Registration Dossier
Registration Dossier
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EC number: 403-830-5 | CAS number: 89331-94-2 B 290; BK 400; CK 34; DIBUTYL-N-102; DX-20; FAT NR. 40391/A; FLUORAN BLACK BD 869; FLUORAN SCHWARZ BD 869; NOIR FLUORANE BD 869; ODB-2; PSD-290; SENOR-2; TG-31; TH-108; WINCON-2
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
No mutagenic potential was observed in a bacterial reverse mutation assay conducted with the test substance. A mouse lymphoma assay was also negative. The test substance did not show any clastogenic activity in a chromosome aberration assay.
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 1988-02-12 to 1988-02-28
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study without detailed documentation
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Deviations:
- yes
- Remarks:
- Only 4 tester strains were used.
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Species / strain / cell type:
- S. typhimurium, other: TA 98, 100, 1535, 1537
- Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- Aroclor induced rat liver (S9 mix)
- Test concentrations with justification for top dose:
- DRF: 5, 50, 500, 5000 µg/plate with or without metabolic activation
Main test: 50, 150, 500, 1500, 5000 µg/plate with or without metabolic activation - Vehicle / solvent:
- - Vehicle used: DMSO
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-Aminoanthracene, 2-AA
- Remarks:
- with S9 mix; 2 µg/plate for strains TA 1535 and TA 1537; 0.5 µ/plate for strain TA 98; 1 µg/plate for strain TA 100
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 2-nitrofluorene
- Remarks:
- 1 µg/plate for strain TA 98, without S9 mix
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- Remarks:
- 80 µg/plate for strain TA 1537, without S9 mix
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- N-ethyl-N-nitro-N-nitrosoguanidine
- Remarks:
- Without S9 mix; 5 µg/plate for strain TA 1535; 3 µg/plate for strain TA 100.
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation)
- Cell density at seeding: approximately 2 x 10^9 organisms per mL which was assessed photometrically.
DURATION
- Exposure duration: 72 h
NUMBER OF REPLICATIONS: 2
DETERMINATION OF CYTOTOXICITY
- Method: reduction in revertant colony counts or by the absence of a complete background bacterial lawn. - Rationale for test conditions:
- According to the guideline
- Evaluation criteria:
- The mean number of revertant colonies for all treatment groups is compared with those obtained for negative and positive control groups. The effect of metabolic activation is assessed by comparing the results obtained both in the presence and absence of the liver microsomal fraction for each treatment group. A Compound is deemed to provide evidence of mutagenic potential if a statistically significant dose-related increase in the number of revertant colonies of at least twice the concurrent solvent control is obtained in two separate experiments.
- Key result
- Species / strain:
- S. typhimurium TA 1538
- Remarks:
- Please refer to study entry of YKG26 for further information
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- 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
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- Concentration of the test substance resulting in precipitation: 1500 μg/plate
- Conclusions:
- The test substance did not show any mutagenic effects in any of the tester strains.
- Executive summary:
In a reverse mutation assay in bacteria according to EU method B.13/14 (Huntingdon, 1988), four strains (TA98, TA100, TA1535, TA1537) of S. typhimurium were exposed to the test substance dissolved in DMSO at concentrations of 50 to 5000 µg/plate in the presence and absence of metabolic activation. The test substance was tested up to a limit concentration of 5000 µg/plate. Precipitation was observed at concentrations of 1500 µg/plate and above. There was no evidence or a concentration related positive response of induced mutant colonies over background. Thus, the test substance is not regarded as mutagenic.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 1988-06-15 to 1988-07-28
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study with acceptable restrictions
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian chromosome aberration test
- Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Remarks:
- K1-BH4
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: BIBRA
- Suitability of cells: The cell line has been frequently used in this test system.
- Methods for maintenance in cell culture: The cells were routinely grown and subcultured in Hams F12 medium (Imperial) supplemented with 5 % foetal calf serum (Gibco) at 37 °C in a humid atmosphere containing 5 % carbon dioxide in 175 cm² plastic tissue culture flasks (Nunc).
- Modal number of chromosomes: 20
MEDIA USED
- Type and identity of media including CO2 concentration: Hams F12 medium (Imperial) 5 %
- Properly maintained: Yes - Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- Aroclor 1254 – induced rat liver (S9 mix)
- Test concentrations with justification for top dose:
- 4, 20 and 40 μg/mL,
At 40 µg/mL, no cytotoxicity was observed in a preliminary toxicity test. However, all final concentrations greater than 40 µg/mL caused precipitate formation in aqueous tissue culture medium. 40 µg/mL is, therefore, considered to be the maximum achievable concentration. - Vehicle / solvent:
- - Vehicle used: DMSO
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- mitomycin C
- Remarks:
- 0.4 µg/L Mitomycin C (without S9 mix)
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- Remarks:
- 20 µg/L Cyclophosphamide (with S9 mix)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
- Cell density at seeding: 8 x 10^4 cells/mL
DURATION
- Exposure duration: 21 hours (without S9 mix), 4 hours (with S9 mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 21 hours
NUMBER OF REPLICATIONS: 2
DETERMINATION OF CYTOTOXICITY
- Method: relative total growth, mitotic index
OTHER EXAMINATIONS:
- Determination of polyploidy: Yes
- Determination of endoreplication: Yes - Evaluation criteria:
- The test article was considered to induce a positive response when the percentage of cells with aberrations are increased in a dose-responding manner with one or more concentrations being statistically elevated relative to the solvent control group (p<0.05).
- Statistics:
- Fisher’s exact test.
- Key result
- Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- Concentrations of above 40 µg/mL led to precipitation of the test substance.
- Conclusions:
- The test substance has shown no evidence of clastogenic activity in this in vitro cytogenetic test system.
- Executive summary:
In a mammalian cell cytogenetics assay (chromosome aberration) according to OECD guideline 473 (Huntingdon, 1989), CHO cell cultures were exposed to the test substance dissolved in DMSO at concentrations of 4, 20 and 40 µg/mL with and without metabolic activation ( Aroclor 1254 – induced rat liver). In a preliminary study the highest dose used did not produce any reduction in mitotic index. The highest dose used (40 µg/mL) was the maximum achievable concentration in the test system. The test substance was tested up to cytotoxic concentrations.
In both the presence and absence of metabolic activation, the test substance caused no statistically significant increase in the proportion of metaphase figures containing chromosomal aberrations at any dose level when compared with the solvent control. Both positive control compounds caused large, statistically highly significant increases in chromosomal damage, demonstrating the sensitivity of this test system and the efficacy of the S9 mix. It is concluded that the test substance has shown no evidence of clastogenic activity in this in vitro cytogenetic test system.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 1991-01-23 to 1991-03-06
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study with acceptable restrictions
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Principles of method if other than guideline:
- Based on the methods of Clive and Spector (1975) and Amacher et al. (1979, 1980)
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian cell gene mutation tests using the thymidine kinase gene
- Target gene:
- thymidine kinase
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- CELLS USED
- Doubling time: ca. 12 h
- Methods for maintenance in cell culture: Cells were stored in polypropylene ampules in a freezing mixture consisting of heat-inactivated horse serum (Imperial Laboratories) containing 10 % dimethylsulphoxide DMSO), at -196 °C.
MEDIA USED
- Type and identity of media including CO2 concentration: RPMI 1640, 5 % CO2
- Properly maintained: Yes
- Periodically 'cleansed' against high spontaneous background: Yes, mutants were eliminated from the cultures by a 24 hour incubation in the presence of methotrexate (0.3 µg/mL), thymidine (9 µg/mL), hypoxanthine (15 µ/mL) and glycine (22.5 µg/mL) followed by 24 hours incubation in similar medium without methotrexate, two days prior to storage at -196 °C. - Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- Aroclor 1254 induced rat liver (S9 mix)
- Test concentrations with justification for top dose:
- Preliminary toxicity test; 0.5, 1, 5, 10, 15, 30, 40, 50 µg/mL
Mutation tests: -S-9 mix
Test 1 1, 2.5, 5, 10, 15, 30, 50 µg/mL
Test 2 1, 2.5, 5, 10, 15, 30, 50 µg/mL
Mutation tests: +S-9 mix
Test 1 0.1, 0.5, 1, 2.5, 5, 10, 15 µg/mL
Test 2 0.1, 0.5, 1, 2.5, 5, 10, 15 µg/mL - Vehicle / solvent:
- - Vehicle used: DMSO
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- ethylmethanesulphonate
- Remarks:
- without S9 mix, 500 µg/mL
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 20-methylcholanthrene
- Remarks:
- with S9 mix, 2.5 µg/mL
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 3 hours
- Expression time (cells in growth medium): 48 hours
- Selection time: 48 hours
- Fixation time: Analysis was performed after 12 days incubation following
selection.
SELECTION AGENT (mutation assays): not indicated
DETERMINATION OF CYTOTOXICITY
- Method: relative total growth - Evaluation criteria:
- The criteria which must be satisfied before a positive response may be claimed are:
1. The induction of at least a two-fold increase in mutant frequency relative to the concurrent control by the test agent.
2. The demonsfration of a statistically significant response.
3. Evidence of a dose-related response.
4. The response must be reproducible.
5. The data are acceptable according to the criteria detaiied in Arlett et al. (1989), in 'Statistica Evaluation of Mutagenicity Test Data', pp. 66 - 101, Cambridge University Press. - Statistics:
- The general method of statistical analysis was by analysis of variance of the mutant frequencies.
- Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- other: Treatment with 1, 15, 30 and 50 μg/mL in tests 1 and 2 produced cell survival levels of 91 - 31 % and 93 - 26 % respectively.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- other: Treatment with 0.5, 5, 10 and 15 μg/mL in test 1 and 0.5, 1, 2.5 and 5 μg/mL in test 2 produced cell survival levels of 86 - 31 % and 95 - 23 % in tests 1 and 2 respectively.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- In the absence of S9-mix treatment with 1 - 50 µg/mL in tests 1 and 2 resulted in relative growth in suspension of 111 - 39 % and 99 - 35 % respectively.
In the presence of S9-mix treatment with 0.1 - 15 µg/mL in tests 1 and 2 resulted in relative growth in suspension of 110 - 31 % and 112 - 5 % respectively. - Conclusions:
- Under the present test conditions, the test substance tested up to cytotoxic concentrations in the absence and presence of metabolic activation in two independent experiments, was negative with respect to the mutant frequency in the L5178Y TK +/- mammalian cell mutagenicity test with S9 mix. The evidence for mutagenic potential of the test substance in the absence of S9 mix is equivocal in this in vitro test system.
- Executive summary:
In a mammalian cell gene mutation assay (TK) according to OECD guideline 476 (Huntingdon, 1991), mouse lymphoma L5178Y cells cultured in vitro were exposed to the test substance dissolved in DMSO at concentrations of 0.5, 1, 5, 10, 15, 30 and 50 µg/mL with and without metabolic activation (S9 mix).
In the absence of S9-mix treatment with 1 - 50 µg/mL in tests 1 and 2 resulted in relative growth in suspension of 111 - 39 % and 99 - 35 %, respectively. In the presence of S9-mix treatment with 0.1 - 15 µg/mL in tests 1 and 2 resulted in relative growth in suspension of 110 - 31 % and 112 - 5 %, respectively.
Statistically significant increases in mutant frequency were observed in both tests in the absence of S9 mix. In test 2 the response was dose-related. Although this data does not fulfil all the criteria for a positive response the detection of a dose-related increase may be indicative of mutagenic potential.
In the presence of S9 mix statistically significant increases in mutant frequency were observed in test 1. However, these increases were small and considered to be not biologically significant.
Therefore, the evidence for mutagenic potential of the test substance in the absence of S9 mix is equivocal in this in vitro test system and there is no evidence for mutagenic potential in the presence of S9 mix using this in vitro test system.
Referenceopen allclose all
Table 1: Summary of the Results, Test 1
Strain |
Dose Level (µg/plate) |
S9 mix |
Mean revertant colony count |
SD |
Individual revertant colony count |
|
TA 1535 |
5000 |
- |
7 |
0.6 |
7P,8P,7P |
|
1500 |
- |
10 |
1.2 |
11P,9P,11P |
||
500 |
- |
13 |
2.9 |
15,15,10 |
||
150 |
- |
10 |
1.0 |
11,9,10 |
||
50 |
- |
16 |
1.5 |
14,16,17 |
||
0 |
- |
12 |
2.1 |
10,11,14 |
||
Solvent |
- |
14 |
2.3 |
15,11,15 |
||
5000 |
+ |
7 |
1.5 |
6P,7P,9P |
||
1500 |
+ |
9 |
3.1 |
12P,10P,6P |
||
500 |
+ |
10 |
1.2 |
11,11,9 |
||
150 |
+ |
12 |
6.1 |
9,19,8 |
||
50 |
+ |
10 |
4.0 |
6,14,11 |
||
0 |
+ |
10 |
1.5 |
12,9,10 |
||
Solvent |
+ |
12 |
4.2 |
9,11,17 |
||
TA 1537 |
5000 |
- |
7 |
2.6 |
6P,5P,10P |
|
1500 |
- |
8 |
1.2 |
7P,9P,9P |
||
500 |
- |
14 |
2.1 |
16,12,15 |
||
150 |
- |
8 |
1.5 |
10,7,8 |
||
50 |
- |
8 |
2.1 |
6,9,10 |
||
0 |
- |
14 |
1.5 |
16,14,13 |
||
Solvent |
- |
16 |
3.5 |
18,12,18 |
||
5000 |
+ |
8 |
1.5 |
6P,9P,8P |
||
1500 |
+ |
9 |
2.0 |
9P,11P,7P |
||
500 |
+ |
11 |
1.5 |
9,11,12 |
||
150 |
+ |
14 |
5.1 |
8,18,15 |
||
50 |
+ |
11 |
3.1 |
8,12,14 |
||
0 |
+ |
10 |
1.2 |
11,11,9 |
||
Solvent |
+ |
11 |
1.5 |
11,10,13 |
||
TA 98 |
5000 |
- |
20 |
2.0 |
20P,22P,18P |
|
1500 |
- |
30 |
5.7 |
35P,32P,24P |
||
500 |
- |
27 |
12.5 |
23,41,17 |
||
150 |
- |
28 |
3.1 |
25,31,29 |
||
50 |
- |
32 |
7.5 |
33,39,24 |
||
0 |
- |
35 |
5.8 |
38,28,38 |
||
Solvent |
- |
30 |
4.0 |
34,31,26 |
||
5000 |
+ |
11 |
1.0 |
12P,10P,11P |
||
1500 |
+ |
19 |
5.3 |
13P,21P,23P |
||
500 |
+ |
21 |
6.1 |
22,14,26 |
||
150 |
+ |
21 |
5.3 |
25,23,15 |
||
50 |
+ |
18 |
2.6 |
20,19,15 |
||
0 |
+ |
19 |
1.0 |
18,20,19 |
||
Solvent |
+ |
19 |
1.2 |
20,20,18 |
||
TA 100 |
5000 |
- |
81 |
2.5 |
81P,78P,83P |
|
1500 |
- |
104 |
15.7 |
97P,122P,93P |
||
500 |
- |
117 |
13.1 |
103,129,119 |
||
150 |
- |
122 |
16.5 |
141,113,112 |
||
50 |
- |
119 |
8.6 |
111,128,117 |
||
0 |
- |
120 |
4.5 |
120,116,125 |
||
Solvent |
- |
113 |
10.0 |
103,114,123 |
||
5000 |
+ |
91 |
11.5 |
79P,92P,102P |
||
1500 |
+ |
103 |
4.6 |
98P,104P,107P |
||
500 |
+ |
135 |
6.1 |
140,128,136 |
||
150 |
+ |
129 |
8.5 |
132,119,135 |
||
50 |
+ |
125 |
15.8 |
139,129,108 |
||
0 |
+ |
132 |
9.1 |
128,142,125 |
||
Solvent |
+ |
121 |
3.1 |
122,124,118 |
||
Positive Control |
||||||
TA 1535 |
ENNG |
5 |
- |
771 |
38.4 |
740,759,814 |
TA 1537 |
9-AA |
80 |
- |
X |
- |
X, X, X, |
TA 98 |
NF |
1 |
- |
107 |
11.4 |
110,116,94 |
TA 100 |
ENNG |
3 |
- |
378 |
25.0 |
361,367,407 |
TA 1535 |
2-AA |
2 |
+ |
100 |
10.6 |
96,92,112 |
TA 1537 |
2-AA |
2 |
+ |
74 |
9.1 |
81,64,78 |
TA 98 |
2-AA |
0.5 |
+ |
135 |
17.1 |
133,119,153 |
TA 100 |
2-AA |
1 |
+ |
348 |
32.1 |
351,378,314 |
P Precipitation SD Standard deviation X Too many colonies for accurate counting ENNG N-ethyl-N'-nitro-N-nitrosoguanidine 9 AC 9-aminoacridine 2-AA 2-aminoanthracene NF 2-nitrofluorene |
||||||
Table 2: Summary of the Results, Test 2
Strain |
Dose Level (µg/plate) |
S9 mix |
Mean revertant colony count |
SD |
Individual revertant colony count |
|
TA 1535 |
5000 |
_ |
7 |
0.6 |
7P,8P,7P |
|
1500 |
- |
10 |
3.0 |
10P,7P,13P |
||
500 |
- |
11 |
1.0 |
12,11,10 |
||
150 |
- |
9 |
3.5 |
13,7,7 |
||
50 |
- |
13 |
2.6 |
10,15,14 |
||
0 |
- |
15 |
1.5 |
16,15,13 |
||
Solvent |
- |
14 |
2.5 |
17,14,12 |
||
5000 |
+ |
5 |
1.5 |
5P,7P,4P |
||
1500 |
+ |
11 |
2.0 |
9P,11P,13P |
||
500 |
10 |
7 |
2.1 |
5,8,9 |
||
150 |
+ |
14 |
4.2 |
15,9,17 |
||
50 |
+ |
7 |
2.5 |
7,4,9 |
||
0 |
+ |
13 |
3.6 |
9,16,14 |
||
Solvent |
+ |
14 |
4.6 |
18,15,9 |
||
TA 1537 |
5000 |
- |
6 |
2.1 |
5P,4P,8P |
|
1500 |
- |
9 |
1.5 |
9P,10P,7P |
||
500 |
- |
12 |
1.5 |
14,11,12 |
||
150 |
- |
9 |
2.5 |
7,12,9 |
||
50 |
- |
9 |
3.2 |
8,13,7 |
||
0 |
- |
12 |
3.6 |
16,11,9 |
||
Solvent |
- |
14 |
0.6 |
14,13,14 |
||
5000 |
+ |
6 |
1.0 |
7P,6P,5P |
||
1500 |
+ |
10 |
0.0 |
10P,10P,10P |
||
500 |
+ |
11 |
2.6 |
16,11,9 |
||
150 |
+ |
10 |
2.1 |
12,8,9 |
||
50 |
+ |
11 |
0.6 |
11,11,10 |
||
0 |
+ |
13 |
2.0 |
11,13,15 |
||
Solvent |
+ |
15 |
1.5 |
13,15,16 |
||
TA 98 |
5000 |
- |
24 |
6.0 |
30P,25P,18P |
|
1500 |
- |
23 |
1.7 |
24P,24P,21P |
||
500 |
- |
34 |
4.0 |
34,30,38 |
||
150 |
- |
29 |
8.3 |
36,20,32 |
||
50 |
- |
31 |
7.0 |
32,24,38 |
||
0 |
- |
37 |
4.4 |
32,39,40 |
||
Solvent |
- |
36 |
8.0 |
28,37,44 |
||
5000 |
+ |
13 |
1.5 |
15P,13P,12P |
||
1500 |
+ |
17 |
3.1 |
20P,16P,14P |
||
500 |
+ |
22 |
6.1 |
15,23,27 |
||
150 |
+ |
20 |
5.3 |
26,18,16 |
||
50 |
+ |
17 |
2.5 |
17,20,15 |
||
0 |
+ |
19 |
2.1 |
17,21,18 |
||
Solvent |
+ |
20 |
1.5 |
19,20,22 |
||
TA 100 |
5000 |
- |
89 |
13.9 |
85P,77P,104P |
|
1500 |
- |
99 |
6.7 |
95P,107P,96P |
||
500 |
- |
135 |
17.0 |
118,134,152 |
||
150 |
- |
133 |
13.5 |
133,120,147 |
||
50 |
- |
138 |
2.1 |
137,140,136 |
||
0 |
- |
142 |
9.1 |
146,132,149 |
||
Solvent |
- |
127 |
18.8 |
117,149,116 |
||
5000 |
+ |
107 |
9.0 |
116P,98P,108P |
||
1500 |
+ |
109 |
19.1 |
102P,95P,131P |
||
500 |
+ |
139 |
7.8 |
141,130,145 |
||
150 |
+ |
137 |
3.5 |
139,133,139 |
||
50 |
+ |
133 |
7.8 |
137,138,124 |
||
0 |
+ |
148 |
10.1 |
153,136,154 |
||
Solvent |
+ |
142 |
13.3 |
157,134,134 |
||
Positive Control |
||||||
TA 1535 |
ENNG |
5 |
- |
231 |
70.6 |
158,235,299 |
TA 1537 |
9-AA |
80 |
- |
2348 |
55.5 |
2292,2403,2350 |
TA 98 |
NF |
1 |
- |
127 |
4.7 |
131,129,122 |
TA 100 |
ENNG |
3 |
- |
357 |
12.1 |
368,344,358 |
TA 1535 |
2-AA |
2 |
+ |
124 |
9.5 |
131,127,113 |
TA 1537 |
2-AA |
2 |
+ |
76 |
7.1 |
77,68,82 |
TA 98 |
2-AA |
0.5 |
+ |
129 |
7.0 |
122,136,129 |
TA 100 |
2-AA |
1 |
+ |
432 |
48.6 |
377,450,469 |
P Precipitation SD Standard deviation X Too many colonies for accurate counting ENNG N-ethyl-N'-nitro-N-nitrosoguanidine 9 AC 9-aminoacridine 2-AA 2-aminoanthracene NF 2-nitrofluorene |
||||||
Table 1: Summary of the Results
Substance |
Conc., µg/mL |
No of cells examined |
No of Aberrations/100 Cells |
Aberrations |
No of Aberrant Cells |
||||||||||||
Exc. Gaps |
Inc. gaps |
BWF |
BF |
I |
SM |
R |
A |
GT |
P |
CHR |
EXC. Gaps |
Mean, % |
Inc Gaps |
%, Mean |
|||
Without S9 mix |
|||||||||||||||||
DMSO (Solvent Control) |
10 µL/mL |
100 |
0 |
0 |
|
|
|
|
|
|
|
|
|
0 |
0.25 |
0 |
0.25 |
100 |
0 |
0 |
|
|
|
|
|
|
|
|
|
0 |
0 |
||||
100 |
1 |
1 |
|
|
|
|
|
1 |
|
|
|
1 |
1 |
||||
100 |
0 |
0 |
|
|
|
|
|
|
|
|
|
0 |
0 |
||||
Test Item |
4 |
100 |
0 |
0 |
|
|
|
|
|
|
|
|
|
0 |
0.0 |
0 |
0.0 |
100 |
0 |
0 |
|
|
|
|
|
|
|
|
|
0 |
0 |
||||
20 |
100 |
1 |
1 |
|
|
|
1 |
|
|
|
|
|
1 |
0.5 |
1 |
0.5 |
|
100 |
0 |
0 |
|
|
|
|
|
|
|
|
|
0 |
0 |
||||
40 |
100 |
1 |
1 |
1 |
|
|
|
|
|
|
|
|
1 |
0.5 |
1 |
0.5 |
|
100 |
0 |
0 |
|
|
|
|
|
|
|
|
|
0 |
0 |
||||
Mitomycin C (pos. Control) |
0.4 |
100 |
27 |
29 |
9 |
|
6 |
1 |
2 |
4 |
4 |
1 |
2 |
18 |
18.7* |
19 |
19.8* |
87 |
28.7 |
29.9 |
4 |
1 |
9 |
4 |
3 |
1 |
2 |
1 |
1 |
17 |
18 |
||||
With S9 mix |
|||||||||||||||||
DMSO (Solvent Control) |
10 µL/mL |
100 |
6 |
6 |
1 |
2 |
1 |
1 |
|
1 |
|
|
|
6 |
4.25 |
6 |
4.25 |
100 |
4 |
4 |
2 |
1 |
|
|
|
1 |
|
|
|
3 |
3 |
||||
100 |
7 |
7 |
1 |
|
3 |
|
|
3 |
|
|
|
5 |
5 |
||||
100 |
3 |
3 |
1 |
|
|
2 |
|
|
|
|
|
3 |
3 |
||||
Test Item |
4 |
100 |
3 |
3 |
|
|
|
1 |
|
1 |
1 |
|
|
3 |
3.0 |
3 |
3.0 |
100 |
3 |
3 |
|
|
|
|
|
3 |
|
|
|
3 |
3 |
||||
20 |
100 |
2 |
2 |
|
|
|
2 |
|
|
|
|
|
2 |
3.0 |
2 |
3.0 |
|
100 |
4 |
4 |
|
|
|
4 |
|
|
|
|
|
4 |
4 |
||||
40 |
100 |
4 |
4 |
|
|
|
|
2 |
2 |
|
|
|
4 |
3.5 |
4 |
3.5 |
|
100 |
4 |
4 |
2 |
|
1 |
1 |
|
|
|
|
|
3 |
3 |
||||
Cyclophosphamide (pos. Control) |
20 |
78 |
97.4 |
107.7 |
14 |
2 |
10 |
24 |
|
224 |
|
|
8 |
43 |
54.4* |
44 |
55.3 |
36 |
113.9 |
116.7 |
5 |
1 |
7 |
7 |
1 |
20 |
2 |
|
1 |
19 |
19 |
||||
Statistical analysis used was Fisher's test * p < 0.001, Otherwise p > 0.05 BWF Chromatid break with fragment, A Acentric fragment, BF Chromatid break without fragment, GT Greater than 10 aberrations, I Interchange, P Pulverised cell, SM Single minute, CHR Chromatid gap, R Ring |
|||||||||||||||||
In the preliminary test:
Treatment with concentrations of 0.5 to 50 μg/mL in the presence and absence of metabolic activation produced relative growth in suspension of 106 to 2 % and 108 to 17 %, respectively.
In the main test:
with metabolic activation:
Treatment with 0.5, 5, 10 and 15 μg/mL in test 1 and 0.5, 1, 2.5, and 5 μg/mL in test 2 produced cell survival levels of 86 - 31 % and 95 - 23 % in tests 1 and 2, respectively.
without metabolic activation:
Treatment with 1, 15, 30 and 50 μg/mL in tests 1 and 2 produced cell survival levels of 91 - 31 % and 93 - 26 %, respectively.
Statistically significant increases in mutant frequency were observed in both tests in the absence of S9 mix. In test 2 the response was dose-related. Although this data does not fulfill all the criteria for a positive response the detection of a dose-related increase may be indicative of mutagenic potential.
In the presence of S9 mix statistically significant increases in mutant frequency were observed in test 1. However, these increases were small and considered to be not biologically significant.
Therefore, the evidence for mutagenic potential of the test substance in the absence of S9 mix is equivocal in this in vitro test system and there is no evidence for mutagenic potential in the presence of S9 mix using this in vitro test system.
Table 1: Summary of the Results, Test 1
Test Item Con. (µg/mL) |
No. of colonies on non-selective plates |
Survival (% control) |
Mean % survival |
No.of colonies on selective plates |
Mutant frequency (x 10^-6)+ |
Mean mutant frequency (x 10^-6) |
|||||||
1 |
2 |
3 |
Total |
Viability (%) |
1 |
2 |
3 |
Total |
|||||
Without S9 mix |
|||||||||||||
0 (DMSO control) |
132 |
139 |
140 |
411 |
100 |
100 |
100 |
49 |
54 |
38 |
141 |
69 |
85 |
131 |
141 |
118 |
390 |
40 |
52 |
37 |
129 |
66 |
|||||
96 |
121 |
102 |
319 |
49 |
69 |
C |
118 |
111 |
|||||
122 |
130 |
109 |
361 |
46 |
53 |
69 |
168 |
93 |
|||||
1.0 |
126 |
115 |
118 |
359 |
97 |
96 |
91 |
60 |
58 |
45 |
171 |
95 |
91 |
106 |
121 |
117 |
344 |
93 |
86 |
43 |
61 |
44 |
148 |
86 |
|||
15.0 |
71 |
85 |
73 |
228 |
62 |
31 |
33 |
53 |
46 |
56 |
455 |
136 |
133* |
79 |
98 |
105 |
282 |
76 |
34 |
62 |
75 |
46 |
183 |
130 |
|||
30.0 |
97 |
63 |
85 |
245 |
66 |
26 |
33 |
47 |
28 |
60 |
135 |
110 |
122* |
105 |
104 |
129 |
38 |
91 |
39 |
74 |
93 |
60 |
227 |
134 |
|||
50.0 |
98 |
100 |
81 |
279 |
75 |
29 |
31 |
60 |
47 |
68 |
175 |
125 |
132* |
105 |
84 |
106 |
295 |
80 |
32 |
70 |
66 |
67 |
203 |
138 |
|||
EMS, 500 |
73 |
81 |
74 |
228 |
62 |
36 |
31 |
189 |
220 |
218 |
627 |
550 |
706*** |
46 |
36 |
63 |
145 |
39 |
25 |
196 |
218 |
2100 |
624 |
861 |
|||
With S9 mix |
|||||||||||||
0 (DMSO control) |
121 |
119 |
130 |
370 |
100 |
100 |
100 |
64 |
58 |
59 |
181 |
98 |
106 |
113 |
122 |
122 |
357 |
61 |
50 |
58 |
169 |
95 |
|||||
129 |
127 |
122 |
378 |
54 |
73 |
73 |
200 |
106 |
|||||
117 |
111 |
105 |
333 |
64 |
70 |
72 |
206 |
124 |
|||||
0.5 |
102 |
107 |
101 |
310 |
86 |
78 |
86 |
61 |
47 |
53 |
161 |
104 |
92 |
111 |
107 |
103 |
321 |
89 |
94 |
40 |
37 |
50 |
127 |
79 |
|||
5.0 |
102 |
95 |
113 |
310 |
86 |
54 |
46 |
41 |
41 |
52 |
134 |
86 |
108 |
84 |
76 |
86 |
246 |
68 |
37 |
52 |
54 |
53 |
159 |
129 |
|||
10.0 |
134 |
130 |
139 |
403 |
112 |
43 |
38 |
91 |
73 |
80 |
244 |
121 |
132 |
106 |
130 |
93 |
329 |
91 |
33 |
73 |
82 |
79 |
234 |
142 |
|||
15.0 |
146 |
128 |
137 |
411 |
1144 |
33 |
31 |
84 |
89 |
99 |
272 |
132 |
137* |
106 |
98 |
100 |
304 |
84 |
28 |
82 |
64 |
72 |
215 |
141 |
|||
20-Methylcholanthrene, 2.5 |
48 |
50 |
49 |
147 |
41 |
20 |
19 |
165 |
172 |
163 |
500 |
680 |
640*** |
56 |
48 |
C |
104(156) |
43 |
17 |
141 |
170 |
C |
311 (467) |
599 |
|||
+ Where replicate determinations of mutant frequency from a single culture were made, the mean mutant frequency for that culture is shown C Contaminated. Figure in brackets is adjustment made for contaminated plates and is used in all calculations * Significantly different from control P < 0.05 *** Significantly different from control P < 0.001 |
|||||||||||||
Table 2: Summary of the Results, Test 2
Test Item Con. (µg/mL) |
No. of colonies on non-selective plates |
Survival (% control) |
Mean % survival |
No.of colonies on selective plates |
Mutant frequency (x 10^-6)+ |
Mean mutant frequency (x 10^-6) |
|||||||
1 |
2 |
3 |
Total |
Viability (%) |
1 |
2 |
3 |
Total |
|||||
Without S9 mix |
|||||||||||||
0 (DMSO control) |
109 |
107 |
C |
216(324) |
100 |
100 |
100 |
53 |
42 |
42 |
137 |
85 |
93 |
109 |
109 |
102 |
320 |
50 |
53 |
59 |
162 |
101 |
|||||
107 |
104 |
110 |
321 |
33 |
38 |
59 |
130 |
81 |
|||||
100 |
111 |
C |
211(317) |
56 |
53 |
C |
109(164) |
103 |
|||||
1.0 |
100 |
118 |
99 |
317 |
99 |
97 |
93 |
29 |
47 |
59 |
135 |
85 |
85 |
87 |
89 |
112 |
288 |
90 |
89 |
47 |
38 |
36 |
121 |
84 |
|||
15.0 |
97 |
85 |
93 |
375 |
86 |
30 |
33 |
62 |
35 |
43 |
140 |
|
103 |
105 |
105 |
78 |
288 |
90 |
35 |
47 |
447 |
54 |
148 |
102 |
|||
30.0 |
102 |
99 |
75 |
376 |
86 |
25 |
27 |
59 |
70 |
77 |
206 |
103 |
138** |
95 |
96 |
C |
191(287) |
90 |
29 |
C |
53 |
68 |
121(182) |
149 |
|||
50.0 |
85 |
78 |
72 |
235 |
73 |
22 |
26 |
41 |
50 |
62 |
153 |
127 |
155** |
71 |
73 |
91 |
235 |
73 |
29 |
68 |
67 |
77 |
212 |
130 |
|||
EMS, 500 |
112 |
105 |
99 |
316 |
99 |
100 |
77 |
259 |
240 |
260 |
759 |
182 |
527*** |
81 |
77 |
73 |
231 |
72 |
53 |
216 |
219 |
228 |
663 |
480 |
|||
With S9 mix |
|||||||||||||
0 (DMSO control) |
148 |
136 |
136 |
410 |
100 |
100 |
100 |
96 |
C |
C |
96(288) |
140 |
113 |
134 |
133 |
C |
267(401) |
82 |
69 |
64 |
215 |
107 |
|||||
143 |
130 |
C |
273(410) |
82 |
70 |
66 |
218 |
106 |
|||||
126 |
111 |
115 |
352 |
59 |
58 |
C |
117(176) |
100 |
|||||
0.5 |
108 |
122 |
115 |
345 |
88 |
95 |
95 |
47 |
56 |
C |
103(155) |
90 |
90 |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
|||
5.0 |
127 |
103 |
105 |
335 |
85 |
83 |
85 |
19 |
42 |
39 |
100 |
60 |
73 |
145 |
111 |
119 |
375 |
95 |
87 |
56 |
52 |
53 |
161 |
86 |
|||
10.0 |
127 |
124 |
116 |
367 |
93 |
65 |
61 |
72 |
58 |
77 |
207 |
113 |
118 |
111 |
100 |
108 |
319 |
81 |
57 |
50 |
79 |
C |
129(194) |
122 |
|||
15.0 |
73 |
52 |
93 |
218 |
55 |
16 |
23 |
39 |
36 |
38 |
113 |
104 |
91 |
93 |
117 |
108 |
318 |
81 |
30 |
39 |
29 |
56 |
124 |
78 |
|||
20-Methylcholanthrene, 2.5 |
41 |
56 |
56 |
153 |
39 |
19 |
19 |
148 |
C |
133 |
281(422) |
552 |
570*** |
47 |
54 |
C |
101(152) |
39 |
18 |
143 |
154 |
C |
297(446) |
587 |
|||
+ Where replicate determinations of mutant frequency from a single culture were made, the mean mutant frequency for that culture is shown C Contaminated. Figure in brackets is adjustment made for contaminated plates and is used in all calculations * Significantly different from control P < 0.05 ** Significantly different from control P<O.Ol *** Significantly different from control P < 0.001 |
|||||||||||||
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
3 key studies are available to cover genetic toxicity. In accordance with Article 25(3) of the REACH Regulation the robust study summaries submitted 12 years previously, are used as supporting information for the purpose of the registration.
Bacterial reverse mutation
Key study
In a reverse mutation assay in bacteria according to EU method B.13/14 (Huntingdon, 1988), strains (TA98, TA100, TA1535, TA1537) of S. typhimurium were exposed to the test substance dissolved in DMSO at concentrations of 50 to 5000 µg/plate in the presence and absence of metabolic activation. The test substance was tested up to a limit concentration of 5000 µg/plate. There was no evidence or a concentration related positive response of induced mutant colonies over background. Thus, the test substance is not regarded as mutagenic.
Supporting study
The results of the key study were supplemented by a bacterial reverse mutation assay (Ames) in the S. typhimurium tester strain TA1538 (Huntingdon, 1990). The bacteria were exposed to the test substance dissolved in DMSO at concentrations of 50 to 5000 µg/plate in the presence and absence of metabolic activation. The test substance was tested up to a limit concentration of 5000 µg/plate. There was no evidence or a concentration related positive response of induced mutant colonies over background. Thus, the test substance is not regarded as mutagenic.
In a reverse mutation assay in bacteria according to EU method B.13/14 (Mitsubishi, ECHA RSS, 2017), strains (TA98, TA100, TA1535, TA1537, E. coli WP uvrA) of S. typhimurium and E. coli were exposed to the test substance dissolved in DMSO at concentrations of 313 to 5000 µg/plate in the presence and absence of metabolic activation. The test substance was tested up to a limit concentration of 5000 µg/plate. There was no evidence or a concentration related positive response of induced mutant colonies over background. Thus, the test substance is not regarded as mutagenic.
Chromosome aberration
Key study
In a mammalian cell cytogenetics assay (chromosome aberration) according to OECD guideline 473 (Huntingdon, 1989), CHO cell cultures were exposed to the test substance dissolved in DMSO at concentrations of 4, 20 and 40 µg/mL with and without metabolic activation (Aroclor 1254 – induced rat liver). In a preliminary study the highest dose used did not produce any reduction in mitotic index. The highest dose used (40 µg/mL) was the maximum achievable concentration in the test system. The test substance was tested up to cytotoxic concentrations.
In both the presence and absence of metabolic activation, the test substance caused no statistically significant increase in the proportion of metaphase figures containing chromosomal aberrations at any dose level when compared to the solvent control. Both positive control compounds caused large, statistically highly significant increases in chromosomal damage, demonstrating the sensitivity of this test system and the efficacy of the S9 mix. It is concluded that the test substance has shown no evidence of clastogenic activity in this in vitro cytogenetic test system.
Supporting study
In a mammalian cell cytogenetics assay (chromosome aberration) according to EU method B.10 (Toxicol. Lab. Ltd., ECHA RSS, 2017), CHO cell cultures were exposed to the test substance dissolved in DMSO at concentrations of 12.5 to 125 µg/mL with and without metabolic activation (Aroclor 1254 – induced rat liver). The highest concentration represented the solubility limit for this substance. A dose-related decrease in the mitotic index occurred both with and without S9 mix. At the highest dose level the decrease was over 40 % compared with control values. The test substance was tested up to cytotoxic concentrations.
In both the presence and absence of metabolic activation, the test substance caused no statistically significant increase in the proportion of metaphase figures containing chromosomal aberrations at any dose level when compared to the solvent control. It is concluded that the test substance has shown no evidence of clastogenic activity in this in vitro cytogenetic test system.
In vitro mammalian cell gene mutation
Key study
In a mammalian cell gene mutation assay (TK) according to OECD guideline 476 (Huntingdon, 1991), mouse lymphoma L5178Y cells cultured in vitro were exposed to the test substance dissolved in DMSO at concentrations of 0.5, 1, 5, 10, 15, 30 and 50 µg/mL with and without metabolic activation (S9 mix).
In the absence of S9-mix treatment with 1 - 50 µg/mL in tests 1 and 2 resulted in a relative cell growth in suspension of 111 - 39 % and 99 - 35 %, respectively. In the presence of S9-mix treatment with 0.1 - 15 µg/mL in tests 1 and 2 resulted in relative growth in suspension of 110 - 31 % and 112 - 5 %, respectively.
Statistically significant increases in mutant frequency were observed in both tests in the absence of S9 mix. In test 2 the response was dose-related. Although this data does not fulfil all the criteria for a positive response, the detection of a dose-related increase may be indicative of mutagenic potential.
In the presence of S9 mix statistically significant increases in mutant frequency were observed in test 1. However, these increases were small and considered to be not biologically significant.
Therefore, the evidence for mutagenic potential of the test substance in the absence of S9 mix is equivocal in this in vitro test system and there is no evidence for mutagenic potential in the presence of S9 mix using this in vitro test system.
Supporting study
In a mammalian cell gene mutation assay (TK) according to OECD guideline 476 (Pharmaco-LSR Ltd., ECHA RSS, 2017), mouse lymphoma L5178Y cells cultured in vitro were exposed to the test substance dissolved in DMSO at concentrations of 2.73 to 730 µg/mL with and without metabolic activation (S9-mix).
Although some toxicity was seen in preliminary tests at low concentrations, no dose-response relationship was apparent. Some precipitation of the test substance was observed at concentrations of 175 µg/mL and above. Cultures exposed to the test substance showed no increase in mutant colony numbers or increase in the mutant frequencies (per 100000 survivors) compared to controls. Thus, no evidence for a mutagenic potential was observed for the test substance.
Justification for classification or non-classification
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on genetic toxicity, the test item is not classified according to Regulation (EC) No 1272/2008 (CLP).
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