Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Based on the read-across data generated on source substance, Reactive Red 024:1 is also considered to be not mutagenic.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Nov 1979
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Justification for type of information:
Refer chapter 13 for the detailed analogue justification.
Qualifier:
according to guideline
Guideline:
other: AMES, B.N., J. McCANN, and E. YAMASAKI (1975), Methods for Detecting Carcinogens and Mutagens with the Salmonella/ Mammalian-Microsome Mutagenicity Test. Mut. Res. 31, 347-364.
Deviations:
not specified
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine auxotrophic strains of Salmonella typhimurium
Species / strain / cell type:
other: Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction of liver from rats induced with Arochlor and cofactors
Test concentrations with justification for top dose:
Pre-test: without and with microsomal activation: 15, 45, 135, 405 and 1215 µg/0.1 ml.
Repetition test: without and with microsomal activation: 25, 75, 225, 6 75 and 2025 µg/0.1 ml
Vehicle / solvent:
phosphate buffer
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
Without metabolic activation: TA 100
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: daunorubicin-HCl
Remarks:
Without metabolic activation: TA 98
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: N-methyl-N1-nitro-N-nitrosoguanidine
Remarks:
Without metabolic activation: TA 1535
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 9(5)aminoacridine hydrochloride monohydrate
Remarks:
Without metabolic activation: TA 1537
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
Without metabolic activation: TA 1538
Details on test system and experimental conditions:
The tests were carried out in accordance with the method described by AMES et al. The bacteria on which the tests were performed were the histidineauxotrophic TA 98, TA 100, TA 1535, TA 15 37 and TA 1538, strains of Salmonella typhimurium.
The test was performed with the following concentrations of the trial substance without and with microsomal activation: 15, 45, 135, 405 and 1215 microg/0.1 ml. A repetition of the experiments was performed with the concentrations of 25, 75, 225, 6 75 and 2025 microg/ 0.1 ml. In these experiments tests on Strain TA 15 38 were included. The substance was dissolved in phosphate buffer. Phosphate buffer alone was used for the negative controls. In the experiments in which the substance was metabolically activated, activation mixture was added also. 1 ml activation mixture contains: 0.3 ml S9 fraction of liver from rats induced with Aroclor 1254 and 0.7 ml of a solution of co-factors.

Positive control experiments were carried out simultaneously with the following substances: 1) for Strain TA 98: daunorubicin-HCl (DAUNOBLASTINR), 5 and 10 microg/0.1 ml phosphate buffer; 2) for Strain TA 100: 4-nitroquinoline-N-oxide, 0.125 and 0.25 microg/0.1 ml phosphate buffer; 3) for Strain TA 1535: N-methyl-N1-nitro-N-nitrosoguanidine, 3 and 5 microg/0.1 ml phosphate buffer; 4) for Strain TA 15 37: 9(5) aminoacridine hydrochloride monohydrate, 50 and 100 microg/0.1 ml DMSO; 5) for Strain TA 15 38: 2-nitrofluorene, 5 and 10 microg/0.1 ml DMSO. The activation mixture was tested with Strain TA 15 35 and cyclophosphamide (ENDOXAN-ASTA ), 250 microg/0.1 ml phosphate buffer.
In the experiments with and without the addition of microsomal activation mixture three Petri dishes were prepared per strain and per group (i.e. per concentration or per control group).
The plates were incubated for about 48 hours at 37 deg C in darkness. When the colonies had been counted, the arithmetic mean was calculated.
A test substance is generally considered to be non-mutagenic if the colony count in relation to the negative control is not doubled at any concentration .

1.AMESf B.N., F.D. LEE, and W.E. DURSTON (1973), An Improved Bacterial Test System for the Detection and Classification of Mutagens and Carcinogens. Proc. Natl. Acad. Sei. USA 70, 782-786.
2.AMES, B.N., W.E. DURSTON, E. YAMASAKI, and F.D. LEE (1973), Carcinogens are Mutagens: A Simple Test System Combining Liver Homogenates for Activation and Bacteria for Detection.
Proc. Natl. Acad. Sei. USA 7_0, 2281-2285.
3.AMES, B.N., J. McCANN, and E. YAMASAKI (1975), Methods for Detecting Carcinogens and Mutagens with the Salmonella/ Mammalian-Microsome Mutagenicity Test. Mut. Res. 31, 347-364.
Rationale for test conditions:
None
Evaluation criteria:
None
Statistics:
None
Key result
Species / strain:
other: TA 1535, TA 1537, TA 98, TA 1538 and 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
Additional information on results:
In the experiments performed with and without microsomal activation, comparison of the number of histidine-prototrophic mutants in the controls and after treatment with FAT 40034/B revealed no marked differences.
Remarks on result:
other: all strains/cell types tested
Conclusions:
FAT 40034/B was determined to be non-mutagenic in the bacterial reverse mutation assay.
Executive summary:

FAT 40034/B was tested for mutagenic effects on histidine-auxotrophic mutants of Salmonella typhimurium which included TA 98, TA 100, TA 1535, TA 15 37 and TA 1538. The investigations were performed with the following concentrations of the trial substance with and without microsomal activation: 15, 45, 135, 405 and 1215 µg/0.1 ml. A repetition of the experiments was performed with the concentrations of 25, 75, 225, 675 and 2025 µg/0.1 ml. In these experiments tests on Strain TA 1538 were included. In the experiments performed with and without microsomal activation, comparison of the number of back-mutant colonies in the controls and the cultures treated with the various concentrations of FAT 40034/B revealed no marked deviations. Based on the findings of the study, no evidence of the induction of point mutations by FAT 40034/B or by the metabolites of the substance formed as a result of microsomal activation was detectable in the strains of S. typhimurium used in these experiments. Hence, FAT 40034/B was determined to be non-mutagenic in the bacterial reverse mutation assay.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010-01-20 till 2010-06-14
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:
other: Japanese Guidelines: "Kanpoan No. 287 - Environmental Agency", "Eisei No. 127 - Ministry of Health & Welfare", and "Heisei 0911 0131 Kikyoku No. 2 - Ministry of International Trade & Industry"
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
German GLP
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: TZ 5978 /BOP 07-09
- Expiration date of the lot/batch: July 31, 2014

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
Target gene:
This in vitro test was performed to assess the potential of test material TE to induce structural chromosome aberrations in V79 cells (cell-line from the lung of the Chinese Hamster) in the absence and presence of metabolic activation.
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media:MEM (minimal essential medium) containing Hank’s salts, 10 % (v/v) fetal bovine serum (FBS), Neomycin (5 µg/mL) and Amphotericin B (2.5 µg/mL)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: no data
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital/meta-naphthoflavone induced rat liver S9 mix
Test concentrations with justification for top dose:
Exposure period 4 h without S9 mix: 19.5, 39.1, 78.1 µg/mL
Exposure period 18 h without S9 mix: 180, 210, 240, 270, 300 µg/mL
Exposure period 4 h with S9 mix (experiment 1): 39.1, 78.1, 156.3 µg/mL
Exposure period 4 h with S9 mix (experiment 2): 100, 150, 210 µg/mL
Vehicle / solvent:
The test item was dissolved in 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:
DURATION
- Exposure duration: 4 h and 18 h
- Expression time (cells in growth medium): after 4 h incubation: 14 h, after 18 h incubation: cells were directly processed at the end of the incubation period
- Fixation time (start of exposure up to fixation or harvest of cells): 18 h

SPINDLE INHIBITOR (cytogenetic assays): Colcemid (0.2 µg/mL)
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: At least 100 well spread metaphases per culture were evaluated for cytogenetic damage on coded slides.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: The number of polyploid cells in 500 metaphases per culture was determined (% polyploid metaphases; in the case of this aneuploid cell line polyploid means a near tetraploid karyotype).
Evaluation criteria:
ACCEPTABILITY OF THE TEST
The chromosome aberration test performed in our laboratory is considered acceptable, if it meets the following criteria:
a) The number of structural aberrations found in the solvent controls falls within the range of the laboratory’s historical control data.
b) The positive control substances produce significant increases in the number of cells with structural chromosome aberrations, which are within the range of the laboratory’s historical control data.

EVALUATION OF RESULTS
A test item is classified as non-clastogenic if:
- the number of induced structural chromosome aberrations in all evaluated dose groups is in the range of the laboratory’s historical control data range, and
- 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 is not in the range of the laboratory’s historical control data range, and
- either a concentration-related or a significant increase of the number of structural chromosome aberrations is observed.

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. 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 criterion is valid:
A test item can be classified as aneugenic if the number of induced numerical aberrations is not in the range of the laboratory’s historical control data range.
Statistics:
Statistical significance was confirmed by means of the Fisher’s exact test (p <0.05).
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
after 18h incubation time
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with
Genotoxicity:
ambiguous
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Neither test item precipitation nor relevant influence of the test item on pH value or osmolarity was observed.
Toxic effects indicated by reduced mitotic indices were observed in Experiment I after treatment with 78.1 µg/mL (35.2 % of control) in the absence of S9 mix and in Experiment II after treatment with 210.0 µg/mL (52.9 % of control) in the presence of S9 mix. In addition, in Experiment I in the absence of S9 mix the cell numbers were reduced to 47.2 % of control after treatment with 78.1 µg/mL. In all other experimental parts no cytotoxicity was observed up to the highest evaluable test item concentrations. However, higher concentrations could not be evaluated in Experiment I in the presence of S9 mix.
In Experiment I in the absence and presence of S9 mix no biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. The number of aberrant cells, excluding gaps (1.5 – 4.0 %) was within the total laboratory´s historical solvent control data range (0.0 – 4.0 % aberrant cells, excluding gaps).
In Experiment II in the absence of S9 mix following continuous treatment, statistically significant increases in the number of aberrant cells, excluding gaps were observed at all evaluated concentrations in the range of 180.0 to 300.0 µg/mL. As all values exceeded the total laboratory´s historical solvent control data range (0.0 – 4.0 % aberrant cells, excluding gaps) this observation is considered to be biologically relevant. In the presence of S9 mix statistically significant increases in the number of aberrant cells, excluding gaps (2.0 %) were observed at all evaluated concentrations. Since the percentages of aberrant cells, excluding gaps were within the range of the laboratory´s historical solvent control data (0.0 – 3.5 % aberrant cells, excluding gaps) this observation is regarded as biologically irrelevant.
No biologically relevant increase in the rate of polyploid metaphases was found after treatment with the test item (1.0 - 3.3 %) as compared to the rates of the solvent controls (2.6 - 4.3 %).
In both experiments, either ethylmethylsulfonate (400 or 1000 µg/mL) or cyclophosphamid A (1.4 µg/mL) were used as positive controls and showed distinct increases in the number of cells with structural chromosome aberrations.
Remarks on result:
other: all strains/cell types tested

Summary of results of the chromosome aberration study with the test material; preparation interval: 18h for all experiments

Test item concentration [µg/mL]

Polyploid cells [%]

Cell numbers [% of control]

Mitotic indices [% of control]

Aberrant cells [%]

incl. gaps *

excl. gaps *

with exchanges

Exposure period 4h, -S9

Solvent control1

2.9

100.0

100.0

3.5

2.5

0.0

Positive control2

1.8

n.t.

105.0

18.0

17.5S

6.5

19.5

3.3

92.5

86.2

3.0

3.0

0.5

39.1

1.0

70.2

85.8

3.5

3.5

0.5

78.1

2.3

47.2

35.2

5.0

4.0

0.0

Exposure period 18h, -S9

Solvent control1

4.3

100.0

100.0

0.0

0.0

0.0

Positive control3

2.3

n.t.

78.5

14.5

14.0S

4.0

180

2.4

90.1

94.8

5.0

4.5S

0.5

210 #

1.6

112.6

79.3

6.0

5.3S

0.0

240

1.5

101.1

109.3

9.5

8.0S

1.0

270

2.2

118.0

84.8

9.5

8.5S

3.0

300 #

2.5

95.3

96.3

7.5

6.3S

1.0

Exposure period 4h, +S9

Solvent control1

2.6

100.0

100.0

1.5

1.5

0.5

Positive control4

2.7

n.t.

69.9

14.5

14.0S

4.5

39.1

2.4

109.8

106.2

2.0

1.5

0.5

78.1

2.7

100.8

104.8

2.5

2.5

1.0

156.3

2.5

102.8

74.2

4.5

4.0

0.5

Solvent control1

2.8

100.0

100.0

0.0

0.0

0.0

Positive control4

3.9

n.t.

71.5

12.0

12.0S

1.0

100

2.6

103.2

99.4

2.5

2.0S

0.5

150

2.9

89.0

67.0

2.5

2.0S

0.5

210

2.4

95.5

52.9

2.5

2.0S

0.5

* Inclusive cells carrying exchanges

# Evaluation of 200 metaphases per culture

n.t. Not tested

SAberration frequency statistically significant higher than corresponding control values

1Deionised water 10.0 % (v/v)

2Ethylmethylsulfonate 1000 µg/mL

3Ethylmethylsulfonate 400 µg/mL

4Cyclophosphamide A 1.4 µg/mL

 

Conclusions:
FAT 40850/A is considered to be clastogenic in the chromosome aberration test in the absence of metabolic activation.
Executive summary:

FAT 40850 was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro according to OECD guideline 473 and EU Method B.10. In each experimental group two parallel cultures were set up. At least 100 metaphases per culture were evaluated for structural chromosome aberrations. The highest applied concentration in Experiment I (5000 µg/mL) was chosen with respect to the current OECD Guideline 473. Purity of the test item was not taken into account for dose selection. Dose selection for the cytogenetic experiments was performed considering the toxicity data. The evaluation of chromosomal aberration was limited by cytotoxicity. In Experiment I in the absence of S9 mix and in Experiment II in the presence of S9 mix clear cytotoxicity of about 50 % was observed at the highest evaluated concentration. In Experiment I in the presence of S9 mix higher concentrations could not be evaluated due to strong test item cytotoxicity. In Experiment I and in Experiment II in the presence of S9 mix no biologically relevant clastogenicity was observed at the concentrations evaluated either with or without metabolic activation. The percentages of aberrant cells, excluding gaps were within the total laboratory´s historical solvent control data range (0.0 – 4.0 % aberrant cells, excluding gaps). In Experiment II in the absence of S9 mix statistically significant increases in the number of aberrant cells, excluding gaps were observed at all evaluated concentrations in the range of 180 to 300 µg/mL. All values exceeded the laboratory´s historical solvent control data range (0.0 – 3.5 % aberrant cells, excluding gaps). Therefore, the occurrence of chromosomal aberrations is considered biologically relevant. No increase in polyploid metaphases was noticed after treatment with the test item as compared to 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 under the experimental conditions reported, the test iteminduced structural chromosome aberrations in V79 cells (Chinese hamster cell line) in vitro. Therefore, FAT 40850/A is considered to be clastogenic in this chromosome aberration test in the absence of metabolic activation.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
HPRT
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010-02-23 till 2010-05-21
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:
other: Japanese Guidelines: "Kanpoan No. 287 - Environment Protection Agency", "Eisei No. 127 - Ministry of Health & Welfare", and "Heisei 09710/31 Kikyoku No. 2 - Ministry of International Trade & Industry"
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
German GLP
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:
SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: TZ 5978 /BOP 07-09
- Expiration date of the lot/batch: July 31, 2014

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
Target gene:
HPRT (hypoxanthine-guanine phosphoribosyl transferase)
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: MEM (minimal essential medium) supplemented with 10 % fetal bovine serum (FBS) and 1 % neomycin.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/β-naphtoflavone induced rat liver S9 mix
Test concentrations with justification for top dose:
Experiment I:
without S9 mix: 5.0; 10.0; 20.0; 40.0; and 60.0 μg/mL
with S9 mix: 20.0; 40.0; 80.0; 160.0; and 240 μg/mL

Experiment II:
without S9 mix: 25.0; 50.0; 100; 200; and 400 μg/mL
with S9 mix: 25.0; 50.0; 100; 200; and 300 μg/mL
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:
7,12-dimethylbenzanthracene
Remarks:
with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: without metabolic activation: 4 and 24h; with metabolic activation: 4h
- Expression time (cells in growth medium): 7d
- Selection time (if incubation with a selection agent): 8d

SELECTION AGENT (mutation assays): 6-thioguanine
STAIN: methylene blue

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
Evaluation criteria:
ACCEPTABILITY OF THE ASSAY
The gene mutation assay is considered acceptable if it meets the following criteria:
- the numbers of mutant colonies per 106 cells found in the solvent controls fall within the laboratory historical control data range.
- the positive control substances must produce a significant increase in mutant colony frequencies.
- the cloning efficiency II (absolute value) of the solvent controls must exceed 50 %.

EVALUATION OF RESULTS
A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points. A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.
A positive response is described as follows:
A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concentrations in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
However, in a case by case evaluation this decision depends on the level of the corresponding solvent control data. If there is by chance a low spontaneous mutation rate within the laboratory´s historical control data range, a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of solvent controls within all experiments of this study was also taken into consideration.
Statistics:
A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT®11 (SYSTAT Software, Inc., 501, Canal Boulevard, Suite C, Richmond, CA 94804, USA) statistics software. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance were considered together.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
No relevant and reproducible increase in mutant colony numbers was observed in the main experiments up to the maximal concentration. The induction factor exceeded the threshold of three times the corresponding solvent control in the first culture of experiment II at 400 μg/mL without metabolic activation. However, this increase was not reproduced in the parallel culture under identical conditions or in the first experiment without metabolic activation. Furthermore, the increase was not dose dependent as indicated by the lacking statistical significance. In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 11.5 up to 30.7 mutants per 10E6 cells; the range of the groups treated with the test item was from 6.9 up to 87.6 mutants per 10E6 cells. No precipitation occurred up to the maximum concentration with and without metabolic activation. There was no relevant shift of pH and osmolarity of the medium even in the stock solution of the test item. EMS (150 μg/mL in experiment I and 130 μg/mL in experiment II) and DMBA (1.1 μg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.
Remarks on result:
other: all strains/cell types tested

Summary of mutagenicity data of V79 cells after incubation with test material

 

Concentration [µg/mL]

S9 mix

Culture I

Culture II

mutant colonies/106 cells

induction factor

mutant colonies/106 cells

induction factor

Experiment 1, 4h treatment

Solvent control1

 

-

30.7

1.0

29.5

1.0

Positive control2

150

-

174.2

5.7

135.3

4.6

test material

5

-

32.7

1.1

31.1

1.1

 

10

-

30.5

1.0

21.7

0.7

 

20

-

35.8

1.2

31.4

1.1

 

40

-

19.3

0.6

27.9

0.9

 

60

-

26.4

0.9

37.3

1.3

Solvent control1

 

+

24.0

1.0

11.5

1.0

Positive control3

1.1

+

754.3

31.4

690.8

60.0

 

20

+

19.6

0.8

24.8

2.2

 

40

+

11.3

0.5

23.4

2.0

 

80

+

23.2

1.0

13.2

1.1

 

160

+

12.7

0.5

12.1

1.1

 

240

+

18.8

0.8

15.9

1.4

Experiment 2, 24h treatment

Solvent control1

 

-

20.1

1.0

20.1

1.0

Positive control2

130

-

383.8

19.1

393.7

19.6

test material

25

-

43.1

2.1

culture not continued#

 

50

-

29.7

1.5

25.3

1.3

 

100

-

17.4

0.9

15.0

0.7

 

200

-

27.4

1.4

33.2

1.7

 

400

-

87.6

4.4

27.9

1.4

 

600

-

culture not continued##

14.8

0.7

Experiment 2, 4h treatment

Solvent control1

 

+

13.2

1.0

15.5

1.0

Positive control3

1.1

+

1391.0

105.5

1384.7

89.6

test material

25

+

13.3

1.0

culture not continued#

 

50

+

18.7

1.4

12.1

0.8

 

100

+

11.4

0.9

16.9

1.1

 

200

+

24.5

1.9

8.5

0.6

 

300

+

27.5

2.1

12.8

0.8

 

400

+

culture not continued##

6.9

0.4

# culture not continued since a minimum of only 4 analysable concentrations is required

## culture not continued due to exceedingly strong toxic effects

1 water

2 ethylmethylsulfonate

3 7.12-dimethylbenzanthracene

 

Conclusions:
FAT 40850/A is considered to be non-mutagenic in this HPRT assay.
Executive summary:

The study was performed to investigate the potential of FAT 40850/A to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster according to OECD Guideline 476 and EU Method B17. The study was performed in two independent experiments, using identical experimental procedures. In the first experiment the treatment period was 4 hours with and without metabolic activation. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.

The cell cultures were evaluated at the following concentrations:

Experiment I: without S9 mix: 5-60 μg/mL, with S9 mix: 20-240 μg/mL;

Experiment II: without S9 mix: 25-400 μg/mL, with S9 mix: 25-300 μg/mL.

No precipitation of the test item was observed up to the maximal concentration in all experiments. No effects of osmolarity or pH were observed. Relevant cytotoxic effects occurred at 40 μg/mL and above in experiment I without metabolic activation. In the second experiment cytotoxic effects were noted at 400 μg/mL and above without metabolic activation and 300 μg/mL and above with metabolic activation. The data are nevertheless acceptable since the corresponding cell density at the first subcultivation after treatment remained above the limit of 10 %. No relevant and reproducible increase in mutant colony numbers/106 cells was observed in the main experiments up to the maximal concentration. The induction factor exceeded the threshold of three times the corresponding solvent control in the first culture of experiment II at 400 μg/mL without metabolic activation. However, this increase was not reproduced in the parallel culture under identical conditions or in the first experiment without metabolic activation. Furthermore, the increase was not dose dependent as indicated by the lacking statistical significance. A linear regression analysis was performed to assess a possible dose dependent increase of mutant frequency. A single significant dose dependent trend of the mutation frequency was determined in the second experiment at culture I with metabolic activation. However, the trend was judged as biologically irrelevant since the mutation frequency did not exceed the threshold described above and all of the individual values remained within the historical range of solvent controls. In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 11.5 up to 30.7 mutants per 106 cells; the range of the groups treated with the test item was from 6.9 up to 87.6 mutants per 106 cells. Ethylmethylsulfonate (150 μg/mL in experiment I and 130 μg/mL in experiment II) and 7.12-dimethylbenzanthracene (1.1 μg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies. In conclusion, it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells and is considered to be non-mutagenic in the HPRT assay.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Read-across substance, FAT 40850 did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse.

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:
2010-07-29 to 2010-09-29
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
as at 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
as at 2008
GLP compliance:
yes (incl. QA statement)
Remarks:
German GLP
Type of assay:
mammalian erythrocyte micronucleus test
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: TZ5978 / BOP 07-09
- Expiration date of the lot/batch: July 31, 2014
-
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
Species:
mouse
Strain:
NMRI
Details on species / strain selection:
The mouse is an animal that has been used for many years as a suitable experimental animal in cytogenetic investigations. There are many data available from such investigations, which may be helpful in the interpretation of results from the micronucleus test. In addition, the mouse is an experimental animal in many physiological, pharmacological and toxicological studies. Data from such experiments also may be useful for the design and the performance of the micronucleus test
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories B.V., Postbus 6174, 5960 AD Horst / The Netherlands
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: males mean value = 36.7 g (SD ± 1.5 g)
- Fasting period before study: no
- Housing: singly in Makrolon Type II/III cages with wire mesh top (EHRET GmbH, 79302 Emmendingen, Germany) and granulated soft wood bedding (Rettenmaier & Söhne GmbH + Co. KG,73494 Rosenberg, Germany)
- Diet: pelleted standard diet, ad libitum(Harlan Laboratories B.V. Postbus 6174, 5960 AD Horst, The Netherlands)
- Water: tap water, ad libitum
- Acclimation period: >= 5 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 45 - 100
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: water
- Source: B. Braun Melsungen AG 34212 Melsungen, Germany
- Catalogue no.: 6724092.00.00
- Justification for choice of solvent/vehicle: chosen for its relative non-toxicity for animals
- Concentration of test material in vehicle: 50, 100, 200 mg/mL
- Amount of vehicle (if gavage or dermal): 10 mL/kg
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in in the vehicle (sterile water).
Duration of treatment / exposure:
single oral gavage on Day 1
Frequency of treatment:
single oral gavage
Post exposure period:
no
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control
Dose / conc.:
500 mg/kg bw/day (nominal)
Remarks:
Low dose
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
Middle dose
Dose / conc.:
2 000 mg/kg bw/day (nominal)
Remarks:
High dose
Dose / conc.:
2 000 mg/kg bw/day (nominal)
Remarks:
Positive Control
No. of animals per sex per dose:
- main test: 7 males per dose (only males were used in the main study as the toxicity pretest showed comparable susceptibility of males and females)
- toxicity pre-test: 2 males and 2 females per dose
Control animals:
yes, concurrent vehicle
Positive control(s):
none; no data; cyclophosphamide (CPA)
- Supplier: Fisher Scientific GmbH, 61130 Nidderau, Germany
- Justification for choice of positive control(s): recommended as positive control substance in the OECD guideline
- Route of administration: oral gavage
- Doses / concentrations: 40 mg/Kg bw (concentration in vehicle (water): 4 mg/mL)
Tissues and cell types examined:
bone marrow cells
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
2000 mg/kg bw is the limit dose according to the OECD guideline. Two lower doses with a spacing factor of 2 were selected.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
- single aoral treatment
- Sampling:
- pre-test: examination for acute toxic symptoms 1 h, 2-4 h, 6 h, 24 h, 30 h, and 48 h after administration
- main test: examination for acute toxic symptoms 1 h, 2-4 h, 6 h, 24 h (and 48 h) after administration; animals of all dose levels sacrificed at 24 h post dosing, an additional high dose group sacrificed 48 post treatment

DETAILS OF SLIDE PREPARATION:
- sacrifices of animals using CO2 followed by bleeding
- removal of the femora, cutting off of epiphyses, marrow flushed out with foetal calf serum using a syringe
- centrifugation of the cell suspension at 1500 rpm (390 x g) for 10 minutes
- discarding of supernatant, resuspesion of the remaining cell pellet and spreading of a small drop of the suspension on a slide
- air drying of the slide and staining with May-Grünwald (Merck, 64293 Darmstadt, Germany)/Giemsa (Merck, 64293 Darmstadt, Germany)
- mounting of cover slips with EUKITT (Kindler, 79110 Freiburg, Germany)
- preparation of at least one slide per each bone marrow sample

METHOD OF ANALYSIS:
- slide analysis using NIKON microscopes with 100x oil immersion objectives
- analysis for micronuclei of 2000 polychromatic erythrocytes (PCE) per animal
- determination of ratio between polychromatic and normochromatic erythrocytes in the same sample for detection of cytotoxicity
- cytotoxicity expressed in polychromatic erythrocytes per 2000 erythrocytes
- slides were coded
- use of samples from all animals per test group

OTHER:
Evaluation criteria:
The study was considered valid as the following criteria are met:
- at least 5 animals per group can be evaluated.
- PCE to erythrocyte ratio should not be less than 20 % of the negative control.
- the positive control shows a statistically significant and biological relevant increase of
micronucleated PCEs compared to the negative control.

Positive result:
If the test item induces either a dose-related increase or a clear increase in the number of micronucleated polychromatic erythrocytes in a single dose group. Statistical methods (nonparametric Mann-Whitney test) will be used as an aid in evaluating the results. However, the primary point of consideration is the biological relevance of the results.

Negative result
If the test item that fails to produce a biological relevant increase in the number of micronucleated polychromatic erythrocytes it is considered non-mutagenic in this system.
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Remarks:
The animals treated with 2000 mg/kg b.w. did not express any toxic reactions. However, discoloured urine was observed (orange) in animals treated with 2000 mg/kg bw both in pre-test and main experiment.
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000 mg/kg bw
- Solubility: no problems with solubility reported
- Clinical signs of toxicity in test animals:
- no adverse effects
- orange discoloration of the urine, indicating bioavailability of the test item which is a red dye
- Evidence of cytotoxicity in tissue analyzed: no
- Rationale for exposure: 2000 mg/kg bw is the limit dose recommended in the OECD guideline.


RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels (for Cytogenetic or SCE assay): not applicable
- Induction of micronuclei (for Micronucleus assay):
- No micronuclei induced in any of the treatment groups
- Micronuclei induced in the positive control group at the expected rate, indicating the sensitivity of the test system
- for details see Tables 2 and 3
- Ratio of PCE/NCE (for Micronucleus assay):
- not significantly changed in any of the treatment groups
- for details see Tables 2 and 3
- Appropriateness of dose levels and route:
- orange discoloration of the urine in the highest dose group, indicating bioavailability of the test item which is a red dye
- testing up to the limit dose
- Statistical evaluation: non-parametric Mann-Whitney test

Table 2: Summary of Micronucleus Test Results

Test group

Dose mg/kg b.w.

Sampling time (h)

PCEs with micronuclei (%)

range

PCE per 2000 erythocytes

Vehicle

0

24

0.079

0 - 5

1219

Test item

500

24

0.186

0 - 8

1264

Test item

1000

24

0.107

1 - 6

1177

Test item

2000

24

0.136

2 - 6

1203

Positive control

40

24

2.979

23 -82

1118

Test item

2000

48

0.057

0 - 2

1233

Table 3: Statistical significance at the five per cent level (p < 0.05, evaluated by means of the non-parametric Mann-Whitney test).

Vehicle control versus test group

Significance

p

500 mg test material/kg b.w.; 24 h

-

0.0758

1000 mg test material/kg b.w.; 24 h

-

0.2287

2000 mg test material/kg b.w.; 24 h

-

0.0807

40 mg CPA/kg b.w.; 24 h

+

0.0003

2000 mg test material/kg b.w.; 48 h

-

n.t.

-         =         not significant

+         =         significant

n.t. = not tested

Table 4: Historical controls from 2003 - 2009

 

Negative Controls Males

Positive Controls (CPA) Males

Mean* ± SD

0.096 ± 0.040

2.332 ± 0.696

Range**

0.01 - 0.22

0.70 - 4.52

No. of Experiments

318

316

*:        mean value (percent micronucleated cells)

**:      range of the mean group values (percent micronucleated cells)

Conclusions:
FAT 40850/A did not induce micronuclei as determined by the in-vivo micronucleus test in the bone marrow cells of the mouse.
Executive summary:

FAT 40850/A was assessed in the in-vivo micronucleus assay for its potential to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse according to OECD TG 474. The test item was formulated in sterile water, which was also used as vehicle control. The volume administered orally was 10 mL/kg b.w. 24 h and 48 h after a single administration of the test item the bone marrow cells were collected for micronuclei analysis.

Seven males per test group were evaluated for the occurrence of micronuclei. Per animal 2000 PCEs were scored for micronuclei. To describe a cytotoxic effect due to the treatment with the test item the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and reported as the number of PCEs per 2000 erythrocytes. The following dose levels of the test item were investigated: 24 h preparation interval: 500, 1000, and 2000 mg/kg bw; 48 h preparation interval: 2000 mg/kg bw. As estimated by a pre-experiment 2000 mg of the test material per kg b.w. (the maximum guideline-recommended dose) was suitable. The mean number of polychromatic erythrocytes was not decreased after treatment with the test item as compared to the mean value of PCEs of the vehicle control indicating that the test material did not have any cytotoxic properties in the bone marrow. However, the animals showed discoloured urine after treatment with 2000 mg/kg b.w. indicating the bioavailability of the test item. 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 the test material were near to the value of the vehicle control group. Additionally all values were within the historical vehicle control database. 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 in-vivo micronucleus test in the bone marrow cells of the mouse.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Bacterial Reverse Mutation Assay:


Data on bacterial reverse mutation assay was not available for the target substance (Reactive Red 24:1). To fill the data gaps, read across approach is adapted using similar substance Reactive Red 024. FAT 40034/B was tested for mutagenic effects on histidine-auxotrophic mutants of Salmonella typhimurium which included TA 98, TA 100, TA 1535, TA 1537 and TA 1538. There was no evidence of the induction of point mutations by FAT 40034/B or by the metabolites of the substance formed as a result of microsomal activation was detectable in the strains of S. typhimurium used in these experiments. Based on the read across data generated from the Ames assay of Reactive Red 024, the target substance (Reactive Red 024:1) is also considered to be non-mutagenic in the bacterial reverse mutation assay.


 


In vitro mammalian cell gene mutation assay with FAT 40850 (read-across)


Data on HPRT assay was not available for the target substance Reactive Red 024:1. To fill the data gaps, read-across approach is adapted using similar substance Reactive Red 286 (FAT 40850/A). Read-across is claimed basis of structural relationship of the target and the source chemicals. Read-across substance have been investigated for mutagenicity potential. The study was performed to investigate the potential of FAT 40850/A to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster according to OECD Guideline 476 and EU Method B17.  


In conclusion, based on the read-across data generated from the HPRT assay with Reactive Red 286 (FAT 40850/A), the target substance Reactive Red 024:1 is also not considered as non-mutagenic in this HPRT assay.


In vitro chromosomal aberration assay with FAT 40850 (read-across)


Data on in vitro chromosome aberration test was not available for the target substance Reactive Red 024:1. To fill the data gaps, read across approach is adapted using similar substance Reactive Red 286 (FAT 40850/A). Read-across is claimed basis of structural relationship of the target and the source chemicals. Read-across substance have been investigated for in vitro clastogenicity potential. FAT 40850 was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro according to OECD guideline 473 and EU Method B.10. In conclusion, it can be stated that under the experimental conditions reported, the test item induced structural chromosome aberrations in V79 cells (Chinese hamster cell line) in vitro. Therefore, based on the read-across data generated from in vitro chromosome aberration test with Reactive Red 286 (FAT 40850/A), the target chemical Reactive Red 024:1 is also considered to be clastogenic in chromosome aberration test in the absence of metabolic activation.


Micronucleus assay with FAT 40850 (read across)


Data on in-vivo genetic toxicity was not available for the target substance (FAT 40034). To fill the data gaps, read-across approach is adapted using similar substance Reactive Red 286 (FAT 40850/A). Read-across is claimed basis of structural relationship of the target and the source chemicals. Read-across substance have been investigated for in vivo genetic toxicity potential. FAT 40850/A was assessed in the in vivo micronucleus assay for its potential to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse according to OECD TG 474. In conclusion, based on the read-across data generated from the in vivo micronucleus test with Reactive Red 286 (FAT 40850/A), the target substance Reactive Red 024:1 is also considered not to induce micronuclei in the bone marrow cells of the mouse.


Additional information


In addition to the above data analysis a QSAR analysis was conducted for the target chemical, Reactive Red 024:1, including OECD Toolbox v 4.4.1, EPA TEST v 4.2.1, Danish QSAR DART and the QSAR model developed especially for dyes in the REACH Dyes Consortium. All models used i.e. OECD Toolbox, EPA TEST, Danish DART and Dyes Consortium QSAR predicted Reactive Red 024:1 to be Ames negative. When read-across was employed in the OECD toolbox for the endpoint ‘bacterial mutation assay with and without metabolic activation’, it also predicted the chemical to be negative based on two similar chemicals with structure similarity of 95 and 94.3 %, respectively.


Summary


In summary, test data investigating genotoxicity in mammalian cells or animals on the target substance are currently not available. Data from closely related substances EC 274-417-3, EC 700-470-3 and EC 277-616-3 displayed no alerts on mutagenicity in Ames test. Considering also the read-across results available the target chemical can be considered as not mutagenic and not clastogenic. Therefore, the target substance is considered to be not genotoxic. For reference the results of the QSAR models run are attached to chapter 13 of this dossier.

Justification for classification or non-classification

Based on the available read-across data, classification for Reactive Red 024:1 does not considered necessary for genotoxicity in accordance to the EU Classification, Labeling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.