12-12 to 2013-03-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

The signature of the management has been omitted in order to harmonize BSL procedures with internationally established procedures, Seeding the cultutures: 5 minutes instead of 3 minutes. Did not influence the quality or integrity of the present study

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

The signature of the management has been omitted in order to harmonize BSL procedures with internationally established procedures, Seeding the cultutures: 5 minutes instead of 3 minutes. Did not influence the quality or integrity of the present study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

yes

Remarks:

The signature of the management has been omitted in order to harmonize BSL procedures with internationally established procedures, Seeding the cultutures: 5 minutes instead of 3 minutes. Did not influence the quality or integrity of the present study

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Specific details on test material used for the study:

Name: FAT 40348/F

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: BOP 01-12 (Lot: BS-1109708)
- Expiration date of the lot/batch: 06 February 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature

Target gene:

hypoxanthine-guanine-phosphoribosyl-transferase (HPRT)

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

-Type and identity of media: MEM
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Liver S9 of Wistar Phenobarbital and ß-Naphthoflavone-induced rat liver S9 mix and Liver S9 of Sprague Dawley Phenobarbital and ß-Naphthoflavone-induced rat liver S9 mix.

Test concentrations with justification for top dose:

Pre-experiment for experiment I (with and without metabolic activation): 5, 10, 25, 50, 100, 250, 500, 1000, 2500, 5000 µg/mL
Pre-experiment for experiment II (only without metabolic activation, 20 h long-term exposure assay): 2.5, 5, 10, 25, 50, 75, 100, 250, 500, 750 µg/mL

Experiment I
without metabolic activation: 2.5, 5, 10, 25, 50, 100, 200, 300 and 400 µg/mL
and with metabolic activation: 0.25, 0.5, 1, 10, 20, 40, 60 and 80 µg/mL

Experiment II
without metabolic activation: 25, 50, 100, 250, 500, 750, 1000, 1400, 1800 and 2200 µg/mL
and with metabolic activation: 0.4, 0.8, 1.5, 3, 6, 12, 25, 50, 75 and 100 µg/mL

Vehicle / solvent:

Vehicle (Solvent) used: For the pre-experiments for experiment I the test item was dissolved in cell culture medium (MEM + 0% FBS).

For the main experiments and the pre-experiment for experiment II a stock solution of the test item in Aqua ad injectabilia was prepared (tenfold) and processed by sterile filtration. The dilution series was prepared in Aqua ad injectabilia. 10% of the dilution series, and/or Aqua ad injectabilia were added to cell culture medium prior to treatment (resulting in the designated concentrations of the test item).

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation; 300 µg/mL

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

with metabolic activation; 1.5 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: dissolved in Aqua ad inj. / medium
DURATION: 4 h (short-term exposure), 20 h (long-term exposure)
Expression time (cells in growth medium): 5 days
Selection time (if incubation with selection agent): about one week

SELECTION AGENT ( mutation assay) 11 µg/mL 6-thioguanine (TG)
NUMBER OF REPLICATIONS: two separate experiments (I+II) with single exposure; 5 individual flasks were seeded and evaluated
NUMBER OF CELLS EVALUATED: 400000 cells per flask
DETERMINATION OF CYTOTOXICITY: Method: relative growth

Evaluation criteria:

A test is considered to be negative if there is no biologically relevant increase in the number of mutants.
There are several criteria for determining a positive result:
- a reproducible three times higher mutation frequency than the solvent control for at least one of the concentrations;
- a concentration related increase of the mutation frequency; such an evaluation may be considered also in the case that a three-fold increase of
the mutant frequency is not observed;
- if there is by chance a low spontaneous mutation rate in the corresponding negative and solvent controls a concentration related increase of the mutations within their range has to be discussed.

Statistics:

No statistical evaluation required because the biological relevance of the results is the criterion for the interpretation of results.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Experiment I without S9: ≥ 25 μg/mL; experiment I with S9: ≥ 10 μg/mL; Experiment II without S9: ≥ 500 μg/mL; Experiment II with S9:≥ 12 μg/mL

Untreated negative controls validity:

valid

Positive controls validity:

valid

Precipitation: No precipitation of the test item was noted in any of the experiments.

 

Toxicity:

A biologically relevant growth inhibition (reduction of relative growth below 70 %) was observed after the treatment with the test item in experiment I and II with and without metabolic activation. In experiment I without metabolic activation the relative growth was 10.9 % for the highest concentration (400 µg/mL) evaluated. The highest biologically relevant concentration evaluated with metabolic activation was 80 µg/mL with a relative growth of 12.8 %.

In experiment II without metabolic activation the relative growth was 15.0 % for the highest concentration (2200 µg/mL) evaluated. The highest concentration evaluated with metabolic activation was 100 µg/mL with a relative growth of 16.7 %.

 

Mutagenicity:

In experiment I without metabolic activation most mutant values of the negative controls, the solvent controls and test item concentrations found were within the historical control data of the test facility BSL BIOSERVICE (about 5-43 mutants per 10⁶ cells). No dose-response relationship could be observed. The mutation frequencies found in the groups treated with the test item did not show a biologically relevant increase as compared to the solvent controls. Mutation frequencies with the negative control were found to be 26.57 and 10.68, of the solvent control 11.11 and 25.00 mutants/10⁶ cells and in the range of 2.40 to 33.18 mutants/10⁶cells with the test item, respectively. The highest mutation rate (compared to the solvent control values) of 1.84 was found at a concentration of 400 µg/mL with a relative growth of 1 0.9 %.

 

With metabolic activation most mutant values of the negative controls, the solvent controls and test item concentrations found were within the historical control data of the test facility BSL BIOSERVICE (about 5-44 mutants per 10⁶ cells). No dose-response relationship could be observed. The mutation frequencies found in the groups treated with the test item did not show a biologically relevant increase as compared to the solvent controls. Mutation frequencies with the negative control were found to be 30.30 and 22.66, of the solvent control 21.67 and 18.60 mutants/10⁶ cells and in the range of 1.91 to 39.80 mutants/10⁶ cells with the test item, respectively. The highest mutation rate (compared to the solvent control values) of 1.98 was found at a concentration of 20 µg/mL/mL with a relative growth of 29.4 %

 

In experiment II without metabolic activation all mutant values of the negative controls, the solvent controls and test item concentrations found were within the historical control data of the test facility BSL BIOSERVICE (about 5-43 mutants per 10⁶ cells). No dose response relationship could be observed. The mutation frequencies found in the groups treated with the test item did not show a biologically relevant increase as compared to the solvent controls. Mutation frequencies with the negative control were found to be 15.38 and 25,20, of the solvent control 21.09 and 10.77 mutants/10⁶ cells and in the range of 8.73 to 31.80 mutants/10⁶ cells with the test item, respectively. The highest mutation rate (compared to the solvent control values) of 2.00 was found at a concentration of 1000 µg/mL /mL with a relative growth of 35.8 %.

 

In experiment II with metabolic activation all mutant values of the negative controls, the solvent controls and test item concentrations found were within the historical control data of the test facility BSL BIOSERVICE (about 5-44 mutants per 10⁶ cells). No dose response relationship could be observed. The mutation frequencies found in the groups treated with the test item did not show a biologically relevant increase as compared to the solvent controls. Mutation frequencies with the negative control were found to be 29.37 and 28.85, of the solvent control 30.71 and 17.67 mutants/10⁶ cells and in the range of 11.07 to 43.03 mutants/10⁶ cells with the test item, respectively. The highest mutation rate (compared to the solvent control values) of 1.78 was found at a concentration of 75 µg /mL with a relative growth of 26.1 %. DMBA (1.5 µg /mL) and EMS (300 µg /mL) were used as positive controls and showed distinct and biologically relevant effects in mutation frequency.

Conclusions:

FAT 40348/F is considered to be non-mutagenic in the HPRT locus using V79 cells of the Chinese Hamster.

Executive summary:

FAT 40348/F was assessed for its potential to induce mutations at the HPRT locus using V79 cells of the Chinese Hamster as per OECD 476, (EC) No 440/2008 and OPPTS 870.5300 guidelines. The selection of the concentrations was based on data from the pre-experiments. Experiment I with and without metabolic activation and experiment II with metabolic activation were performed as a 4 h short-term exposure assay. Experiment II without metabolic activation was performed as 20 h long time exposure assay. In this mammalian cell gene mutation assay (HPRT locus),V79 cells cultured in-vitro were exposed to FAT 40348/F TE at concentrations of

- 2.5, 5, 10, 25, 50, 100, 200, 300 and 400 µg/mL (without metabolic activation, Experiment I)

- 0.25, 0.5, 1, 10, 20, 40, 60 and 80 µg/mL (with metabolic activation, Experiment I)

- 25, 50, 100, 250, 500, 750, 1000, 1400, 1800 and 2200 µg/mL (without metabolic activation, Experiment II)

- 0.4, 0.8, 1.5, 3, 6, 12, 25, 50, 75 and 100 µg/mL (with metabolic activation, Experiment II).

No precipitation of the test item was noted in the experiments. Biologically relevant growth inhibition was observed in experiment I and II with and without metabolic activation. In experiment I without metabolic activation the relative growth was 10.9 % for 400 µg/mL evaluated. The highest biologically relevant concentration evaluated with metabolic activation was 80 µg/mL with a relative growth of 12.8 %. In experiment II without metabolic activation the relative growth was 15.0 % for the highest concentration (2200 µg/mL) evaluated. The highest concentration evaluated with metabolic activation was 100 µg/mL with a relative growth of 16.7 %. In experiment I without metabolic activation the highest mutation rate (compared to the solvent control values) of 1.84 was found at a concentration of 400 µg/mL with a relative growth of 10.9 %. In experiment I with metabolic activation the highest mutation rate (compared to the solvent control values) of 1.98 was found at a concentration of 20 µg/mL with a relative growth of 29.4 %. In experiment II without metabolic activation the highest mutation rate (compared to the solvent control values) of 2.00 was found at a concentration of 1000 µg/mL with a relative growth of 35.8 %. In experiment II with metabolic activation the highest mutation rate (compared to the solvent control values) of 1.78 was found at a concentration of 75 µg/mL with a relative growth of 26.1 %. The positive controlsdidinduce the appropriate response. There was no evidence of a concentration related positive response of induced mutant colonies over background. The mutation frequencies found in the groups treated with the test item did not show a biologically relevant increase as compared to the solvent controls. In conclusion, FAT 40348/F is considered to be non-mutagenic in the HPRT locus using V79 cells of the Chinese Hamster.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Reactive Red 245 is considered to be not clastogenic in the micronucleus assay.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

September 11, 1990 to January 21, 1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5395 (In Vivo Mammalian Cytogenics Tests: Erythrocyte Micronucleus Assay)

GLP compliance:

yes

Remarks:

The OECD Principles of Good Laboratory Practice", Paris 1981

Type of assay:

mammalian erythrocyte micronucleus test

Specific details on test material used for the study:

Name: FAT 40406/A
Purity: 89.3 %
Batch No.: SR 6947/14
Aggregate State at RT: solid
Colour: dark red
Storage: room temperature
Expiration Date: July 1995

SOURCE OF TEST MATERIAL
- Source and lot/batch No. of test material: SR 6947/14
- Expiration date of the lot/batch: July 1995

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: In vehicle: in water for at least 48 h

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: BRL Tierfarm Füllinsdorf, CH-4414 Füllinsdorf/Basel, Switzerland
- Age at start of acclimatization: minimum 10 weeks
- Weight at study initiation: approximately 30 g
- Fasting period before study: 18 hours
- Housing: Individually in Makrolon type-1 cages with wire mesh top (EHRET GmbH, D-7830 Emmendingen) with granulated soft wood bedding (ALTROMIN, D-4937 Lage/Lippe)
- Diet: pelleted standard diet (ALTROMIN, D-4937 Lage/Lippe)
- Water: tap water, ad libitum (Gemeindewerke, D-6101 Roßdorf)
- Acclimation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: The vehicle was chosen to its non-toxicity for the animals.
- Concentration of test material in vehicle: 4000 mg/kg bw
- Amount of vehicle (if gavage or dermal): 20 mL/kg bw

Details on exposure:

On the day of the experiment, the test article was dissolved in aqua dest..

Frequency of treatment:

Single treatment

Post exposure period:

24, 48 and 72 h

Dose / conc.:

4 000 mg/kg bw (total dose)

Remarks:

On the basis of pre-experiments 4000 mg/kg was estimated to be the maximum tolerated dose.

No. of animals per sex per dose:

6

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide;
- Route of administration: orally
- Doses: 30 mg/kg bw
- Volume administrated: 10 mL/kg bw
- Vehicle: physiological saline

Tissues and cell types examined:

Bone marrow (Normochromatic and polychromatic erythrocytes)

Details of tissue and slide preparation:

DETAILS OF SLIDE PREPARATION:
The animals were sacrificed by cervical dislocation. The femora were removed, the epiphyses were cut off and the marrow was flushed out with fetal calf serum, using a 5 mL syringe. The cell suspension was centrifuged at 1,500 rpm for 5 minutes and the supernatant was discarded. A small drop of the resuspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald (Merck, D-6100 Darmstadt)/Giemsa (Gurr, BDH Limited Poole, Great Britain). Cover slips were mounted with EUKITT (KINDLER, D-7800 Freiburg). At least one slide was made from each bone marrow sample.

Evaluation criteria:

A test article is classified as mutagenic if it induces either a statistically significant dose-related increase in the number of micronucleated polychromatic erythrocytes or a reproducible statistically significant positive response for at least one of the test points. A test article producing neither a statistically significant dose-related increase in the number of micronucleated polychromatic erythrocytes nor a statistically significant and reproducible positive response at any one of the test points is considered non-mutagenic in this system.

METHOD OF ANALYSIS:
Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. 1000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in normochromatic erythrocytes per 1000 PCEs. The analysis was performed with coded slides. Five animals per sex and group were evaluated as described. The remaining animal of each test group was evaluated in case an animal had died in its test group spontaneously or due to gavage error.

Statistics:

Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- In the first pre-experiment 4 animals (2 males, 2 females) received orally a single dose of 5000 mg/kg b.w., FAT 40406/A dissolved in aqua dest. The volume administered was 20 mL/kg b.w. In the males, a reduction in sponteaneous activity was observed after one hour. One female died after 24 h and one male died after 48 h. After 24 h in one male, reduction in spontaneous activity, eyelid closure, apathy, and abdominal position was observed.
- In the second pre-experiment 4 animals (2 males, 2 females) per dose received orally a single dose of 3000 mg/kg bw or 4000 mg/kg bw FAT 40406/A dissolved in aqua dest. The volume administered was 20 mL/kg b.w. In the 3000 mg/kg bw group reduction of spontaneous activity was observed up to 24 h. Eyelid closure was observed after six hours in one male and one female mouse. Apathy was observed in one female mouse after six hours. In the 4000 mg/kg bw group reduction of spontaneous activity was observed up to 48 h. Eyelid closure was observed after six hours in one male and one female mouse. Apathy was observed in one female and one male mouse after six hours. On the basis of these results 4000 mg/kg b.w. FAT 40'406/A was estimated to be the maximum tolerated dose.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei: In comparison to the corresponding negative controls there was no significant enhancement in the frequency of the detected micronuclei at any preparation interval after application of the test article. The mean values of micronuclei observed after treatment with FAT 40406/A were in the same range as compared to the negative control groups.
- Ratio of PCE/NCE: The mean number of normochromatic erythrocytes was increased after treatment with the test article at preparation interval 48 hours as compared to the mean values of NCEs of the corresponding negative control, indicating that FAT 40406/A had cytotoxic properties.
- 30 mg/kg b.w. cyclophosphamide administered per os was used as positive control which showed a distinct increase of induced micronucleus frequency.

Conclusions:

FAT 40406/A did not induce micronuclei in bone marrow cells of the mouse and is considered to be non-mutagenic in this micronucleus assay.

Executive summary:

In a GLP-compliant erythrocyte micronucleus test performed according to OECD guideline 474 to investigate the potential of FAT 40406/A to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. The test article was dissolved in aqua dest. This solvent was used as negative control. The volume administered orally was 20 ml/kg b.w. 24 h, 48 h and 72 h after a single application of the test article the bone marrow cells were collected for micronuclei analysis. Ten animals (5/sex) per test group, with the exception of the test article group at preparation interval 72 h in which the preparations of two females could not be scored, were evaluated for the occurrence of micronuclei. 1000 polychromatic erythrocytes (PCE) per animal were scored for micronuclei. To describe a cytotoxic effect due to the treatment with the test article the ratio between polychromatic and normochromatic erythrocytes (NCE) was determined in the same sample and reported as the number of NCE per 1000 PCE. The following dose level of the test article was investigated: 24 h, 48 h, and 72 h preparation interval: 4000 mg/kg b.w. In pre-experiments this dose level was estimated to be the maximum tolerated dose. The animals expressed toxic reactions. After treatment with the test article the ratio between PCEs and NCEs was affected as compared to the corresponding negative control at preparation interval 48 hours thus indicating cytotoxic effects. In comparison to the corresponding negative controls there was no significant enhancement in the frequency of the detected micronuclei at any preparation interval after application of the test article. An appropriate reference mutagen was used as positive control which showed a distinct increase of induced micronucleus frequency. In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test article did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse. Therefore, FAT 40406/A is considered to be non-mutagenic in this micronucleus assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro:

In a GLP-compliant Ames test, performed according to OECD guideline 471, 5 Salmonella typhimurium strains (TA 1535, TA 1537, TA 1538, TA 98, and TA 100) were used to the test the mutagenic potential of the test substance (10, 100, 333.3, 1000, 2500, 5000 µg per plate), both with and without metabolic activation (CCR 1990). Cytotoxicity was observed in strain TA 1537 without S9 mix and TA 1538 with S9 mix at the highest concentration tested (5000 µg/plate) in experiment 1. Up to the highest investigated dose, neither a significant and reproducible increase of the number of revertants was found in any strain as compared to the solvent control nor a concentration-dependent enhancement of the revertant number exists. The presence of liver microsomal activation did not influence these findings. Therefore, it was concluded that test substance did not show any mutagenic activity in any strain. 

In a GLP-compliant chromosome aberration test, tested according to OECD guideline 473, Chinese hamster lung fibroblast (V79) were exposed to the test substance with and without metabolic activation (CCR 1991). Different exposure times (up to 28 h) and dosage (up to 5 mg/mL) were chosen in this study. The mitotic index was reduced after treatment with the highest concentrations (with and without S9 mix) indicating that the test substanc ehad cytotoxic properties.There was no relevant increase in cells with structural aberrations after treatment with the test article at any fixation interval either without or with metabolic activation by S9 mix. Therefore, it was stated that the test substance did not induce structural chromosome aberrations in the V79 Chinese hamster cell line. No studies on the mutagenicity in mammalian cells of Reactive Red 245 were available. However, a HPRT gene mutation test is available for the structural analogue Reactive Red 238. 

In a GLP-compliant mammalian cell gene mutation assay, tested according to OECD guideline 476, Chinese hamster V79 cells were exposed to the test substance with and without metabolic activation and the potential to induce mutations at the HPRT locus was assessed (BSL Bioservice 2013). The selection of the concentrations was based on data from the pre-experiments. Experiment I with and without metabolic activation and experiment II with metabolic activation were performed as a 4h short-term exposure assay. Experiment II without metabolic activation was performed as 20h long time exposure assay. The following concentrations were used. Experiment I without metabolic activation: 2.5, 5, 10, 25, 50, 100, 200, 300, and 400µg/mL; Experiment I with metabolic activation: 0.25, 0.5, 1, 10, 20, 40, 60, and 80 µg/mL; Experiment II without metabolic activation: 25, 50, 100, 250, 500, 750, 1000, 1400, 1800 and 2200 µg/mL; Experiment II with metabolic activation: 0.4, 0.8, 1.5, 3, 6, 12, 25, 50, 75 and 100µg/mL. No precipitation was noted in the experiments. Biologically relevant growth inhibition was observed in experiment I and II with and without metabolic activation. In experiment I without metabolic activation the relative growth was 10.9 % for the highest concentration (400 µg/mL) evaluated. The highest biologically relevant concentration evaluated with metabolic activation was 80 µg/mL with a relative growth of 12.8 %. In experiment II without metabolic activation the relative growth was 15.0 % for the highest concentration (2200 µg/mL) evaluated. The highest concentration evaluated with metabolic activation was 100 µg/mL with a relative growth of 16.7 %. In both experiments no biologically relevant increase of mutants was found after treatment with the test item (with and without metabolic activation). No dose-response relationship was observed. The positive controls showed distinct biologically relevant effects in mutation frequency. In conclusion, the test substance is considered to be non-mutagenic in the HPRT locus using V79 cells of the Chinese hamster.

 

In vivo:

In a GLP-compliant erythrocyte micronucleus test, tested according to OECD guideline 474, 6 NMRI mice per sex were treated once by oral gavage with the test substance (4000 mg/kg bw) dissolved in water followed by a 24, 48 and 72 hours post exposure period. This study was performed to investigate the potential of FAT 40406/A to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. The test article was dissolved in aqua dest. This solvent was used as negative control. The volume administered orally was 20 ml/kg b.w. 24 h, 48 h and 72 h after a single application of the test article the bone marrow cells were collected for micronuclei analysis. Ten animals (5 males, 5 females) per test group, with the exception of the test article group at preparation interval 72 h in which the preparations of two females could not be scored, were evaluated for the occurrence of micronuclei. 1000 polychromatic erythrocytes (PCE) per animal were scored for micronuclei. In pre-experiments this dose level was estimated to be the maximum tolerated dose. To describe a cytotoxic effect due to the treatment with the test article the ratio between polychromatic and normochromatic erythrocytes (NCE) was determined in the same sample and reported as the number of NCE per 1000 PCE. The following dose level of the test article was investigated: 24 h, 48 h, and 72 h preparation interval: 4000 mg/kg b.w. In pre-experiments this dose level was estimated to be the maximum tolerated dose. The animals expressed toxic reactions. After treatment with the test article the ratio between PCEs and NCEs was affected as compared to the corresponding negative control at preparation interval 48 hours thus indicating cytotoxic effects. In comparison to the corresponding negative controls there was no significant enhancement in the frequency of the detected micronuclei at any preparation interval after application of the test article. An appropriate reference mutagen was used as positive control which showed a distinct increase of induced micronucleus frequency. In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test article did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse. Therefore, FAT 40406/A is considered to be non-clastogenic in this micronucleus assay.

Short description of key information:
Reactive Red 245 was found to have negative outcpme in the in vitro chromosomal aberration test and Ames test. Furthermore, the in vivo micronucleus test did not show any evidence of clastogenic activity. The source substance Reactive Red 238 was non-mutagenic in the HPRT mutation assay. Thus using the principles of read across, the target substance Reactive Red 245 is also considered to be not mutagenic in mammalian cells as well. In conclusion, Reactive Red 245 is to be considered as not genotoxic.

Justification for classification or non-classification

Based on the available genotoxicity studies, Reactive Red 245 does not need to be classified for genotoxicity according to the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008