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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Reactive Red 065 is mutagenic in Reverse Mutation Assay using Bacteria (Salmonella Typhimurium) and in in vitro Mammalian Chromosome Aberration Test. However, in In vitro Mammalian Cell Gene Mutation Test (HPRT-Locus) in Chinese Hamster V79 Cells test substance is considered to be non-mutagenic. Therefore, it has been proposed to conduct in vivo Mammalian Erythrocyte Micronucleus test.

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Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014-07-03 to 2014-07-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: 274-437-2
- Expiration date of the lot/batch: 274-437-2

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ≤-20 °C
- Solubility of the test substance in the solvent/dispersant/vehicle/test medium: 60 g/L in water at 20 °C
Target gene:
His
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
rat S9 liver microsomal fraction
Test concentrations with justification for top dose:
3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate
Vehicle / solvent:
Vehicle(s)/solvent(s) used: Aqua destillata (distilled water)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
TA 100 and TA 1535 at 10 µg/plate- without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-NOPD (4-nitro-o-phenylene-diamine)
Remarks:
TA 98 at 10 µg/plate and TA 1537 at 40 µg/plate- without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
TA 102 at 1 µL/plate- without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanththracene
Remarks:
TA 98, TA 100, TA 1535 and TA 1537 at 2.5 µg/plate and TA 102 at 10 µg/plate- with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
-For the plate incorporation method the following materials were mixed in a test tube and poured over the surface of a minimal agar plate:
100 µL Test solution at each dose level, solvent control, negative control or reference mutagen solution (positive control),
500 µL S9 mix (for testing with metabolic activation) or S9 mix substitution buffer (for testing without metabolic activation),
100 µL Bacteria suspension (cf. Preparation of Bacteria, pre-culture of the strain),
2000 µL Overlay agar.
After solidification the plates were inverted and incubated at 37 °C for at least 48 h in the dark.

NUMBER OF REPLICATIONS: For each strain and dose level, including the controls, three plates were used.

DETERMINATION OF CYTOTOXICITY
- Method: clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately ≤0.5 in relation to the solvent control
Evaluation criteria:
The Mutation Factor is calculated by dividing the mean value of the revertant counts through the mean values of the solvent control (the exact and not the rounded values are used for calculation).
A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as follows:
- if in tester strains TA 98, TA 100 and TA 102 the number of reversions is at least twice as high
- if in tester strains TA 1535 and TA 1537 the number of reversions is at least three times higher than the reversion rate of the solvent control.

Evaluation of Cytotoxicity:
Cytotoxicity can be detected by a clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately ≤0.5 in relation to the solvent control.
Statistics:
According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
No precipitation of the test item was observed in any tester strain used in experiment I and II (with and without metabolic activation). Toxic effects of the test item were noted only in tester strain TA 102 at a concentration of 5000 µg/plate (without metabolic activation). No biologically relevant increases in revertant colony numbers were noted in tester strains TA 98, TA 1535 and TA 102. Biologically relevant increases of revertant colony numbers were observed in tester strain TA 100 at concentrations of 1000 µg/plate and higher (without metabolic activation) and at a concentration of 5000 µg/plate (with metabolic activation). The threshold value of 2.0 was exceeded and a maximum mutation factor of 3.6 was reached at a concentration of 5000 µg/plate (without metabolic activation). Biologically relevant increases of revertant colony numbers were also observed in tester strain TA 1537 at a concentration of 5000 µg/plate (with metabolic activation). The threshold value of 3.0 was exceeded and a maximum mutation factor of 4.0 was reached at a concentration of 5000 µg/plate (with metabolic activation). Moreover, a dose-response relationship was found in tester strain TA 100 (with and without metabolic activation) and in tester strain TA 1537 (with metabolic activation).
Remarks on result:
other: all strains/cell types tested
Conclusions:
FAT 40062/C TE is considered to be mutagenic in this bacterial reverse mutation assay.
Executive summary:

FAT 40062/C TE was investigated for its potential to induce gene mutations according to the plate incorporation test with the Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 102. Several concentrations of the test item were used. The assay was conducted with and without metabolic activation. The concentrations, including the controls, were tested in triplicate. The following concentrations of the test item were prepared and used:

311.6, 100, 316, 1000, 2500 and 5000 µg/plate. No precipitation of the test item was observed in any tester strain used (with and without metabolic activation). Toxic effects of the test item were noted only in tester strain TA 102 at a concentration of 5000 µg/plate (without metabolic activation). The reduction in the number of revertants down to a mutation factor of 0.5 found in tester strain TA 102 at a concentration of 1000 µg/plate (with and without metabolic activation) was regarded as not biologically relevant due to lack of a dose-response relationship and lack of concomitant clearing of the background lawn. No biologically relevant increases in revertant colony numbers were noted in tester strains TA 98, TA 1535 and TA 102. Biologically relevant increases of revertant colony numbers were observed in tester strain TA 100 at concentrations of 1000 µg/plate and higher (without metabolic activation) and at a concentration of 5000 µg/plate (with metabolic activation). The threshold value of 2.0 was exceeded and a maximum mutation factor of 3.6 was reached at a concentration of 5000 µg/plate (without metabolic activation). Biologically relevant increases of revertant colony numbers were also observed in tester strain TA 1537 at a concentration of 5000 µg/plate (with metabolic activation). The threshold value of 3.0 was exceeded and a maximum mutation factor of 4.0 was reached at a concentration of 5000 µg/plate (with metabolic activation). Moreover, a dose-response relationship was found in tester strain TA 100 (with and without metabolic activation) and in tester strain TA 1537 (with metabolic activation). The reference mutagens induced a distinct increase of revertant colonies indicating the validity of the experiment. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, FAT 40062/C TE caused gene mutations by base pair changes and frameshifts in the genome of the tester strain TA 100 and TA 1537. Therefore, FAT 40062/C TE is considered to be mutagenic in this bacterial reverse mutation assay.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: other: clastogenicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014-07-31 - 2014-11-26
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:
yes
Remarks:
Concerning study plan- Final concentration of CPA from 0.83 μg/mL to 1.11 μg/mL.
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes (incl. QA statement)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany
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: SBL/027/LRB (India)
- Expiration date of the lot/batch: 05 May 2019

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ≤-20 °C
- Solubility of the test substance in the solvent/dispersant/vehicle/test medium: 60 g/L in water at 20 °C
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
Metabolic activation:
with and without
Metabolic activation system:
Liver S9 of Wistar phenobarbital and ß-naphthoflavone-induced rat liver S9 mix
Test concentrations with justification for top dose:
Pre-experiment:
with and without metabolic activation: 10, 25, 50, 100, 150, 250, 500, 1000, 2500 and 5000 µg/mL

The following concentrations were selected in the main experiments for the microscopic analyses:
Experiment I:
without metabolic activation: 75, 100, 200 and 300 µg/mL
with metabolic activation: 50, 100 and 150 µg/mL
Experiment II:
without metabolic activation: 50, 100 and 200 µg/mL
with metabolic activation: 60,120 and 160 µg/mL


Vehicle / solvent:
-Solvent used: cell culture medium
-Justification for choice of solvent: Due to the nature of the test item it was solved in cell culture medium (MEM) .
The solvent was compatible with the survival of the cells and the S9 activity.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
were treated the same way as all dose groups
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation (400 and 900 µg/mL)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
were treated the same way as all dose groups
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with metabolic activation (1.11 µg/mL)
Details on test system and experimental conditions:
TREATMENT TIME:
4 h (Experiment I with and without metabolic activation, experiment II with metabolic activation)
20 h (Experiment II without metabolic activation)

FIXATION INTERVAL: 20 h (ExperimE4 - 5 x 10E4 cells
NUMBER OF CULTURES: two cultures per concentration
NUMBER OF CELLS SCORED: 200 cells per concentration (100 cells per culture), except experiment I without mettbolic activation at concentrations of 100, 200 and 300 µg/mL and experiment II without metabolic activation at concentrations of 50, 100 and 200 µg/mL 400 cells per concentration were scored.
DETERMINATION OF CYTOTOXICITY: Mitotic index, cell count
Evaluation criteria:
There are several criteria for determining a positive result:
- a clear and dose-related increase in the number of cells with aberrations,
- a biologically relevant response for at least one of the dose groups, which is higher than the laboratory negative control range (0.0 % - 4.0 % aberrant cells without metabolic activation and 0.0 % - 4.3 % aberrant cells with metabolic activation).
Statistics:
A statistical evaluation was used as an aid for interpretation of the results. Statistical significance at the 5 % level (p < 0.05) was evaluated by the Fischer´s exact test. The p value was used as a limit in judging for significance levels in comparison with the corresponding negative control.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid

Table1:          Summary: Experiment I and II, without metabolic activation

 

Dose Group

Concentration [µg/mL]

Relative Mitotic Index [%]

Relative Cell Count
[%]

Mean %

Aberrant Cells

Historical Laboratory Negative Control Range

Precipi-tationa

Statistical Signifi-canceb

incl. Gaps

excl. Gaps

Experiment I

4 h treatment,

20 h preparation interval

C

0

100

100

3.5

2.0

0.0 % - 4.0 % aberrant cells

-

-

3

75

96

79

4.5

1.5

-

-

4

100

69

82

5.0

3.0

-

-

5

200

57

72

6.3

4.3

-

-

6

300

48

56

8.5

6.0

-

+

 

EMS

900

87

78

10.0

7.5

-

+

 

 

 

Experiment II

20 h treatment, 20 h preparation interval

C

0

100

100

2.5

1.0

0.0 % - 4.0 % aberrant cells

-

-

3

50

102

109

5.5

3.0

-

-

5

100

93

101

2.8

1.5

-

-

6

200

51

66

12.8

8.0

-

+

EMS

400

83

98

20.0

13.5

-

+

The mitotic index was determined in 1000 cells per culture of each test group.

The cell count was determined by a cell counter per culture for each test group.

The relative values of the mitotic index and cell count are related to the negative controls.

 

C:       Negative Control (Culture Medium)

EMS:   Ethylmethanesulfonate

a:        - without precipitation, + with precipitation

b:       statistical significant increase compared to negative controls (Fisher’s exact test, p< 0.05), + significant; - not significant

Table2:          Summary: Experiment I and II, with metabolic activation

 

Dose Group

Concentration [µg/mL]

Relative Mitotic Index
 [%]

Relative Cell Count
[%]

Mean %

Aberrant Cells

Historical Laboratory Negative Control Range

Precipi-tationa

Statistical Signifi-canceb

incl. Gaps

excl. Gaps

Experiment I

4 h treatment,

20 h preparation interval

C

0

100

100

1.5

1.5

0.0 % - 4.3 % aberrant cells

-

-

2

50

105

81

4.5

2.0

-

-

3

100

114

72

4.0

2.5

-

-

4

150

66

49

4.5

3.0

-

-

CPA

1.11

74

85

8.0

6.0

-

+

 

 

 

Experiment II

4 h treatment, 20 h preparation interval

C

0

100

100

7.0

4.0

0.0 % - 4.3 % aberrant cells

-

-

2

60

102

79

6.0

3.5

-

-

4

120

120

63

4.5

2.0

-

-

5

160

95

39

9.5

7.5

-

-

CPA

1.11

110

83

17.0

15.0

-

+

The mitotic index was determined in 1000 cells per culture of each test group.

The cell count was determined by a cell counter per culture for each test group.

The relative values of the mitotic index and cell count are related to the negative controls.

 

C:       Negative Control (Culture Medium)

CPA:   Cyclophosphamide

a:        - without precipitation, + with precipitation

b:        statistical significant increase compared to negative controls (Fishers exact test, p< 0.05), + significant; - not significant

Conclusions:
FAT 40062/C TE is considered to be clastogenic in this chromosome aberration test.
Executive summary:

An in vitro Mammalian Chromosome Aberration Test was carried out with FAT 40062/C TE according to OECD guideline 473 and EU Methos B.10 to assess its potential to induce structural chromosome aberrations in Chinese hamster V79 cells. Independent experiments were carried out with and without the addition of liver S9 mix from rats (exogenous metabolic activation). The metaphases were prepared at approximately 20 h after start of treatment with the test item. The treatment interval was 4 h without and with metabolic activation in experiment I. In experiment II, the treatment interval was 20 h without and 4 h with metabolic activation. Duplicate cultures were treated at each concentration and 100 metaphases per culture were scored for structural chromosomal aberrations (for exceptions, see Tables). The metaphases were prepared around 20 h after start of treatment with the test item. Based on the results of the solubility test MEM cell culture medium was used as solvent (MEM + 0 % FBS). The following concentrations were evaluated for the microscopic analysis of chromosomal aberrations:

Experiment I:

without metabolic activation: 75, 100, 200 and 300 µg/mL

with metabolic activation: 50, 100 and 150 µg/mL

Experiment II:

without metabolic activation: 50, 100 and 200 µg/mL

with metabolic activation: 60, 120 and 160 µg/mL

In all experiments no precipitation of the test item was detected without and with metabolic activation at all concentrations evaluated. In experiment I without metabolic activation, cytotoxic effects of the test item were determined at concentrations of 100 µg/mL and higher considering the relative mitotic index. The cell count was biologically relevant decreased at the highest evaluated concentration of 300 µg/mL. With metabolic activation cytotoxic effects of the test item were determined at the highest test item concentration of 150 µg/mL considering the relative mitotic index and cell count. In experiment II without metabolic activation, cytotoxic effects of the test item were observed at the highest test item concentration of 200 µg/mL considering the relative mitotic index and cell count. With metabolic activation, no cytotoxic effects of the test item were observed considering the relative mitotic index. However, considering the relative cell count cytotoxic effects were observed at concentrations of 120 µg/mL and higher. In experiment I and II, concentration-related and/or reproducible increases of aberrant cells were observed at evaluated concentrations in comparison to the negative control. These increases of aberrant cells were considered as biologically relevant. Statistical methods were used as an aid in evaluating the test results and the analysis confirms a significantly increases of aberrant cells in experiment I and II compared to the negative control. In all experiments vehicle controls gave frequencies of aberrations within the range expected for the V79 cell line. Both positive controls, EMS and CPA induced distinct and biologically relevant increases in the number of cells containing structural chromosomal aberrations. No biologically relevant increase in the frequencies of polyploid cells was determined after treatment with the test item both without or with metabolic activation. According to the results of the present study, the test substance did lead to a biologically relevant increase in the number of cells with chromosome aberration. The positive controls induced the appropriate responses. So, FAT 40062/C TE induced structural chromosomal aberrations in the V79 Chinese hamster cell line and therefore, FAT 40062/C TE is considered to be clastogenic in this chromosome aberration test.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014-07-23 to 2014-09-19
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:
SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: SBL/027/LRB (India)
- Expiration date of the lot/batch: 05 May 2019

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ≤-20 °C
- Solubility of the test substance in the solvent/dispersant/vehicle/test medium: 60 g/L at 20 °C
Target gene:
hypoxanthine-guanine-phosphoribosyl-transferase (HPRT)
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
-Type and identity of media: MEM
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
Liver S9 of Wistar Phenobarbital and ß-Naphthoflavone-induced rat liver S9 mix
Test concentrations with justification for top dose:
Pre-experiment for experiment I (with and without metabolic activation):
10, 25, 50, 100, 250, 500, 1000, 2000, 3500 and 5000 µg/mL
Pre-experiment for experiment II (only without metabolic activation, 20 h long-term exposure assay):
25, 50, 100, 250, 500, 750, 1000, 1250, 2500 and 5000 µg/mL
Experiment I
without metabolic activation: 5, 10, 20, 50, 100, 200, 300 and 400 µg/mL
and with metabolic activation: 10, 20, 50, 100, 200, 300, 400, 500 und 750 µg/mL
Experiment II
without metabolic activation: 25, 50, 100, 150, 200, 225, 250, 300 and 350 µg/mL
and with metabolic activation: 15, 30, 60, 120, 200, 280, 360, 440, 520 and 600 µg/mL
Vehicle / solvent:
Vehicle (Solvent) used: Cell culture medium (MEM + 0 % FBS 4h treatment; MEM + 10 % FBS 20 h treatment) was used as solvent.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation; 300 µg/mL
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
with metabolic activation; 0.8 and 1.0 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: dissolved in 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: ≥ 200 μg/mL; Experiment I with S9: ≥ 300 μg/mL; Experiment II without S9: ≥ 150 μg/mL; Experiment II with S9:≥ 200 μg/mL
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Conclusions:
FAT 40062/C TE is considered to be non-mutagenic in the HPRT locus using V79 cells of the Chinese Hamster.
Executive summary:

FAT 40062/C TE was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster according to OECD 476 guideline and EU Method B.17. The main experiments were carried out with and without metabolic activation. The experiments with metabolic activation were performed by including liver microsomes and NADP for efficient detection of a wide variety of carcinogens requiring metabolic activation. In experiment I, 400 μg/mL (without metabolic activation) and 750 μg/mL (with metabolic activation) were selected as the highest concentrations. In experiment II, 350 μg/mL (without metabolic activation) and 600 μg/mL (with metabolic activation) were selected as the highest concentrations. 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 was performed as 20 h long time exposure assay (without metabolic activation). The pH-value detected with the test item was within the physiological range (7.0 ± 0.4).  

The test item was investigated at the following concentrations:

Experiment I

Without metabolic activation:

5, 10, 20, 50, 100, 200, 300 and 400 μg/mL

and with metabolic activation:

10, 20, 50, 100, 200, 300, 400, 500 und 750 μg/mL

 

Experiment II

Without metabolic activation:

25, 50, 100, 150, 200, 225, 250, 300 and 350 μg/mL

And with metabolic activation:

15, 30, 60, 120, 200, 280, 360, 440, 520 and 600 μg/mL

 

Precipitation:

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

 

Toxicity:

A biologically relevant growth inhibition (reduction of relative growth below 70 %) was observed after the treatment with the test item in experiment I and II with and without metabolic activation. In experiment I without metabolic activation, the relative growth was 11.6 % for the highest concentration (400 μg/mL) evaluated. The highest biologically relevant concentration evaluated with metabolic activation was 750 μg/mL with a relative growth of 12.8%.

In experiment II without metabolic activation, the relative growth was 13.3 % for the highest concentration (350 μg/mL) evaluated. The highest concentration evaluated with metabolic activation was 600 μg/mL with a relative growth of 15.5%.

 

Mutagenicity:

In experiment I without metabolic activation most mutant values of the negative controls and test item concentrations found were within the historical control data of the test facility BSL BIOSERVICE (about 2-43 mutants per 106 cells). No dose-response relationship could be observed. The mutation frequencies found in the groups treated with the test item did not show a biologically relevant increase as compared to the negative controls. Mutation frequencies with the negative control were found to be 7.37, 28.44 and 17.79 mutants/106 cells and in the range of 15.43 to 47.67 mutants/106 cells with the test item, respectively. The highest mutation rate (compared to the negative control values) of 2.67 was found at a concentration of 200 μg/mL with a relative growth of 45.4 %. With metabolic activation most mutant values of the negative controls, and test item concentrations found were within the historical control data of the test facility BSL BIOSERVICE (about 5-44 mutants per 106 cells). No dose-response relationship could be observed. The mutation frequencies found in the groups treated with the test item did not show a biologically relevant increase as compared to the negative controls. Mutation frequencies with the negative control were found to be 20.56, 27.07 and 32.00 mutants/106 cells and in the range of 14.61 to 54.60 mutants/106 cells with the test item, respectively. The highest mutation rate (compared to the negative control values) of 2.06 was found at a concentration of 750 μg/mL with a relative growth of 12.8 %. In experiment II without metabolic activation, most mutant values of the negative controls and test item concentrations found were within the historical control data of the test facility BSL BIOSERVICE (about 2-43 mutants per 106cells). No dose-response relationship could be observed. The mutation frequencies found in the groups treated with the test item did not show a biologically relevant increase as compared to the negative controls. Mutation frequencies with the negative control were found to be 29.81 and 29.28 mutants/106 cells and in the range of 25.45 to 57.79 mutants/10E6 cells with the test item, respectively. The highest mutation rate (compared to the negative control values) of 1.96 was found at a concentration of 350 μg/mL with a relative growth of 13.3 %.

In experiment II with metabolic activation, most mutant values of the negative controls, and test item concentrations found were within the historical control data of the test facility BSL BIOSERVICE (about 5-44 mutants per 106 cells). No dose-response relationship could be observed. The mutation frequencies found in the groups treated with the test item did not show a biologically relevant increase as compared to the negative controls. Mutation frequencies with the negative control were found to be 33.33 and 28.07 mutants/106 cells and in the range of 11.11 to 68.83 mutants/106 cells with the test item, respectively. The highest mutation rate (compared to the negative control values) of 2.24 was found at a concentration of 440 μg/mL with a relative growth of 17.1 %. DMBA (0.8 and 1.0 μg/mL) and EMS (300 μg/mL) were used as positive controls and showed distinct and biologically relevant effects in mutation frequency. In conclusion, in the described in vitro cell gene mutagenicity test under the experimental conditions reported, the test item FAT 40062/C TE is considered to be non-mutagenic in the HPRT locus using V79 cells of the Chinese Hamster.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Testing proposal for in-vivo micronucleus test.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study planned
Study period:
6 months after ECHA's final decision
Justification for type of information:
TESTING PROPOSAL ON VERTEBRATE ANIMALS
Hazard endpoint for which vertebrate testing was proposed: Genetic toxicity in vivo with the registered substance.

NON-CONFIDENTIAL NAME OF SUBSTANCE:
- Name of the substance on which testing is proposed to be carried out: Reactive Red 65

CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
- Available GLP studies: There are no available GLP studies on the substance or on read-across analogues suitable to fill the endpoint.

- Available non-GLP studies: There are no available non-GLP studies on the substance or on read-across analogues suitable to fill the endpoint.

- Historical human data: There is no historical human data on the substance or on read-across analogues suitable to fill the endpoint.

- (Q)SAR: (Q)SAR analysis is not sufficient to fill the endpoint. There are no adequate models to address this endpoint.

- In vitro methods: Already available, but further in vivo data needed. Available study: OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

- Weight of evidence: There is not sufficient data on the substance or read across analogues to be able to establish a weight of evidence argument.

- Grouping and read-across: There is not sufficient data on the substance or read-across analogues to be able to group or propose read-across.

- Substance-tailored exposure driven testing [if applicable]: Not applicable
- Approaches in addition to above [if applicable]: Not applicable
- Other reasons [if applicable]: Not applicable

CONSIDERATIONS THAT THE SPECIFIC ADAPTATION POSSIBILITIES OF ANNEXES VI TO X (AND COLUMN 2 THEREOF) OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
- Test proposal is fully in line with ECHA guidance document*, and can neither be replaced by in vitro testing nor by using other data from other substances.
* Chapter R.7a: Endpoint specific guidance Version 4.1 – October 2015

FURTHER INFORMATION ON TESTING PROPOSAL IN ADDITION TO INFORMATION PROVIDED IN THE MATERIALS AND METHODS SECTION:
- Details on study design / methodology proposed: OECD Guideline 474: Mammalian Erythrocyte Micronucleus Test
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Executive summary:

The Registrant proposes to conduct the following test by oral route using the indicated test method:

EU Method B.12 Mutagenicity -In Vivo Mammalian Erythrocyte Micronucleus test or the equivalent OECD Guideline 474 – Mammalian Erythrocyte Micronucleus test

The Registrant has assessed genotoxicity of FAT 40062/B TE in three in vitro systems.

In Reverse Mutation Assay using Bacteria (Salmonella Typhimurium) FAT 40062/B exerted a mutagenic effect in this test system.

In vitro Mammalian Chromosome Aberration Test, FAT 40062/B TE induced structural chromosome aberrations in the V79 Chinese hamster cell line in the absence and the presence of metabolic activation.

In vitro Mammalian Cell Gene Mutation Test (HPRT-Locus) in Chinese Hamster V79 Cells, FAT 40062/B TE and its metabolites did not show any mutagenic activity in this forward mutation system.

According to REACH Annex IX 8.4 Column 2, if there is a positive result in any of the in vitro genotoxicity studies in Annex VII or VIII and there are no results available from an in vivo study already, an appropriate in vivo somatic cell genotoxicity study shall be proposed by the Registrant. The information on this endpoint is not available for the registered substance but needs to be present in the technical dossier to meet the data requirements. Consequently, there is a data gap and it is necessary to generate the data for this endpoint.

Endpoint conclusion
Endpoint conclusion:
no study available (further information necessary)

Additional information

Genotoxicity of FAT 40062/C TE was assessed in three in vitro systems. In Reverse Mutation Assay using Bacteria (Salmonella Typhimurium) FAT 40062/C TE caused gene mutations by base pair changes and frameshifts in the genome of the tester strain TA 100 and TA 1537. In a Chromosomal Aberration test in Chinese Hamster V79 cells, FAT 40062/C TE induced structural chromosomal aberrations in the V79 cell line. However, in an in vitro Mammalian Cell Gene Mutation Test (HPRT-Locus) in Chinese Hamster V79 Cells, FAT 40062/C is non-mutagenic. According to REACH Annex IX 8.4 Column 2, if there is a positive result in any of the in vitro genotoxicity studies in Annex VII or VIII and there are no results available from an in vivo study already, an appropriate in vivo somatic cell genotoxicity study shall be proposed by the Registrant. The information on this endpoint is not available for the registered substance but needs to be present in the technical dossier to meet the data requirements. Consequently, there is a data gap and it is necessary to generate the data for this endpoint.

Justification for classification or non-classification

Based on the absence of adverse effect in the in-vitro genetic toxicity studies, Reactive Red 065 is considered not to classify for genotoxicity in accordance to the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008. However, a testing proposal to perform an in vivo micronucleus study is made. The final classification will be proposed after completion of this test. According to REACH Annex IX 8.4 Column 2, if there is a positive result in any of the in vitro genotoxicity studies in Annex VII or VIII and there are no results available from an in vivo study already, an appropriate in vivo somatic cell genotoxicity study shall be proposed by the Registrant. Therefore, the registrant proposes to conduct the following tests using the indicated test method: In Vivo Mammalian Erythrocyte Micronucleus test (EU Method B.12 Mutagenicity) or the equivalent Mammalian Erythrocyte Micronucleus test OECD Guideline 474).