trisodium 3-amino-4-[4-[4-(2-(2-ethenylsulfonylethoxy)ethylamino)-6-fluoro-1,3,5-triazine-2-ylamino]-2-sulfophenylazo]-5-hydroxynaphthalene-2,7-disulfonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item was found positive in Chromosome aberration test, but was found negative in Ames and HPRT In vitro study.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Study initiation date - 09 March 1999; Experiment start date - 17 March 1999; Experiment completion date - 06 April 1999; Study completion date - 26 April 1999.

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

"Kanpoan No. 287 ~ Environment Protection Agency"
"Eisei No. 127 ~ Ministry of Health & Welfare"
"Heisei 09/10/31 Kikyoku No. 2 ~ Ministry of International Trade & Industry"

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Identity: FAT 40574/B
Batch: WP 23/99
Purity: Approx. 75 %
Appearance: Solid, dark-red powder
Storage: At room temperature at about 20 °C
Expiration Date: 08 February 2006

Target gene:

Histidine

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Regular checking of the properties of the strains regarding the membrane permeability and ampicillin resistance as well as spontaneous mutation rates is performed in RCC Cytotest Cell Research according to Ames et al. In this way it was ensured that the experimental conditions set down by Ames were fulfilled.
The bacterial strains TA 1535, TA 98, and TA 100 were obtained from Dr. B.N. Ames (University of California, 94720 Berkeley, U.S.A.). The bacterial strain TA 1537 was obtained from BASF (D-67063 Ludwigshafen). The bacterial strain WP2 uvrA was obtained from Dr. Heinz Träger, Knoll AG, D-67008 Ludwigshafen.

Metabolic activation:

with and without

Metabolic activation system:

S9 liver fractions of rats induced with phenobarbital/ ß-naphthoflavone.
The S9 liver microsomal fraction was obtained from the livers of 8 - 12 weeks old male rats, strain Wistar Hanlbm (BRL, CH-4414 Füllinsdorf; weight approx. 220 - 320 g) which received daily applications of 80 mg/kg b.w. Phenobarbital i.p. dissolved in aqua deionised (Desitin; D-22335 Hamburg) and ß-Naphthoflavone orally dissolved in corn oil (Aldrich, D-89555 Steinheim) on three subsequent days. The livers were prepared 24 hours after the last treatment. After decapitation of the anaesthetised animals the livers of the animals were removed, washed in 150 mM KCl and homogenised. The homogenate was diluted 1+3 in KCl and centrifuged cold at 9,000 g for 10 minutes at 4 °C. A stock of the supernatant containing the microsomes was frozen in ampoules and stored at -80° C. Small numbers of the ampoules are kept at - 20 °C for up to one week before use. The protein content was determined using an analysis kit of Bio-Rad Laboratories, D-80939 München (Bio-Rad protein assay, Catalogue No. 5000006). The protein concentration in the S9 preparation was 30 mg/ml (lot no. 280199) in the pre-experiment and in experiment I, and 35.9 mg/ml (lot no. 260299) in experiment II.

S9Mix
Before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 co-factor solution. The amount of S9 supernatant was 15 % v/v in the cultures. The concentrated co-factor solution yields the following concentrations in the S9 mix:
8 mM MgCl2
33 mM KCl
5 mM Glucose-6-phosphate
5 mM NADP
in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.
During the experiment the S9 mix was stored in an ice bath. The S9 mix preparation was performed according to Ames et al.

Test concentrations with justification for top dose:

Nominal concentration: 33; 100; 333; 1000; 2500; and 5000 µg/plate

Vehicle / solvent:

Solvent: deionised water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Deionised water

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

With metabolic activation: S.typhimurium TA 1535, TA 100

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene-diamine, 4-NOPD

Remarks:

With metabolic activation: S.typhimurium TA 1537, TA 98

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Deinonised water

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

With metabolic activation: E.coli WP2 uvrA

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene, 2-AA

Remarks:

Without metabolic activation: TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Triplicate
- Number of independent experiments: Two

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium: Experiment I : In agar (plate incorporation); Experiment II: Preincubation

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: Yes Experiment II.
- Exposure duration/duration of treatment: 60 min

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Background growth inhibition

METHODS FOR MEASUREMENTS OF GENOTOXICIY : Dose related and reproducible increase in the number of revertants or a biologically relevant and reproducible increase for at least one test concentration is induced.

Evaluation criteria:

Acceptability of the Assay:
The Salmonella typhimurium and Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
- regular background growth in the negative and solvent control
- the spontaneous reversion rates in the negative and solvent control are in the range of our historical data
- the positive control substances should produce a significant increase in mutant colony frequencies

Evaluation of Results:
A test article is considered positive if either a dose related and reproducible increase in the number of revertants or a biologically relevant and reproducible increase for at least one test concentration is induced.
A test article producing neither a reproducible and dose related increase in the number of revertants, nor a biologically relevant and reproducibly positive response at any one of the test points is considered non-mutagenic in this system.
A mutagenic response is described as follows:
A test article is considered mutagenic if in the strains TA 98, TA 100, and WP2 the number of reversions will be at least twice as high and in the strains TA 1535 and TA 1537 at least three times higher as compared to the spontaneous reversion rate (3,4).
Also, a dose-dependent and reproducible 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.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

True negative controls validity:

not examined

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

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

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

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

No relevant toxic effects, evident as a reduction in the number of revertants, occurred in the est groups with and without metabolic activation. The plates incubated with the test article showed normal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with FAT 40574/B at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

Conclusions:

The test article did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Executive summary:

A GLP-study was performed according to OECD test guideline 471 to investigate the potential of FAT 40574/B to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and in addition the Escherichia coli strain WP2 uvrA. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test article was tested at the following concentrations: 33; 100; 333; 1000; 2500 and 5000 µg/plate. No relevant toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation. Slight, seemingly toxic effects occurred in strain TA 100 with metabolic activation in the first experiment. However, this effect is based upon a high solvent control value (105 colonies mean value versus 85 of the corresponding solvent control) and does represent statistical fluctuations rather than a true toxic effect. Another slight toxic effect was observed in strain TA 98 without metabolic activation in the second experiment. Since this effect was not really dose dependent, it was again judged as caused by statistical fluctuations. The plates incubated with the test article showed normal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with FAT 40574/B at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Study initiation date - 09 March 1999; Experiment start date - 29 March 1999; Experiment completion date - 21 June 1999; Study completion date - 24 June 1999.

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

"Kanpoan No. 287 ~ Environment Protection Agency"
"Eisei No. 127 -- Ministry of Health & Welfare"
"Heisei 09/10/31 Kikyoku No. 2 -- Ministry of International Trade & Industry".

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

Identity: FAT 40574/B
Batch: WP 23/99
Purity: Approx. 75 %
Appearance: Solid, dark-red powder
Storage: At room temperature at about 20 °C
Expiration Date: 08 February 2006

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

Large stocks of the V79 cell line (supplied by Laboratory for Mutagenicity Testing, LMP, Technical University Darmstadt, D-64287 Darmstadt) were stored in liquid nitrogen in the cell bank of RCC-CCR allowing the repeated use of the same cell culture batch in experiments. Before freezing each batch was screened for mycoplasm contamination and checked for karyotype stability. Consequently, the parameters of the experiments remain similar because of standardized characteristics of the cells.

Thawed stock cultures were propagated at 37 °C in 80 cm plastic flasks (GREINER, D-72632 Frickenhausen). About 5 x 10E5 cells per flask were seeded into 15 ml of MEM (Minimal Essential Medium; SEROMED; D-12247 Berlin) supplemented with 10 % fetal calf serum (FCS; Boehringer Mannheim, D-68261 Mannheim). The cells were subcultured twice weekly. The cell cultures were incubated at 37 °C in a humidified atmosphere with 4.5 % carbon dioxide (95.5 % air).

Metabolic activation:

with and without

Metabolic activation system:

The S9 liver microsomal fraction was obtained from the livers of 8 - 12 weeks old male rats, strain Wistar Hanlbm (BRL, CH-4414 Füllinsdorf; weight approx. 220 - 320 g) which received daily applications of 80 mg/kg b.w. phenobarbital i.p. dissolved in deionised water (Desitin; D-22335 Hamburg) and ß-naphthoflavone orally dissolved in corn oil (Aldrich, D- 89555 Steinheim) on three subsequent days. The livers were prepared 24 hours after the last treatment. After decapitation of the anaesthetised animals, the livers of the animals were removed, washed in 150 mM KCl and homogenized. The homogenate, diluted 1+3 with KCl was centrifuged at 9000 g for 10 minutes (4 °C). Aliquotes of the supernatant containing the microsomal fraction were frozen and stored in ampoules at -80 °C. Small numbers of the ampoules were kept at -20 °C for up to one week. The protein content was determined using the analysis kit of Bio-Rad Laboratories, D-80939 München: Bio-Rad protein assay, Catalogue No. 5000006. The protein concentration in the S9 preparation is usually between 20 and 45 mg/ml. The protein concentration was 30 mg/ml (Lot. No.: 280199) in the pre-test and 35.3 mg/ml (Lot. No.: 180399) in the main experiments.

S9 Mix
An appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0.75 mg/ml in the cultures. Cofactors were added to the S9 mix to reach the following concentrations:
8 mM MgCl2
33 mM KCl
5 mM glucose-6-phosphate
4 mM NADP
in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.
During the experiment the S9 mix was stored in an ice bath. The S9 mix preparation was performed according to Ames et al.

Test concentrations with justification for top dose:

Concentration range in the main test (with metabolic activation): Experiment I (4 h): 75, 150, 300, 600, 900, 1200 µg/ml
Concentration range in the main test (with metabolic activation): Experiment II (4 h: 62.5, 125, 250, 500, 750, 1000 µg/ml
Concentration range in the main test (without metabolic activation): Experiment I (4 h): 250, 500, 1000, 2000, 3000, 4000 µg/ml
Concentration range in the main test (without metabolic activation): Experiment II (18 h): 187.5, 375, 750, 1500, 2250, 3000 µg/ml
Concentration range in the main test (without metabolic activation): Experiment II (28 h): 750, 1500, 2250, 3000 µg/ml

Vehicle / solvent:

Deionised water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Without metabolic activation

Untreated negative controls:

yes

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:

Exposure period (with metabolic activation): 4 hours
Exposure period (without metabolic activation): 28 hours

Expression time:
----

Selection time:
----

Fixation time:
18 and 28 hours

Evaluation criteria:

Acceptability of the Assay:
The chromosome aberration assay performed in our laboratory is considered acceptable if it meets the following criteria:
a) The number of structural aberrations found in the negative and/or solvent controls falls within the range of our historical laboratory control data: 0.00 % - 4.00 %.
b) The positive control substances should produce significant increases in the number of cells with structural chromosome aberrations, which are within the range of the laboratories historical control data

Evaluation of Results:
A test article is classified as non-mutagenic if:
- the number of induced structural chromosome aberrations in all evaluated dose groups are in the range of our historical control data (0.0 - 4.0 % aberrant cells exclusive gaps).
- no significant increase of the number of structural chromosome aberrations are observed.
A test article is classified as mutagenic if:
- the number of induced structural chromosome aberrations are not in the range of our historical control data (0.0 - 4.0 % aberrant cells exclusive gaps).
- either a concentration-related or a significant increase of the number of structural chromosome aberrations are observed.

Statistics:

Statistical significance was confirmed by means of the Fischer's exact test (p <0.05). However, both biological and statistical significance should be considered together. If the a.m. criteria for the test article are not clearly met, the classification with regard to the historical data and the biological relevance is discussed and/or a confirmatory experiment is performed.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

with 600 µg/ml (50 % of control)

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In exp I, after 4 h treatment with 2000 µg/ml (47 % of control). In exp II, after continuous treatment with 1500 µg/ml (47 % of control) at interval 18 h only

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In the presence of S9 mix, in experiment II at the 28 h preparation interval after treatment with 250 µg/ml the chromosome aberration rate was increased. 7 % aberrant cells exclusive gaps were observed. This aberration rate exceeds the upper limit of the laboratory's historical negative control range (0 - 4 % aberrant cells exclusive gaps) and was prnved to be statistically significant. In addition, the number of cells carrying exchanges was increased. At the other experimental points, no increased aberration frequency was observed, neither in the absence, nor in the presence of S9 mix.

In a range finding pre-test on toxicity cell numbers 24 h after start of treatment were scored as indicator for cytotoxicity. Concentrations between 39.1 and 5000 µg/ml were applied. Toxic effects were observed after 4 h treatment with 2500 µg/ml and above in the absence of S9 mix and with 1250 µg/ml and above in the presence of S9 mix. In addition, continuous treatment with 2500 µg/ml and above in the absence of S9 mix induced strong toxic effects. Neither precipitation of the test article in culture medium nor influence of the test article on the pH value or osmolarity was observed (solvent control: 282 mOsm, pH 7.5 versus 298 mOsm and pH 7.5 at 5000 µg/ml). The mitotic indices of the evaluated experimental points were slightly reduced at the top concentration. However, reduced cell numbers <50 % of control were observed in experiment I in the presence of S9 mix after 4 h treatment with 600 µg/ml (50 % of control) and in the absence of S9 mix after 4 h treatment with 2000 µg/ml (47 % of control). In experiment II the cell numbers were reduced in the absence of S9 mix after continuous treatment with 1500 µg/ml (47 % of control) at interval 18 h only. In the presence of S9 mix 250 µg/ml could be evaluated as top concentration, showing slightly reduced cell numbers (61 % of control). The next higher concentration, exhibiting sufficient toxicity, could not be evaluated for cytogenetic damage since nucleolus fragmentation was observed. In the presence of S9 mix, in experiment II at the 28 h preparation interval after treatment with 250 µg/ml the chromosome aberration rate was increased. 7 % aberrant cells exclusive gaps were observed. This aberration rate exceeds the upper limit of the laboratory's historical negative control range (0 - 4 % aberrant cells exclusive gaps) and was proved to be statistically significant. In addition, the number of cells carrying exchanges was increased. At the other experimental points, no increased aberration frequency was observed, neither in the absence, nor in the presence of S9 mix. At the 28 h interval in the presence of S9 mix, additional observations for a clastogenic potential were remarked. Fragmentation of the nuclei and micronuclei were observed after treatment with 250 and 500 µg/ml, giving additional evidence for a clastogenic potential. In both experiments, no biologically relevant increase in the rate of polyploid metaphases was found after treatment with the test article (exp. I: 3.2 % - 5.7 %; exp. II: 2.5 % - 5.4 %) as compared to the rates of the solvent controls (exp. I: 4.0 % - 4.2 %; exp. II: 2.4 % - 3.3 %). In both experiments, EMS (600 and 1000 µg/ml, respectively) and CPA (0.71 µg/ml) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

Conclusions:

FAT 40574/B did induce structural chromosome aberrations in V79 cells (Chinese hamster cell line).

Executive summary:

The test article FAT 40'574/B, dissolved in deionised water, was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in two independent experiments. This study was conducted according to OECD 473 in a GLP-certified laboratory.

	Without S9 mix			With S9 mix
	Exp I	Exp II		Exp I	Exp II
Exposure period	4h	18h	28h	4h	4h
Recovery	14h	-	-	14h	24h
Preparation interval	18 h	18 h	28 h	18 h	28 h
conc (µg/ml)	-	187.5	-
conc (µg/ml)	1000	375	-	150	62.5
conc (µg/ml)	2000	750	-	300	125
conc (µg/ml)	3000	1500	750	600	250

 

In each experimental group two parallel cultures were set up. Per culture 100 metaphase plates were scored for structural chromosome aberrations. The highest applied concentration in the pre-test on toxicity (5000 µg/ml) was chosen with regard to current OECD Guideline 473. Dose selection of the cytogenetic experiments was performed considering the toxicity data of the pre-test. The chosen treatment concentrations and the rational are described above. Reduced cell numbers were observed after 4 treatment with 2000 µg/ml and above in the absence of S9 mix and 600 µg/ml in the presence of S9 mix. After continuous exposure in the absence of S9 mix (exp. II) reduced cell numbers were observed after treatment with 1500 µg/ml and above. In the presence of S9-mix 250 µg/ml could be evaluated as top concentration, showing slightly reduced cellnumbers (61 % of control). The next higher concentration, exhibiting sufficient toxicity,could not be evaluated for cytogenetic damage since nucleolus fragmentation was observed. A significant and biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed in the presence of S9 mix at interval 28 h after treatment with 250 µg/ml. 7 % aberrant cells exclusive gaps were observed. This aberration rate exceeds the upper limit of the laboratory's historical negative control range (0 - 4 % aberrant cells exclusive gaps) and was proved to be statistically significant. In addition, the number of cells carrying exchanges was increased. At the other experimental points, no increased aberration frequency was observed, neither in the absence, nor in the presence of S9 mix. At the 28 h interval in the presence of S9 mix, additional observations for a clastogenic potential were remarked. Fragmentation of the nuclei and micronuclei were observed after treatment with 250 and 500 µg/ml, giving additional evidence for a clastogenic potential. No increase in the frequencies of polyploid metaphases was found after treatment with the test article as compared to the frequencies of the controls. Appropriate mutagens were used as positive controls. They induced statistically significant increases (p <0.05) 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 induced structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro. Therefore, FAT 40574/B is considered to be mutagenic in this chromosome aberration test.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Study initiation date - 08 January 2013; Experiment start date - 22 January 2013; Experiment completion date - 26 April 2013; Study completion date - 16 May 2013.

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:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Germany)

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:

Identity: FAT 40574/C
Batch: BOP 01-12 (15 February 2012)
Purity: Sum of all coloured substance: 86.2 %; main constituents: 73.9 %
Appearance: Solid, dark-red powder
Storage: At room temperature
Expiration Date: 20 February 2017.

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 Sprague Dawley Phenobarbital and ß-Naphthoflavone-induced rat liver S9 mix:
The S9 liver microsomal fraction was obtained from Trinova Biochem GmbH, Giessen, Germany. Male SD rats were induced with phenobarbital/ ß-Naphthoflavone. A stock of the supernatant containing the microsomes is frozen in aliquots of 5 mL and stored at <75 °C.

S9 Mix:
An appropriate quantity of the S9 supernatant was thawed and mixed with S9 Cofactor solution to result in a final protein concentration of 0.75 mg/mL in the cultures. Cofactors were added to the S9 mix to reach the concentration below;
8 mM Mg Cl2
33 mM KCl
5 mM Glucose-6-phosphate
5 mM NADP
in 100 mM sodium phosphate buffer pH 7.4. During the experiment the S9 mix was stored on ice.

Test concentrations with justification for top dose:

Pre-experiment for experiment I (with and without metabolic activation):
10, 25, 50, 100, 250, 500, 1000, 1750, 2500, 5000 µg/mL

Experiment I
without metabolic activation:
3.16, 10.0, 31.6, 100, 250, 500, 1500, 2000, 2500 and 2750 µg/mL
and with metabolic activation:
50, 100, 250, 500, 1000, 1500, 2000, 3000 and 5000 µg/mL

Experiment II
without metabolic activation:
25, 50, 100, 250, 500, 1500, 2500, 3000, 4000 and 5000 µg/mL
and with metabolic activation:
31.6, 100, 316, 1000, 1600, 2200, 2900, 3600, 4300 and 5000 µg/mL

Vehicle / solvent:

Vehicle (Solvent) used: For the pre-experiment the test item was dissolved in cell culture medium (MEM + 0 % FBS). For the main experiments a stock solution of the test item in aqua ad injectionem was prepared (tenfold) and processed by sterile filtration. The dilution series was prepared in aqua ad injectionem. 10% of the dilution series, and/or aqua ad injectionem 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

Remarks:

Medium (MEM)

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

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

With metabolic activation; 0.8 and 1.0 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.

Key result

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: 2750 μg/mL; experiment I with S9: 5000 μg/mL; Experiment II without S9: 5000 μg/mL; Experiment II with S9: 5000 μg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Conclusions:

In conclusion, in the described mutagenicity test under the experimental conditions reported, the test item FAT 40574/C is considered to be non-mutagenic in the HPRT locus using V79 cells of the Chinese Hamster.

Executive summary:

In a mammalian cell gene mutation assay (HPRT locus) conducted according to OECD test guideline 476, V79 cells cultured in vitro were exposed to FAT 40574/C at concentrations of

- 3.16, 10.0, 31.6, 100, 250, 500, 1500, 2000, 2500 and 2750 µg/mL (without metabolic activation, Experiment I)

- 50, 100, 250, 500, 1000, 1500, 2000, 3000 and 5000 µg/mL (with metabolic activation, Experiment I)

- 25, 50, 100, 250, 500, 1500, 2500, 3000, 4000 and 5000 µg/mL (without metabolic activation, Experiment II)

- 31.6, 100, 316, 1000, 1600, 2200, 2900, 3600, 4300 and 5000 µg/mL (with metabolic activation, Experiment II).

FAT 40574/C was tested up to cytotoxic concentrations. 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 11.1% for the highest concentration (2750 µg/mL) evaluated. The highest biologically relevant concentration evaluated with metabolic activation was 5000 µg/mL with a relative growth of 32.5%. In experiment II without metabolic activation the relative growth was 29.6% for the highest concentration (5000 µg/mL) evaluated. The highest concentration evaluated with metabolic activation was 5000 µg/mL with a relative growth of 39.8 %. 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 1500 µg/mL with a relative growth of 37.2 %. In experiment I with metabolic activation the highest mutation rate (compared to the solvent control values) of 1.50 was found at a concentration of 3000 µg/mL with a relative growth of 64.0 %. In experiment II without metabolic activation the highest mutation rate (compared to the solvent control values) of 2.09 was found at a concentration of 250 µg/mL with a relative growth of 75.7%.  In experiment II with metabolic activation the highest mutation rate (compared to the solvent control values) of 2.29 was found at a concentration of 2200 µg/mL with a relative growth of 76.6 %. The positive controls did induce the appropriate response. There was no evidence of a concentration related positive response of induced mutant colonies over background. This study is classified as acceptable. This study satisfies the requirement for Test Guideline OPPTS 870.5300, OECD 476 for in vitro mutagenicity (mammalian forward gene mutation) data.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

The test item was considered to be non-mutagenic in this 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:

Study initiation date - 01 December 1999; Experiment start date - 01 December 1999; Experiment completion date - 17 December 1999; Study completion date - 17 December 1999.

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:

other: EEC Directive 92/69, L 383, Annexe V, B 12, dated December 29, 1992.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian bone marrow chromosome aberration test

Specific details on test material used for the study:

Identity: FAT 40574/B
Batch: WP 23/99
Purity: Approx. 75 %
Appearance: Solid, dark-red powder
Storage: At room temperature at about 20 °C
Expiration Date: 08 February 2006

Species:

mouse

Sex:

male/female

Details on test animals or test system and environmental conditions:

Source: BRL, CH-4414 Füllinsdorf
Initial Age at Start of Acclimatization: 8 - 12 weeks
Acclimatization: minimum 5 days
Initial Body Weight at Start of Treatment: males mean value 34.3 g (SD ± 2.5 g); females mean value 27.8 g (SD ± 1.9 g)
Housing: single
Cage Type: Makrolon Type I, with wire mesh top (EHRET GmbH, D-79302 Emmendingen)
Bedding: granulated soft wood bedding (ALTROMIN, D-32791 Lage/Lippe)
Feed: Pelleted standard diet, ad libitum
Water: Tap water, ad libitum
Environment: Temperature 21 ± 3 °C; Relative humidity 25 - 54 %; Artificial light 6.00 a.m. - 6.00 p.m.

Route of administration:

oral: gavage

Vehicle:

Deionised water

Details on exposure:

On the day of the experiment, the test item was formulated in deionised water. The vehicle was chosen to its non-toxicity for the animals. All animals received a single standard volume of 10 ml/kg body weight orally.

Frequency of treatment:

Once

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Vehicle - Group 1

Dose / conc.:

200 mg/kg bw/day (nominal)

Remarks:

Low dose group - Group 2

Dose / conc.:

670 mg/kg bw/day (nominal)

Remarks:

Mid dose group - Group 3

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Remarks:

High dose group - Group 4

No. of animals per sex per dose:

Male: 200 mg/kg; No. of animals: 5; Sacrifice time: 24 hours
Male: 670 mg/kg; No. of animals: 5; Sacrifice time: 24 hours
Male: 2000 mg/kg; No. of animals: 5; Sacrifice time: 24 hours
Male: 2000 mg/kg; No. of animals: 5; Sacrifice time: 48 hours
Female: 200 mg/kg; No. of animals: 5; Sacrifice times: 24 hours
Female: 670 mg/kg; No. of animals: 5; Sacrifice times: 24 hours
Female: 2000 mg/kg; No. of animals: 5; Sacrifice times: 24 hours
Female: 2000 mg/kg; No. of animals: 5; Sacrifice times: 48 hours

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide (CPA)

Tissues and cell types examined:

The marrow of the femora;
Two types of erythrocytes were observed in the bone marrow smears: normochromatic (mature red blood cells about to pass into the blood stream) and polychromatic (immature red blood cells).

Details of tissue and slide preparation:

Preparation of the Animals:
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 syringe. The cell suspension was centrifuged at 1500 rpm (390 x g) for 10 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-64293 Darmstadt)/Giemsa (Gurr, BDH Limited Poole, Great Britain). Cover slips were mounted with EUKITT (KINDLER, D-79110 Freiburg). At least one slide was made from each bone marrow sample.

Analysis of Cells:
Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. 2000 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 2000 the PCEs. The analysis was performed with coded slides.

Evaluation criteria:

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

Statistics:

Non-parametric Mann-Whitney test.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Doses producing toxicity: In the pretest: 2000 mg/kg (reduction of spontaneous activity)

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Micronuclei in polychromatic erythrocytes (PCE) and relationship PCE/ total erythrocytes scoring 24 and 48 hours after treatment

Test group	Dose (mg/kg b.w)	Sampling time (h)	PCEs with micronuclei (%)	Range	PCE/NCE
Vehicle	0	24	0.105	1-5	2000/1610
Test article	200	24	0.095	0-7	2000/1734
Test article	670	24	0.055	0-3	2000/1750
Test article	2000	24	0.040	0-4	2000/1860
Cyclo-phosphamide	40	24	1.580	11 - 43	2000/2008
Test article	2000	48	0.100	0-6	2000/1661

Conclusions:

The test item did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse.

Executive summary:

This study was performed to investigate the potential of FAT 40'574/B to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. This study was conducted according to OECD test guideline 474 in a GLP-certified laboratory. The test item was formulated in deionised water. Deionised water was used as vehicle control. The volume administered orally was 10 ml/kg bw.. 24 h and 48 h after a single administration of the test item the bone marrow cells were collected for micronuclei analysis. Ten animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei. 2000 polychromatic erythrocytes (PCEs) per animal were scored for micronuclei. To describe a cytotoxic effect due to the treatment with the test item the ratio between polychromatic and normochromatic erythrocytes (NCE) was determined in the same sample and reported as the number of NCEs per 2000 PCEs. The following dose levels of the test item were investigated:

24 h preparation interval: 200, 670, and 2000 mg/kg b.w..

48 h preparation interval: 2000 mg/kg b.w..

The highest dose (2000 mg/kg; maximum guideline-recommended dose) was estimated by a pre-experiment to be suitable. After treatment with the highest dose of the test item (2000 mg/kg b.w.) the number of NCEs was increased as compared to the mean value of NCEs of the vehicle control at preparation interval 24 hours thus indicating that FAT 40'574/B had cytotoxic effectiveness in the bone marrow. In comparison to the corresponding vehicle controls there was no enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test item and with any dose level used. 40 mg/kg b.w. cyclophosphamide was used as positive control which showed a substantial increase of induced micronucleus frequency. In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test item did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse. Therefore, FAT 40574/B is considered to be non-mutagenic in this micronucleus assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro genotoxicity studies:

Ames

A GLP-study was performed according to OECD test guideline 471 to investigate the potential of FAT 40'574/B to induce gene mutations according to the plate incorporation test (experiment I) and the preincubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and in addition the Escherichia coli strain WP2 uvrA. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test article was tested at the following concentrations: 33; 100; 333; 1000; 2500; and 5000 μg/plate. No relevant toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation. Slight, seemingly toxic effects occurred in strain TA 100 with metabolic activation in the first experiment. However, this effect is based upon a high solvent control value (105 colonies mean value versus 85 of the corresponding solvent control) and does represent statistical fluctuations rather than a true toxic effect. Another slight toxic effect was observed in strain TA 98 without metabolic activation in the second experiment. Since this effect was not really dose dependent, it was again judged as caused by statistical fluctuations. The plates incubated with the test article showed normal background growth up to 5000 μg/plate with and without metabolic activation in both independent experiments. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with FAT 40574/B at any dose level, neither in the presence nor absence of concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

 

Chromosome aberration V79

The test article FAT 40574/B, dissolved in deionised water, was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in two independent experiments. This study was conducted according to OECD 473 in a GLP-certified laboratory. In each experimental group two parallel cultures were set up. Per culture 100 metaphase plates were scored for structural chromosome aberrations. The highest applied concentration in the pre-test on toxicity (5000 μg/ml) was chosen with regard to current OECD Guideline 473. A significant and biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed in the presence of S9 mix at interval 28 h after treatment with 250 µg/ml. 7 % aberrant cells exclusive gaps were observed. This aberration rate exceeds the upper limit of the laboratory's historical negative control range (0 - 4 % aberrant cells exclusive gaps) and was proved to be statistically significant. In addition, the number of cells carrying exchanges was increased. At the other experimental points, no increased aberration frequency was observed, neither in the absence, nor in the presence of S9 mix. At the 28 h interval in the presence of S9 mix, additional observations for a clastogenic potential were remarked. Fragmentation of the nuclei and micronuclei were observed after treatment with 250 and 500 µg/ml, giving additional evidence for a clastogenic potential.In conclusion, it can be stated that in the study described and under the experimental conditions reported, the test article induced structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro. Therefore, FAT 40'574/B is considered to be mutagenic in this chromosome aberration test.

HPRT

In a mammalian cell gene mutation assay (HPRT locus) conducted according to OECD test guideline 476, V79 cells cultured in vitro were exposed to FAT 40574/C at concentrations of - 3.16, 10.0, 31.6, 100, 250, 500, 1500, 2000, 2500 and 2750 μg/mL (without metabolic activation, Experiment I)

- 50, 100, 250, 500, 1000, 1500, 2000, 3000 and 5000 μg/mL (with metabolic activation, Experiment I)

- 25, 50, 100, 250, 500, 1500, 2500, 3000, 4000 and 5000 μg/mL (without metabolic activation, Experiment II)

- 31.6, 100, 316, 1000, 1600, 2200, 2900, 3600, 4300 and 5000 μg/mL (with metabolic activation, Experiment II). FAT 40574/C was tested up to cytotoxic concentrations.

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 11.1 % for the highest concentration (2750 μg/mL) evaluated. The highest biologically relevant concentration evaluated with metabolic activation was 5000 μg/mL with a relative growth of 32.5 %. In experiment II without metabolic activation the relative growth was 29.6 % for the highest concentration (5000 μg/mL) evaluated. The highest concentration evaluated with metabolic activation was 5000 μg/mL with a relative growth of 39.8 %. 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 1500 μg/mL with a relative growth of 37.2%. In experiment I with metabolic activation the highest mutation rate (compared to the solvent control values) of 1.50 was found at a concentration of 3000 μg/mL with a relative growth of 64.0 %. In experiment II without metabolic activation the highest mutation rate (compared to the solvent control values) of 2.09 was found at a concentration of 250 μg/mL with a relative growth of 75.7 %. In experiment II with metabolic activation the highest mutation rate (compared to the solvent control values) of 2.29 was found at a concentration of 2200 μg/mL with a relative growth of 76.6 %. The positive controls did induce the appropriate response. There was no evidence of a concentration related positive response of induced mutant colonies over background.

 

In vivo micronucleus test

This study was performed to investigate the potential of FAT 40574/B to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. This study was conducted according to OECD test guideline 474 in a GLP-certified laboratory. The test item was formulated in deionised water. Deionised water was used as vehicle control. The volume administered orally was 10 ml/kg bw. 24 h and 48 h after a single administration of the test item the bone marrow cells were collected for micronuclei analysis. Ten animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei. 2000 polychromatic erythrocytes (PCEs) per animal were scored for micronuclei. To describe a cytotoxic effect due to the treatment with the test item the ratio between polychromatic and normochromatic erythrocytes (NCE) was determined in the same sample and reported as the number of NCEs per 2000 PCEs. The following dose levels of the test item were investigated:

24 h preparation interval: 200, 670, and 2000 mg/kg b.w..

48 h preparation interval: 2000 mg/kg b.w..

The highest dose (2000 mg/kg; maximum guideline-recommended dose) was estimated by a pre-experiment to be suitable. After treatment with the highest dose of the test item (2000 mg/kg b.w.) the number of NCEs was increased as compared to the mean value of NCEs of the vehicle control at preparation interval 24 hours thus indicating that FAT 40574/B had cytotoxic effectiveness in the bone marrow. In comparison to the corresponding vehicle controls there was no enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test item and with any dose level used. 40 mg/kg b.w. cyclophosphamide was used as positive control which showed a substantial increase of induced micronucleus frequency. In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test item did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse. Therefore, FAT 40574/B is considered to be non-mutagenic in this micronucleus assay.

 

Conclusion on genotoxicity:

Reactive Red 271 did not elicit a positive response in the bacterial reverse mutation assay and the in vitro mammalian cell gene mutations assay with V79 cells. In the mammalian chromosomal aberration assay, structural chromosomal aberrations were observed in the presence of S9 mix at 28 h interval after treatment with top concentration which had acceptable cell viability. However, the substance was not able to reproduce the clastogenic response in the in vivo micronucleus assay. Hence, Reactive Red 271 was considered to be neither mutagenic nor clastogenic.

Justification for classification or non-classification

Based on the assessment of three in vitro mutagenicity studies and one in vivo study, the test substance is not subject to classification for mutagenicity according to CLP (Regulation EC No 1272/2008).