Registration Dossier
Registration Dossier
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EC number: 944-953-9 | CAS number: 73326-59-7
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
The test item is considered to be non-mutagenic in the Salmonella typhimurium and Escherichia coli reverse mutation assay (OECD 471).
In addition, the test item is considered to be non-mutagenic in this in vitro micronucleus test (OECD 487)
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:
- 08 September 2016 - 16 November 2016
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- 1997
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Version / remarks:
- 2008
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
- Metabolic activation:
- with and without
- Metabolic activation system:
- Phenobarbital/β-naphthoflavone induced rat liver S9
- Test concentrations with justification for top dose:
- Pre-Experiment/Experiment I & Ia:
3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate
Experiment II:
TA 1537 and TA 100: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate
All remaining strains: 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate - Vehicle / solvent:
- - Vehicle/solvent used: DMSO
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- sodium azide
- methylmethanesulfonate
- other: 4-nitro-o-phenylene-diamine / 2-aminoanthracene
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: plate incorporation test (experiment I & Ia), pre-incubation test (experiment II)
DURATION
- Preincubation period: 60 minutes
- Exposure duration: ≥ 48 hours
NUMBER OF REPLICATIONS: 3 (two independent experiments)
DETERMINATION OF CYTOTOXICITY
- Method: counting numbers of revertants using a validated computer system - Evaluation criteria:
- A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant. - Statistics:
- no statistical analysis
- 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 1537
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at 5000 µg/plate (experiment II)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at ≥ 2500 µg/plate (experiment II)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at ≥ 2500 µg/plate (experiment II)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at 5000 µg/plate (experiment II)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at ≥ 2500 µg/plate (experiment II)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at ≥ 1000 µg/plate (experiment I), at ≥ 2500 µg/plate (experiment II)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at 5000 µg/plate (experiment II)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: The test item precipitated in the overlay agar in the test tubes from 2500 to 5000 μg/plate in experiment I & Ia and at 5000 μg/plate in experiment II. No precipitation of the test item in the overlay agar on the incubated agar plates was observed. - Conclusions:
- The test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, the test item is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.
- Executive summary:
A bacterial reverse mutation assay (OECD 471) was performed to investigate the potential of the test item to induce gene mutations using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2uvrA. A plate incorporation test (experiment I and Ia: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate) and a pre-incubation test (experiment II: TA 1537 and TA 100: 3; 10; 33; 100; 333; 1000; 2500; and 5000μg/plate All remaining strains: 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate) was performed.
The assay was performed in three independent experiments with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate.
The test item precipitated in the overlay agar in the test tubes from 2500 to 5000 μg/plate in experiment I & Ia and at 5000 μg/plate in experiment II. No precipitation of the test item in the overlay agar on the incubated agar plates was observed.
In experiment Ia the plates incubated with the test item showed reduced background growth from 2500 to 5000 μg/plate in the absence of metabolic activation (in strain WP2uvrA).
Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in all strains except strain TA 1535.
No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.
The controls confirmed the validity of the study.
- Endpoint:
- in vitro cytogenicity / micronucleus study
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 14 September 2016 - 30 January 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
- Version / remarks:
- 2016
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian cell micronucleus test
- Species / strain / cell type:
- lymphocytes: human
- Details on mammalian cell type (if applicable):
- healthy non-smoking donors
Experiment I: male donor, 24 years old
Experiment II: female donor, 28 years old - Cytokinesis block (if used):
- Cytochalasin B
- Metabolic activation:
- with and without
- Metabolic activation system:
- Phenobarbital/β-naphthoflavone induced rat liver S9
- Test concentrations with justification for top dose:
- Concentrations used:
Experiment I (with and without S9 mix): 12.1; 21.2; 37.0; 64.8; 113; 199; 347; 608; 1064 and 1862 µg/mL)
Experiment II (without S9 mix): 163; 245; 368; 552; 828; 1241 and 1862 µg/mL)
Top dose: 1862 μg/mL (approx. 10 mM), was chosen with regard to the molecular weight of the test item and with respect to the current OECD Guideline 487 - Vehicle / solvent:
- - Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures with respect to the current OECD Guideline 487 - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- mitomycin C
- other: Demecolcin
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: Experiment I: 4 hours; Experiment II: 20 hours
NUMBER OF REPLICATIONS: 2
METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: The slides were prepared by dropping the cell suspension in fresh fixative onto a clean microscope slide. The cells were stained with Giemsa.
NUMBER OF CELLS EVALUATED: at least 1000 binucleate cell per culture
CRITERIA FOR MICRONUCLEUS IDENTIFICATION: The micronuclei were counted in cells showing a clearly visible cytoplasm area.
DETERMINATION OF CYTOTOXICITY
- Method: Cytokinesis-block proliferation index (CBPI)
- Any supplementary information relevant to cytotoxicity: CBPI was determined in 500 cells per culture. - Evaluation criteria:
- The micronuclei have to be stained in the same way as the main nucleus. The area of the micronucleus should not extend the third part of the area of the main nucleus. At least 1000 binucleate cells per culture were scored for cytogenetic damage on coded slides.
- Statistics:
- Statistical significance was confirmed by the Chi square test (α < 0.05), using a validated test script of “R”, a language and environment for statistical computing and graphics. Within this test script a statistical analysis was conducted for those values that indicated an increase in the number of cells with micronuclei compared to the concurrent solvent control.
- Key result
- Species / strain:
- lymphocytes: human
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- moderate cytotoxicity was observed at the highest evaluated concentration (experiment II)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none
- Effects of osmolality: none
- Precipitation: Experiment I: precipitation (with S9 mix) at ≥ 1064 μg/mL
- Definition of acceptable cells for analysis: The micronuclei were counted in cells showing a clearly visible cytoplasm area.
- Other confounding effects: phase separation: Experiment I (without S9 mix) at ≥ 1064 μg/mL Experiment II (without S9 mix) at ≥ 828 μg/mL
RANGE-FINDING/SCREENING STUDIES: yes
NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: 1000 binucleate cells per culture (in total 2000 binucleated cells per concentration and controls)
ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: CBPI - Conclusions:
- The test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes. Therefore, the test item is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to phase separating, precipitating or the highest evaluable concentrations.
- Executive summary:
The test item dissolved in DMSO, was assessed for its potential to induce micronuclei in human lymphocytes in vitro in two independent experiments.
In Experiment I, the exposure period was 4 hours with and without S9 mix. In Experiment II, the exposure period was 20 hours without S9 mix. The cells were prepared 40 hours after start of treatment with the test item.
In each experimental group two parallel cultures were analyzed. 1000 binucleate cells per culture were scored for cytogenetic damage on coded slides. To determine a cytotoxic effect the CBPI was determined in 500 cells per culture and cytotoxicity is described as % cytostasis. As highest treatment concentration in this study 1862 μg/mL (approx. 10 mM) was chosen.
In Experiment I, in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration, which showed phase separation or precipitation. In Experiment II in the absence of S9 mix, moderate cytotoxicity was observed at the highest evaluated concentration. Concentrations showing clear cytotoxic effects, however, were not evaluable for cytogenetic damage.
In the absence and presence of S9 mix, no relevant increases in the number of micronucleate cells were observed after treatment with the test item.
The controls confirmed the validity of the study.
Referenceopen allclose all
Table 1: Summary of Experiment I
Metabolic Activation |
Test Group |
Dose Level (per plate) |
Revertant Colony Counts (Mean ± SD) |
|||
TA 1535 |
TA 1537 |
TA 98 |
TA 100 |
|||
Without Activation |
DMSO |
|
17 ± 4 |
13 ± 2 |
22 ± 9 |
150 ± 2 |
Untreated |
|
12 ± 2 |
11 ± 5 |
26 ± 2 |
148 ± 8 |
|
PI 27319 |
3 µg |
14 ± 7 |
9 ± 3 |
20 ± 5 |
139 ± 25 |
|
10 µg |
17 ± 5 |
14 ± 4 |
26 ± 3 |
135 ± 17 |
||
33 µg |
18 ± 3 |
15 ± 1 |
23 ± 4 |
136 ± 5 |
||
100 µg |
12 ± 3 |
12 ± 2 |
25 ± 8 |
149 ± 13 |
||
333 µg |
14 ± 5 |
10 ± 3 |
23 ± 8 |
132 ± 7 |
||
1000 µg |
15 ± 1 |
10 ± 6 |
24 ± 2 |
66 ± 9 |
||
2500 µg |
10 ± 4 |
7 ± 2 |
23 ± 6 |
65 ± 3 |
||
5000 µg |
10 ± 5 |
7 ± 2 |
18 ± 3 |
66 ± 16 |
||
NaN3 |
10 µg |
1400 ± 12 |
|
|
2126 ± 51 |
|
4-NOPD |
10 µg |
|
|
430 ± 46 |
|
|
4-NOPD |
50 µg |
|
85 ± 11 |
|
|
|
With Activation |
DMSO |
|
13 ± 2 |
12 ± 2 |
29 ± 9 |
108 ± 7 |
Untreated |
|
15 ± 5 |
14 ± 4 |
33 ± 9 |
146 ± 13 |
|
PI 27319 |
3 µg |
14 ± 7 |
14 ± 6 |
38 ± 2 |
102 ± 4 |
|
10 µg |
9 ± 2 |
12 ± 6 |
34 ± 4 |
102 ± 11 |
||
33 µg |
11 ± 3 |
12 ± 3 |
38 ± 6 |
100 ± 5 |
||
100 µg |
14 ± 3 |
14 ± 2 |
40 ± 7 |
93 ± 5 |
||
333 µg |
11 ± 2 |
14 ± 3 |
42 ± 6 |
105 ± 12 |
||
1000 µg |
14 ± 4 |
16 ± 3 |
32 ± 6 |
94 ± 24 |
||
2500 µg |
14 ± 3 |
12 ± 6 |
36 ± 4 |
93 ± 10 |
||
5000 µg |
11 ± 3 |
12 ± 3 |
32 ± 8 |
55 ± 18 |
||
2-AA |
2.5 µg |
329 ± 27 |
308 ± 40 |
3613 ± 479 |
3697 ± 101 |
|
NaN3: sodium azide, 2 -AA: 2-aminoanthracene, 4 -NOPD: 4-nitro-o-phenylene-diamine
Table 2 Summary of Experiment Ia
Metabolic Activation |
Test Group |
Dose Level (per plate) |
Revertant Colony Counts (Mean ± SD) |
WP2 uvrA |
|||
Without Activation |
DMSO |
|
37 ± 9 |
Untreated |
|
39 ± 9 |
|
PI 27319 |
3 µg |
41 ± 13 |
|
10 µg |
40 ± 8 |
||
33 µg |
42 ± 8 |
||
100 µg |
41 ± 6 |
||
333 µg |
36 ± 6 |
||
1000 µg |
32 ± 5 |
||
2500 µg |
27 ± 7R |
||
5000 µg |
24 ± 6R |
||
NaN3 |
2.0 µL |
1024 ± 27 |
|
With Activation |
DMSO |
|
41 ± 7 |
Untreated |
|
46 ± 15 |
|
PI 27319 |
3 µg |
48 ± 2 |
|
10 µg |
40 ± 4 |
||
33 µg |
45 ± 4 |
||
100 µg |
48 ± 4 |
||
333 µg |
44 ± 7 |
||
1000 µg |
42 ± 6 |
||
2500 µg |
33 ± 5 |
||
5000 µg |
28 ± 3 |
||
2-AA |
10.0 µg |
452 ± 34 |
MSS: methyl methane silfonate, 2-AA: 2-aminoanthracene, R: Reduced background growth
Table 3 Summary of Experiment II
Metabolic Activation |
Test Group |
Dose Level (per plate) |
Revertant Colony Counts (Mean ± SD) |
||||
TA 1535 |
TA 1537 |
TA 98 |
TA 100 |
WP2 uvrA |
|||
Without Activation |
DMSO |
|
12 ± 4 |
10 ± 3 |
25 ± 5 |
127 ± 19 |
46 ± 7 |
Untreated |
|
9 ± 4 |
14 ± 4 |
26 ± 6 |
164 ± 8 |
49 ± 9 |
|
PI 27319 |
3 µg |
|
11 ± 2 |
|
127 ± 20 |
|
|
10 µg |
15 ± 4 |
11 ± 4 |
27 ± 3 |
146 ± 10 |
48 ± 5 |
||
33 µg |
12 ± 7 |
11 ± 1 |
24 ± 4 |
127 ± 8 |
40 ± 9 |
||
100 µg |
11 ± 4 |
12 ± 3 |
28 ± 7 |
132 ± 14 |
40 ± 4 |
||
333 µg |
14 ± 2 |
9 ± 2 |
29 ± 8 |
121 ± 6 |
43 ± 5 |
||
1000 µg |
11 ± 6 |
9 ± 3 |
17 ± 5 |
103 ± 11 |
41 ± 5 |
||
2500 µg |
11 ± 1 |
3 ± 2 |
15 ± 1 |
20 ± 6 |
24 ± 6 |
||
5000 µg |
16 ± 1 |
0 ± 1 |
0 ± 0 |
0 ± 1 |
18 ± 7 |
||
NaN3 |
10 µg |
1481 ± 130 |
|
|
2526 ± 39 |
|
|
4-NOPD |
10 µg |
|
|
453 ± 10 |
|
|
|
4-NOPD |
50 µg |
|
74 ± 4 |
|
|
|
|
MMS |
2.0 µL |
|
|
|
|
890 ± 50 |
|
With Activation |
DMSO |
|
13 ± 3 |
11 ± 5 |
37 ± 11 |
119 ± 8 |
61 ± 8 |
Untreated |
|
9 ± 3 |
8 ± 3 |
45 ± 9 |
177 ± 13 |
53 ± 10 |
|
PI 27319 |
3 µg |
|
12 ± 5 |
|
96 ± 7 |
|
|
10 µg |
14 ± 2 |
9 ± 5 |
33 ± 7 |
109 ± 15 |
60 ± 4 |
||
33 µg |
13 ± 5 |
13 ± 4 |
33 ± 8 |
107 ± 10 |
54 ± 2 |
||
100 µg |
13 ± 2 |
11 ± 5 |
31 ± 4 |
96 ± 13 |
53 ± 5 |
||
333 µg |
12 ± 3 |
8 ± 3 |
36 ± 4 |
114 ± 11 |
55 ± 8 |
||
1000 µg |
11 ± 4 |
7 ± 3 |
28 ± 2 |
81 ± 13 |
49 ± 1 |
||
2500 µg |
10 ± 3 |
8 ± 2 |
9 ± 4 |
52 ± 6 |
29 ± 4 |
||
5000 µg |
11 ± 1 |
0 ± 1 |
1 ± 1 |
2 ± 1 |
20 ± 5 |
||
2-AA |
2.5 µg |
394 ± 6 |
100 ± 16 |
3884 ± 271 |
4828 ± 603 |
|
|
2-AA |
10.0 µg |
|
|
|
|
456 ± 10 |
|
NaN3: sodium azide, 2-AA: 2-aminoanthracene,4 -NOPD: 4-nitro-o-phenylene-diamine
Historical Data
Strain |
|
without S9 mix |
with S9 mix |
||||||
Mean |
SD |
Min |
Max |
Mean |
SD |
Min |
Max |
||
TA 1535 |
Solvent control |
11 |
2.15 |
7 |
23 |
12 |
2.14 |
7 |
21 |
Untreated control |
12 |
2.97 |
6 |
24 |
12 |
2.71 |
7 |
26 |
|
Positive control |
1090 |
123.80 |
334 |
1372 |
392 |
62.85 |
176 |
549 |
|
TA 1537 |
Solvent control |
10 |
1.83 |
6 |
18 |
13 |
3.27 |
7 |
27 |
Untreated control |
10 |
2.29 |
6 |
20 |
14 |
3.72 |
7 |
25 |
|
Positive control |
83 |
12.28 |
55 |
131 |
175 |
44.44 |
82 |
327 |
|
TA 98 |
Solvent control |
24 |
3.75 |
16 |
36 |
33 |
5.55 |
18 |
51 |
Untreated control |
26 |
4.72 |
15 |
43 |
36 |
5.83 |
17 |
56 |
|
Positive control |
344 |
51.13 |
211 |
599 |
3822 |
857.83 |
319 |
5048 |
|
TA 100 |
Solvent control |
155 |
24.19 |
84 |
194 |
145 |
31.81 |
81 |
204 |
Untreated control |
174 |
21.92 |
90 |
206 |
170 |
23.62 |
93 |
212 |
|
Positive control |
1956 |
279.93 |
658 |
2528 |
3606 |
676.07 |
722 |
4940 |
|
WP2 uvrA |
Solvent control |
41 |
5.72 |
27 |
63 |
51 |
6.91 |
37 |
72 |
Untreated control |
42 |
6.01 |
31 |
63 |
53 |
7.05 |
38 |
88 |
|
Positive control |
732 |
161.66 |
322 |
1066 |
362 |
72.26 |
212 |
858 |
|
Mean = mean value of revertants/plate, SD = standard deviation, Min = minimal value, Max = maximal value
Table 1: Number of micronucleated cells; exposure period 4 hrs without S9 mix, Experiment I
Treatment group |
Conc. per mL |
S9 mix |
Exposure / preparation |
Micronucleated cells |
|||||||||
Binucleate cells withnmicronuclei culture 1 |
sum culture 1 |
Binucleate cells withnmicronuclei culture 2 |
sum culture 2 |
sum in 2000 binucleate cells |
[%] |
||||||||
1 |
2 |
>2 |
1 |
2 |
>2 |
||||||||
Solv. control# |
0.5 % |
- |
4 / 40 hrs |
7 |
0 |
0 |
7 |
3 |
0 |
0 |
3 |
10 |
0.50 |
Pos. control## |
1.0 µg |
- |
4 / 40 hrs |
85 |
7 |
1 |
93 |
159 |
32 |
1 |
192 |
285 |
14.25 |
Test item |
347 µg |
- |
4 / 40 hrs |
4 |
0 |
0 |
4 |
3 |
0 |
0 |
3 |
7 |
0.35 |
Test item |
608 µg |
- |
4 / 40 hrs |
4 |
0 |
0 |
4 |
3 |
0 |
0 |
3 |
7 |
0.35 |
Test item |
1064 µg |
- |
4 / 40 hrs |
5 |
0 |
0 |
5 |
3 |
0 |
0 |
3 |
8 |
0.40 |
# DMSO
## MMC
Table 2: Number of micronucleated cells; exposure period 4 hrs with S9 mix, Experiment I
Treatment group |
Conc. per mL |
S9 mix |
Exposure / preparation |
Micronucleated cells |
|||||||||
Binucleate cells withnmicronuclei culture 1 |
sum culture 1 |
Binucleate cells withnmicronuclei culture 2 |
sum culture 2 |
sum in 2000 binucleate cells |
[%] |
||||||||
1 |
2 |
>2 |
1 |
2 |
>2 |
||||||||
Solv. control# |
0.5 % |
+ |
4 / 40 hrs |
8 |
0 |
0 |
8 |
14 |
1 |
0 |
15 |
23 |
1.15 |
Pos. control## |
17.5 µg |
+ |
4 / 40 hrs |
68 |
7 |
0 |
75 |
51 |
8 |
0 |
59 |
134 |
6.70 |
Test item |
347 µg |
+ |
4 / 40 hrs |
9 |
0 |
0 |
9 |
9 |
3 |
0 |
12 |
21 |
1.05 |
Test item |
608 µg |
+ |
4 / 40 hrs |
12 |
1 |
0 |
13 |
3 |
0 |
0 |
3 |
16 |
0.80 |
Test item |
1064 µg |
+ |
4 / 40 hrs |
2 |
0 |
0 |
2 |
2 |
0 |
0 |
2 |
4 |
0.20 |
# DMSO
## CPA
Table 3: Number of micronucleated cells; exposure period 20 hrs without S9 mix, Experiment II
Treatment group |
Conc. per mL |
S9 mix |
Exposure / preparation |
Micronucleated cells |
|||||||||
Binucleate cells withnmicronuclei culture 1 |
sum culture 1 |
Binucleate cells withnmicronuclei culture 2 |
sum culture 2 |
sum in 2000 binucleate cells |
[%] |
||||||||
1 |
2 |
>2 |
1 |
2 |
>2 |
||||||||
Solv. control# |
0.5 % |
- |
20 / 40 hrs |
6 |
2 |
0 |
8 |
6 |
0 |
0 |
6 |
14 |
0.70 |
Pos. control## |
100 ng |
- |
20 / 40 hrs |
30 |
7 |
2 |
39 |
22 |
3 |
1 |
26 |
65 |
3.25 |
Test item |
245 µg |
- |
20 / 40 hrs |
5 |
0 |
0 |
5 |
4 |
0 |
0 |
4 |
9 |
0.45 |
Test item |
368 µg |
- |
20 / 40 hrs |
3 |
1 |
0 |
4 |
1 |
0 |
0 |
1 |
5 |
0.25 |
Test item |
552 µg |
- |
20 / 40 hrs |
3 |
0 |
0 |
3 |
2 |
1 |
0 |
3 |
6 |
0.30 |
# DMSO
## Demecolcin
Table 4: Percentage of micronucleated cells in human lymphocyte cultures (2014-2015)
|
Solvent Control without S9 (Micronucleated cells in %) |
Solvent Control with S9 (Micronucleated cells in %) |
|
Pulse treatment (4/40) |
Continuous treatment (20/40) |
Pulse treatment (4/40) |
|
No. of experiments |
50* |
54** |
67*** |
Mean |
0.61 |
0.55 |
0.64 |
95% Ctrl limit |
0.07 – 1.15 |
0.05 – 1.05 |
0.08 – 1.20 |
|
|
|
|
1x SD |
0.27 |
0.25 |
0.28 |
2x SD |
0.54 |
0.50 |
0.56 |
Min |
0.15 |
0.05 |
0.15 |
Max |
1.25 |
1.43 |
1.35 |
* Aqueous solvents – 23 Experiments; Organic solvents – 27 Experiments
** Aqueous solvents – 24 Experiments; Organic solvents – 30 Experiments
*** Aqueous solvents – 24 Experiments; Organic solvents – 43 Experiments
Aqueous solvents: DMEM/Ham’s F12, Deionised water (10% v/v)
Organic solvents: DMSO (0.5 or 1.0%), Acetone, Ethanol and THF (0.5%)
Table 5: Percentage of micronucleated cells in human lymphocyte cultures (2014-2015), continued
Positive Control without S9 (Micronucleated cells in %) |
Positive Control with S9 (Micronucleated cells in %) |
||
Pulse treatment (4/40) MMC |
Continuous treatment (20/40) Demecolcin |
Pulse treatment (4/40) CPA |
|
No. of experiments |
50 |
54 |
81 |
Mean |
11.66 |
3.55 |
4.80 |
95% Ctrl limit |
1.48 – 21.85 |
1.69 – 5.41 |
0.88 – 8.73 |
|
|
|
|
1x SD |
5.09 |
0.93 |
1.96 |
2x SD |
10.18 |
1.86 |
3.92 |
Min |
4.15 |
2.10 |
2.25 |
Max |
24.00 |
6.40 |
11.30 |
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Key study- Ames test
A bacterial reverse mutation assay (OECD 471) was performed to investigate the potential of the test item to induce gene mutations using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA. A plate incorporation test (experiment I and Ia: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate) and a pre-incubation test (experiment II: TA 1537 and TA 100: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate All remaining strains: 10; 33; 100; 333; 1000; 2500; and 5000μg/plate) was performed.
The assay was performed in three independent experiments with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate.
The test item precipitated in the overlay agar in the test tubes from 2500 to 5000 μg/plate in experiment I & Ia and at 5000 μg/plate in experiment II. No precipitation of the test item in the overlay agar on the incubated agar plates was observed.
In the experiment the plates incubated with the test item showed reduced background growth from 2500 to 5000 μg/plate in the absence of metabolic activation (in strain WP2uvrA).
Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in all strains except strain TA 1535.
No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. The controls confirmed the validity of the study.
Key study - MNT
The test item dissolved in DMSO, was assessed for its potential to induce micronuclei in human lymphocytes in vitro in two independent experiments.
In Experiment I, the exposure period was 4 hours with and without S9 mix. In Experiment II, the exposure period was 20 hours without S9 mix. The cells were prepared 40 hours after start of treatment with the test item.
In each experimental group two parallel cultures were analyzed. 1000 binucleate cells per culture were scored for cytogenetic damage on coded slides. To determine a cytotoxic effect the CBPI was determined in 500 cells per culture and cytotoxicity is described as % cytostasis. As highest treatment concentration in this study 1862 μg/mL (approx. 10 mM) was chosen.
In Experiment I, in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration, which showed phase separation or precipitation. In Experiment II in the absence of S9 mix, moderate cytotoxicity was observed at the highest evaluated concentration. Concentrations showing clear cytotoxic effects, however, were not evaluable for cytogenetic damage.
In the absence and presence of S9 mix, no relevant increases in the number of micronucleate cells were observed after treatment with the test item. The controls confirmed the validity of the study.
Justification for classification or non-classification
Classification,
Labelling, and Packaging Regulation (EC) No 1272/2008
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), as amended for the thenth time in
Regulation (EU) No 2017/776.
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