2-{4-[4-[4-fluoro-6-(2-(2-vinylsulfonylethoxy)ethylamino)-1,3,5-triazin-2-ylamino]phenylazo]phenylazo}naphthalene-4,6,8-trisulfonate, trisodium salt

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Reactive Yellow 210 did not induce mutations in bacterial as well as mammalian cells in vitro. It was found to induce chromosomal aberrations in vitro when tested with V79 CHO cells. However, it failed to reproduce similar effect in the in vivo micronucleus assay, hence was considered to be not clastogenic. Thus, Reactive Yellow 210 is considered to be not genotoxic.

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:

09 August 2001 to 25 October 2001

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:

not specified

Qualifier:

according to guideline

Guideline:

other: Japanese Guideline: Kanpoan No. 287 - Environment Protection Agency

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

other: Japanese Guideline: Eisei No. 127 - Ministry of Health & Welfare

Qualifier:

according to guideline

Guideline:

other: Japanese Guideline: Heisei 09/10/31 Kikyoku No. 2 - Ministry of International Trade & Industry

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Identification: FAT 40800/B
Description: dark orange powder
Batch number: REM1
Purity: approx. 70 %
Stability of test item: Stable under storage conditions;
Expiration date: February 14, 2007
Stability in solvent: 24 hours in water, saline, polyethylene glycol, CMC, Vaseline, and FCA
Storage conditions: at room temperature

Target gene:

Histidine for the S. typhimurium strains and Tryptophan for the E.coli strain

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital/beta-naphthoflavone induced rat liver S9 mix

Test concentrations with justification for top dose:

33; 100; 333; 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:

sodium azide

Remarks:

Strain 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:

Strain TA 1537, TA 98, without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Strain WP2, without metabolic activation

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, with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar

DURATION
- Preincubation period: 60 minutes at 37 °C
- After solidification the plates were incubated upside down for at least 48 hours at 37 °C in the dark.

DETERMINATION OF CYTOTOXICITY: Toxic effects were evidenced by a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.

NUMBER OF REPLICATIONS: Two independent experiments performed in triplicate

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed. A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration. An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment. A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

No statistical evaluation of the data is required.

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 1537

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 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 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

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

Additional information on results:

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in both experiments. No relevant toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation.
No substantial and reproducible increase in revertant colony numbers of any of the five tester strains was observed following treatment with FAT 40800/A at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). A moderate but not strictly dose dependent increase of the number of revertant colonies was observed in strain TA 98 in the first experiment with metabolic activation. The colony count exceeded the historical range of negative and solvent controls somewhat at 33 - 333 µg/plate. At 333 µg/plate the threshold of twice the colony count of the corresponding solvent control was just exceeded (factor of 2.1 versus a threshold of 2.0) However, compared to the corresponding negative control a factor of only 1.5 was calculated. Furthermore this effect was not reproduced in the second assay. Therefore, this irreproducible increase was judged to be based upon fluctuations rather than indicating a possible mutagenic potential. There was also no reproducible 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. The positive control somewhat exceeded the historical range of positive controls in strains TA 1535 and TA 100 of the first experiment without metabolic activation. However, this effect indicates the sensitivity of the strains rather than compromising the validity of the assay. The positive control of strain TA 1535 in the second experiment with metabolic activation fell just short of the lower border of the historical range (61 colonies versus 68-511). Since the threshold of thrice the corresponding solvent control was clearly exceeded this minor deviation was also judged as biologically irrelevant.

Conclusions:

FAT 40800/A did not show any mutagenic activity in any strain.

Executive summary:

In a GLP-compliant Ames test, performed according to OECD guideline 471, 4 Salmonella typhimurium strains (TA 1535, TA 1537, TA 98, and TA 100) and one E. Coli strain (WP2 uvrA) were used to test the mutagenic potential of the test substance (33, 100, 333, 1000, 2500, and 5000 µg per plate), in two independent experiments (each in triplicate), both with and without metabolic activation. The test substance did not show any mutagenic activity in any strain and no toxic effects were observed. Therefore, the test substance is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 December 2001 to 01 March 2002

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:

not specified

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

other: Japanese Guideline: Kanpoan No. 287 - Environment Protection Agency

Qualifier:

according to guideline

Guideline:

other: Japanese Guideline: Eisei No. 127 - Ministry of Health & Welfare

Qualifier:

according to guideline

Guideline:

other: Japanese Guideline: Heisei 09/10/31 Kikyoku No. 2 - Ministry of International Trade & Industry

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

Identification: FAT 40800/B
Description: dark orange powder
Batch number: REM1'
Purity: approx. 70 %
Stability of test item: Stable under storage conditions;
Expiration date: August 14, 2007
Stability in solvent: 24 hours in water, saline, polyethylene glycol, CMC, Vaseline, and FCA
Storage conditions: at room temperature

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Before freezing each batch was screened for mycoplasm contamination and checked for karyotype stability.

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital/beta-naphthoflavone induced rat liver S9 mix

Test concentrations with justification for top dose:

See 'any other information on materials and methods'

Vehicle / solvent:

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:

METHOD OF APPLICATION: in medium

DURATION: Two independent experiments were performed. ln Experiment I, the exposure period was 4 hrs with and without metabolic activation. ln Experiment II the exposure period was 4 hrs with S9 mix and 18 hrs and 28 hrs without S9 mix. The chromosomes were prepared 18 hrs (Exp. I and II) and 28 hrs (Exp. II) after start of treatment with the test item.

NUMBER OF CELLS EVALUATED: 100 well spread metaphase plates per culture were scored for cytogenetic damage on coded slides. Only metaphases with characteristic chromosome numbers of 22 ± 1 were included in the analysis.

DETERMINATION OF CYTOTOXICITY
- To describe a cytotoxic effect the mitotic index (% cells in mitosis) was determined. In addition, the number of polyploid cells was determined (% polyploid metaphases; in the case of this aneuploid cell line polyploid means a near tetraploid karyotype).

Evaluation criteria:

A test item is classified as non-clastogenic 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).
and/or
- no significant increase of the number of structural chromosome aberrations is observed.
A test item is classified as clastogenic 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).
and
- either a concentration-related or a significant increase of the number of structural chromosome aberrations is observed.
If the criteria mentioned above for the test item 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. Although the inclusion of the structural chromosome aberrations is the purpose of this study, it is important to include the polyploids and endoreduplications. The following criteria is valid: A test item can be classified as aneugenic if:
- the number of induced numerical aberrations are not in the range of our historical control data (0.0 % - 8.5 % polyploid cells).

Statistics:

Statistical significance was confirmed by means of the Fisher's exact test (p < 0.05). However, both biological and statistical significance should be considered together.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS GENOTOXICITY:
- In both experiments, in the absence and presence of S9 mix, nor statistically significant neither biologically relevant increases in the number of cells carrying structural chromosome aberrations were observed, except after 18 hrs and 28 hrs continuous treatment in the absence of S9 mix. In the presence of S9 mix, the aberration rates of the cells after treatment with the test item (0.5 % - 2.5 % aberrant cells, exclusive gaps) were near the solvent control values (1.5 % aberrant cells, exclusive gaps) and within the range of our historical control data: 0.0 % - 4.0 % aberrant cells exclusive gaps. In the absence of S9 mix, the aberration rates of the cells after 4 hrs and 18 hrs treatment with the test item (0.0 % - 2.5 % aberrant cells, exclusive gaps) were near the range of the solvent control values (0.0 % - 1.0 % aberrant cells, exclusive gaps) and within the range of our historical control data: 0.0 % - 4.0 % aberrant cells exclusive gaps. However, after 18 hrs continuous treatment in the absence of S9 mix a single statistically significant value without biological relevance was observed with 250 µg/mL. At preparation interval 28 hrs in the absence of S9 mix the aberration rate (12.5%) was statistically significant and biologically relevant increased after 28 hrs continuous treatment with 250 µg/ml as compared to the corresponding solvent control (0.5 %). Also, the number of cells carrying exchanges was distinctly increased (7.0 %) as compared to the corresponding solvent control (0.0 %). These observations give additional evidence for a clastogenic potential of the test item. Therefore, these findings have to be regarded as biologically relevant.
- In both experiments, no biologically relevant increase in the rate of polyploid metaphases was found after treatment with the test item (1.1 % - 4.1 %) as compared to the rates of the solvent controls (1.0 % - 3.3 %), except in experiment II after 28 hrs continuous treatment in the absence of S9 mix with 250 µg/mL distinctly increased numbers of polyploid metaphases (9.3 %) were scored as compared to the corresponding solvent control (3.1 %). The rate of polyploid metaphases slightly exceeded our historical control range (0.0 % - 8.5 % polyploid cells). In addition, after 28 hrs continuous treatment with 250 µg/mL numerous micronuclei and nucleus fragments were observed. These findings give evidence for an aneugenic potential of the test item.

TEST-SPECIFIC CONFOUNDING FACTORS:
-In the pre-experiment, precipitation of the test item in culture medium was observed 4 hrs after start of treatment with 900 µg/mL and above in the absence of S9 mix and with 225 µg/mL and above in the presence of S9 mix.
- In the main experiments, precipitation of the test item 4 hrs after start of treatment was observed in the absence of S9 mix with 500 µg/mL and above (exp. II -18 hrs treatment-) or 750 µg/mL and above (exp. I & exp. II -28 hrs treatment-). In the presence of S9 mix precipitation occured with 62.5 µg/mL and above at 18 hrs preparation interval and with 125 µg/mL and above at 28 hrs preparation interval.
- In main experiment I no influence of the test item on the pH value or osmolarity was observed (solvent control 286 mOsm, pH 7.4 versus 302 mOsm, pH 7.2 at 1000 µg/mL).

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- In a range finding pre-test on toxicity cell numbers 24 hrs after start of treatment were scored as an indicator for cytotoxicity. Concentrations between 56.3 and 7200 µg/mL were applied. Clear toxic effects were observed after 4 hrs treatment with 450 µg/mL and above in the absence and with 900 µg/mL and above in the presence of S9 mix. In addition, 24 hrs continuous treatment with 450 µg/mL and above in the absence of S9 mix induced strong toxic effects.
- In the cytogenetic experiments, clear toxic effects indicated by reduced cell numbers below 50 % of control were observed in the experimental parts with continuous treatment in the absence of S9 mix. In detail, reduced cell numbers were observed in the absence of S9 mix after 18 hrs treatment with 375 µg/mL and 28 hrs treatment with 250 µg/mL (38 % and 40 % of control). Additionally, the mitotic indices were slightly reduced at these test groups (56 % and 53 % of control, respectively). Slight cytotoxicity indicated by decreased cell numbers was observed at the concentrations evaluated after 4 hrs treatment (62 % of control) in the absence of S9 mix and after 4 hrs treatment at 18 hrs and 28 hrs preparation interval (79 % and 63 % of control, respectively) in the presence of S9 mix. Due to strong test item precipitation on the slides higher concentrations in the respective experimental parts were not evaluable.

Conclusions:

FAT 40800/A is considered to be clastogenic in the chromosomal aberration test.

Executive summary:

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. 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 (7200 µg/ml) was chosen according to the current OECD Guideline with respect to the molecular weight and the purity (approx. 70 %) of FAT 40'800/B. Dose selection of the cytogenetic experiments was performed considering the toxicity data and the occurrence of precipitation. Clear toxic effects indicated by reduced cell numbers were observed after 18 hrs and 28 hrs continuous treatment with the test item in the absence of S9 mix. No relevant toxicity was observed at the concentrations evaluated after 4 hrs treatment in the absence of S9 mix and after 4 hrs treatment at 18 hrs and 28 hrs preparation interval in the presence of S9 mix. Due to strong test item precipitation on the slides higher concentrations in the respective experimental parts were not evaluable. In experiment II, in the absence of S9 mix, a statistically significant and biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after 28 hrs continuous treatment with the test item. Also a distinct increase in the frequencies of polyploid metaphases was found in the absence of S9 mix after 28 hrs continuous treatment. Appropriate mutagens were used as positive controls. They induced statistically significant increases (p <0.05) in cells with structural chromosome aberrations. In conclusion, the test item induced structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro and therefore, FAT 40800/B is considered to be clastogenic in this chromosome aberration test in the absence of S9 mix.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 December 2012 to 28 March 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:

mammalian cell gene mutation assay

Specific details on test material used for the study:

Name: FAT 40800/A TE
Batch No.: WP 2/01
Physical State: solid
Colour: dark orange
Storage Conditions: at room temperature 7.37
Purity: 84.79 % sum of all coloured substances, mam constituent: 62.18 %
Expiry Date: 12 July 2012

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

Test concentrations with justification for top dose:

Pre-experiment for experiment I (with and without metabolic activation): 50, 100, 250, 500, 750, 1000, 1500, 2000, 2500 and 5000 µg/mL
Pre-experiment for experiment II (only without metabolic activation, 20 h long-term exposure assay): 25, 50, 75, 100, 250, 500, 750 and 1000 µg/mL
Experiment I without metabolic activation: 0.5, 1.0, 2.5, 5.0, 10.0, 20.0, 40.0, 60.0 and 80.0 µg/mL
Experiment I with metabolic activation: 200, 300, 400, 500, 600, 700, 800, 850 and 900 µg/mL
Experiment II without metabolic activation: 50, 100, 200, 300, 400, 450, 500, 550, 600 and 700 µg/mL
Experiment II with metabolic activation: 35, 70, 145, 290, 650, 790, 860 and 930 µg/mL

Vehicle / solvent:

Vehicle (Solvent) used: For the pre-experiment the test item was dissolved in cell culture medium (MEM + 0% FBS 4h treatment).

For the main experiments and the pre-experiment for experiment II (without metabolic activation) 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

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

with metabolic activation; 1.0 and 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.

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: ≥ 10.0 μg/mL; experiment I with S9: ≥ 500 μg/mL; Experiment II without S9: ≥ 300 μg/mL; Experiment II with S9:≥ 650 μg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

FAT 40800/A is considered to be non-mutagenic in the HPRT locus using V79 cells of the Chinese Hamster.

Executive summary:

In GLP-compliant study, FAT 40800/A TE was assessed for its potential to induce mutations at the HPRT locus using V79 cells of the Chinese Hamster according to OECD guideline 476. The selection of the concentrations was based on data from the pre-experiments. Experiment I without and with 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. The test item was investigated at the following concentrations:

Experiment I

without metabolic activation:

0.5, 1.0, 2.5, 5.0, 10.0, 20,0, 40,0, 60.0 and 80.0 µg/mL

and with metabolic activation:

200, 300, 400, 500, 600, 700, 800, 850 and 900 µg/mL

Experiment II

without metabolic activation:

50, 100,200,300,400,450,500,550,600 and 700 µg/mL

and with metabolic activation:

35, 70, 145, 290, 650, 790, 860 and 930 µg/mL

 

No precipitation of the test item was noted in the experiments. Biologically relevant growth inhibition was observed in experiment I and II without and with metabolic activation. In experiment I without metabolic activation, the relative growth was 13.4 % for the highest concentration (80.0 µg/mL) evaluated. The highest biologically relevant concentration evaluated with metabolic activation was 900 µg/mL with a relative growth of 18.1 %. In experiment II without metabolic activation, the relative growth was 18.6% for the highest concentration (700 µg/mL) evaluated. The highest concentration evaluated with metabolic activation was 930 µg/mL with a relative growth of 14.5 %. In both experiments no biologically relevant increase of mutants was found after treatment with the test item (without and with metabolic activation). No dose-response relationship was observed. DMBA and EMS were used as positive controls and showed distinct and biologically relevant effects in mutation frequency. In conclusion, FAT 40800/A 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

The test item did not induce micronuclei in the bone marrow cells of the mouse in the in-vivo 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:

10 April 2001 to 02 July 2001

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)

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Specific details on test material used for the study:

Identification: FAT 40800/B
Description: dark orange powder
Batch number: REM1
Purity: approx. 70 %
Stability of test item: Stable under storage conditions;
Expiration date: August 14, 2007
Stability in solvent: 24 hours in water, saline, polyethylene glycol, CMC, Vaseline, and FCA
Storage conditions: at room temperature

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: RCC Ltd., Biotechnology and Animal Breeding Division; CH-4414 Füllinsdorf
- Age at study initiation: 8-10 weeks
- Weight at study initiation: Males 34.4g (SD ± 3.1 g), Females: 26.7 g (SD ± 2.2 g)
- Assigned to test groups randomly: yes
- Fasting period before study: 18 hours
- Housing: Individually, in Makrolon Type I, with wire mesh top (EHRET GmbH, D-79302 Emmendingen) with granulated soft wood bedding (ALTROMIN, D-32791 Lage/Lippe).
- Diet: pelleted standard diet, ad libitum (ALTROMIN 1324, D-32791 Lage/Lippe)
- Water: tap water, ad libitum, (Gemeindewerke, D-64380 Roßdorf)
- Acclimation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 4
- Humidity (%): 22 - 70
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

water

Frequency of treatment:

Single treatment

Post exposure period:

24 hours for all doses, 48 hours for the 2000 mg/kg bw dose group.

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

Negative control

Dose / conc.:

500 mg/kg bw/day (actual dose received)

Remarks:

Low dose

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

Medium dose

Dose / conc.:

2 000 mg/kg bw/day (actual dose received)

Remarks:

High dose

No. of animals per sex per dose:

6

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide;
- Route of administration: orally
- Doses: 40 mg/kg bw
- Volume administrated: 10 ml/kg bw

Tissues and cell types examined:

Normochromatic and polychromatic erythrocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
- A preliminary study on acute toxicity was performed with two animals per sex under identical conditions as in the mutagenicity study concerning: starvation period, animal strain; vehicle; route, frequency, and volume of administration. The animals were treated orally with the test item and examined for acute toxic symptoms at intervals of around 1 h, 2-4 h, 6 h, 24 h, 30 h, and 48 h after administration of the test item.
- The maximum tolerated dose level is determined to be the dose that causes toxic reactions without having major effects on survival within 48 hours.
- Three adequate spaced dose levels spaced by a factor of 2 were applied at the central sampling interval 24 h after treatment. For the highest dose level an additional sample was taken at 48 h after treatment.

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 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-Griinwald (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.

METHOD OF ANALYSIS:
- Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. At least 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 PCEs. The analysis was performed with coded slides.
Ten animals (5 males, 5 females) per test group were evaluated as described.

Evaluation criteria:

A test item is classified as mutagenic if it induces either a dose-related increase or a clear increase in the number of micronucleated polychromatic erythrocytes in a single dose group. A test item that fails to produce a biological relevant increase in the number of micronucleated polychromatic erythrocytes is considered non-mutagenic in this system.

Statistics:

Statistical methods (nonparametric Mann-Whitney test) will be used as an aid in evaluating the results. However, the primary point of consideratrion is the biological relevance of the results.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

animals treated with 2000 mg/kg b.w.

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

The mean number of normochromatic erythrocytes was not increased after treatment with the test item as compared to the mean value of NCEs of the vehicle control indicating that FAT 40800/B had no cytotoxic properties in the bone marrow. In comparison to the corresponding vehicle controls there was no statistically significant or biologically relevant enhancement in the frequency of the detected micronuclei at any preparation interval and dose level after administration of the test item. The mean values of micronuclei observed after treatment with FAT 40800/B were below or near to the value of the vehicle control group. 40 mg/kg b.w. cyclophosphamide administered orally was used as positive control which showed a statistically significant increase of induced micronucleus frequency.

In pre-experiment the animals treated with 2000 mg/kg b.w. expressed toxic reactions ruffed fur and on the basis of these data 2000 mg/kg b.w. were estimated to be suitable. In the main experiment for the highest dose group 24 animals (12 males, 12 females) received orally a single dose of 2000 mg /kg b.w. FAT 40800/B formulated in deionized water. The volume administered was 10 ml/kg b.w.. The animals treated with 2000 mg /kg b.w. expressed toxic reactions such as reduction of spontaneous activity, abdominal position, eyelid closure, ruffled fur and apathy. 

Conclusions:

The test substance did not induce micronuclei in bone marrow cells of the mouse.

Executive summary:

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 (500, 1000, 2000 mg/kg bw) dissolved in water followed by a 24 or 48 hours post exposure period. Ten animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei and at least 2000 polychromatic erythrocytes (PCEs) per animal were scored. To describe a cytotoxic effect due to the treatment with the test item the ratio between polychromatic and normochromatic erythrocytes (NCEs) was determined in the same sample and reported as the number of NCEs per 2000 PCEs. As estimated by a pre-experiment 2000 mg FAT 40800/B per kg b.w. (the maximum guideline-recommended dose) was suitable. The mean number of normochromatic erythrocytes was not increased after treatment with the test item as compared to the mean value of NCEs of the vehicle control indicating that FAT 40800/B had no cytotoxic properties in the bone marrow. In comparison to the corresponding vehicle controls there was no statistically significant or biologically relevant enhancement in the frequency of the detected micronuclei at any preparation interval and dose level after administration of the test item. The mean values of micronuclei observed after treatment with FAT 40800/B were below or near to the value of the vehicle control group. 40 mg/kg b.w. cyclophosphamide administered orally was used as positive control which showed a statistically significant increase of induced micronucleus frequency. In conclusion, the test item did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro:

In a GLPcompliant Ames test, performed according to OECD guideline 471, 4 Salmonella typhimurium strains (TA 1535, TA 1537, TA 98, and TA 100) and one E. Coli strain (WP2 uvrA) were used to test the mutagenic potential of the test substance (33, 100, 333, 1000, 2500, and 5000 µg per plate), in two independent experiments (each in triplicate), both with and without metabolic activation (RCC 2001). The test substance did not show any mutagenic activity in any strain and no toxic effects were observed.

 

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 (RCC 2002). In the absence of S9 mix, a statistically significant and biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after 28 hrs continuous treatment with the test substance. Also a distinct increase in the frequencies of polyploid metaphases was found in the absence of S9 mix after 28 hrs continuous treatment. In conclusion, it can be stated that under the experimental conditions reported, the test substance is considered to be clastogenic in this chromosomal aberration test.

 

In a mammalian cell HPRT gene mutation assay, tested according to OECD guideline 476 and GLP, Chinese hamster lung fibroblast (V79) were exposed to the test substance with and without metabolic activation (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 4 h short-term exposure assay. Experiment II without metabolic activation was performed as 20 h long time exposure assay. The test item was investigated at the following concentrations: Experiment I without metabolic activation: 0.5, 1.0, 2.5, 10.0, 20.0, 40.0, 60.0 and 80.0 µg/mL; Experiment I with metabolic activation: 200, 300, 400, 500, 600, 700, 800, 850 and 900 µg/mL; Experiment II without metabolic activation: 50, 100, 200, 300, 400, 450, 500, 550, 600, and 700 µg/mL; Experiment II with metabolic activation: 35, 70, 145, 290, 650, 790, 860 and 930 µg/mL. No precipitation was noted. 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 13.4 % for 80 µg/mL evaluated. The highest biologically relevant concentration evaluated with metabolic activation was 900 µg/mL with a relative growth of 18.1 %. In experiment II without metabolic activation the relative growth was 18.6 % for the highest concentration (700 µg/mL) evaluated. The highest concentration evaluated with metabolic activation was 930 µg/mL with a relative growth of 14.5 %. 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 and biologically relevant effects in mutation frequency. Therefore, the test substance is considered to be non-mutagenic in the mammalian cell gene mutation assay.

 

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 (500, 1000, 2000 mg/kg bw) dissolved in water followed by a 24 or 48 hours post exposure period (RCC 2002). Ten animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei and at least 2000 polychromatic erythrocytes (PCEs) per animal were scored. The mean number of normochromatic erythrocytes (NCEs) was not increased after treatment with the test substance as compared to the mean value of NCEs of the vehicle control indicating that the test substance has no cytotoxic properties in the bone marrow. In comparison to the corresponding vehicle controls there was no statistically significant or biologically relevant enhancement in the frequency of the detected micronuclei at any preparation interval and dose level after administration of the test substance. The mean values of micronuclei observed after treatment with the test substance were below or near to the value of the vehicle control group. Therefore, it can be stated that during the study described and under the experimental conditions reported, the test item did not induce micronuclei in the bone marrow cells of the mouse.

 

Conclusion:

Reactive Yellow 210 did not induce mutations in bacterial as well as mammalian cells in vitro. It was found to induce chromosomal aberrations in vitro when tested with V79 CHO cells. However, it failed to reproduce similar effect in the in vivo micronucleus assay, hence was considered to be not clastogenic. Thus, Reactive Yellow 210 is considered to be not genotoxic.

Justification for classification or non-classification

Based on the findings of the genetic toxicity studies, the test substance does not considered to be classified according to the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.