Registration Dossier
Registration Dossier
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EC number: 236-715-1 | CAS number: 13466-20-1
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
In vitro:
Gene mutation (Bacterial reverse mutation assay / Ames test): negative with and without metabolic activation (OECD 471)
Gene mutation (Forward mutation assay / Mouse Lymphoma test): negative with and without metabolic activation (OECD 490)
Cytogenicity (Micronucleous test): negative with and without metabolic activation (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
- 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
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Behörde für Gesundheit und Verbraucherschutz, Hamburg, Germany
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- his operon
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
- Metabolic activation:
- with and without
- Metabolic activation system:
- cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254
- Test concentrations with justification for top dose:
- 31.6, 100, 316, 1000, 3160, 5000 µg/plate
concentrations used up to maximum recommended test concentration - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: 0.05M HCl solution
- Justification for choice of solvent/vehicle: not specified - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- 2-nitrofluorene
- sodium azide
- benzo(a)pyrene
- mitomycin C
- other: 2-amino-anthracene (TA 100, TA 1535 with S9 mix)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation) and preincubation
DURATION
- Preincubation period: 20 minutes
- Exposure duration: 48 to 72 hours
NUMBER OF REPLICATIONS: 3
DETERMINATION OF CYTOTOXICITY
- Method: reduction of the number of colonies by more than 50% compared to the vehicle control and/or scarce background lawn - Evaluation criteria:
- A test item is considered to show a positive response if
- the number of revertants is significantly increased (p ≤ 0.05, U-test according to MANN and WHITNEY) compared to the solvent control to at least 2-fold of the solvent control for TA 98, TA 100, TA1535 and TA 1537 and 1.5-fold of the solvent control for TA 102 in both independent experiments.
- in addition, a significant (p ≤ 0.05) concentration (log value)-related effect (Spearman's rank correlation coefficient) is observed;
- positive results have to be reproducible and the histidine independence of the revertants has to be confirmed by streaking random samples on histidine-free agar plates.
Biological relevance of the results should be considered first.
A test item for which the results do not meet the above mentioned criteria is considered as non-mutagenic in the AMES test. - Key result
- Species / strain:
- S. typhimurium, other: TA 98, TA 100, TA 1535, TA 1537, TA 102
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- RANGE-FINDING/SCREENING STUDIES: Barium bis(dihydrogenorthophosphate) was examined in two preliminary cytotoxicity tests (plate incorporation test without and with metabolic activation) in test strain TA 100. Ten concentrations ranging from 0.316 to 5000 μg/plate were tested. No signs of cytotoxicity were noted up to the top concentration of 5000 μg Barium bis(dihydrogenorthophosphate)/plate.
HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: see table 3
- Negative (solvent/vehicle) historical control data: see table 3 - Conclusions:
- Under the test conditions of this study the test substance was not mutagenic in bacteria with and without metabolic activation.
- Endpoint:
- in vitro cytogenicity / micronucleus study
- Type of information:
- experimental study
- Adequacy of study:
- key study
- 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)
- Remarks:
- Behörde für Gesundheit und Verbraucherschutz, Hamburg, Germany
- Type of assay:
- in vitro mammalian cell micronucleus test
- Target gene:
- not applicable
- Species / strain / cell type:
- lymphocytes: human peripheral lymphocytes
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: human peripheral blood
- Suitability of cells: suitable
- Sex, age and number of blood donors if applicable: male or female, 18 - 35 years old, healthy, non-smoking individuals with no known recent exposures to genotoxic chemicals or radiation
- Whether whole blood or separated lymphocytes were used: not specified
MEDIA USED
- Type and identity of media including CO2 concentration: chromosome complete culture medium with phytohemagglutinin and 1% penicillin/streptomycin - Additional strain / cell type characteristics:
- not applicable
- Cytokinesis block (if used):
- cytochalasin B (cytoB) at 5 µg/mL
- Metabolic activation:
- with and without
- Metabolic activation system:
- cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254
- Test concentrations with justification for top dose:
- 62.5, 125, 250 and 500 µg/mL
Based on preliminary cytotoxoicty test done with concentrations of 3.162, 10.0, 31.62, 100, 316.2, 1000 or 2000 μg/mL. Cytotoxicity was noted starting at concentrations of 316.2 or 1000 μg/mL medium in the experiments without and with metabolic activation (24-hour or 4-hour exposure, respectively). - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: 0.05 M HCl
- Justification for choice of solvent/vehicle: not specified - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- mitomycin C
- other: colchicine
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 4 and 24 hours
- Expression time (cells in growth medium): 20 hours
SPINDLE INHIBITOR (cytogenetic assays): cytoB 5 µg/mL
STAIN (for cytogenetic assays): 10% Giemsa
NUMBER OF REPLICATIONS: 2
NUMBER OF CELLS EVALUATED: at least 2000 binucleated cells per concentration (at least 1000 binucleated cells per culture)
DETERMINATION OF CYTOTOXICITY
- Method: Cytokinesis-Block Proliferation Index - Evaluation criteria:
- A test chemical is considered to be clearly positive if, in any of the experimental conditions examined:
• at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control
• the increase is dose-related in at least one experimental condition when evaluated with an appropriate trend test
• any of the results are outside the distribution of the historical negative control data (Poisson-based 95% control limits)
When all of these criteria are met, the test chemical is then considered able to induce chromosome breaks and/or gain or loss in this test system.
A test chemical is considered clearly negative if, in all experimental conditions examined:
• none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
• there is no concentration-related increase when evaluated with an appropriate trend test,
• all results are inside the distribution of the historical negative control data (Poisson-based 95% control limits).
The test chemical is then considered unable to induce chromosome breaks and/or gain or loss in this test system. - Key result
- Species / strain:
- lymphocytes: human peripheral lymphocytes
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at 500 µg/mL
- 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: No relevant changes.
- Effects of osmolality: No relevant changes.
- Precipitation: none
RANGE-FINDING/SCREENING STUDIES: In a preliminary experiment without and with metabolic activation concentrations of 3.162, 10.0, 31.62, 100, 316.2, 1000 or 2000 μg test substance/mL medium were employed. Cytotoxicity was noted starting at concentrations of 316.2 or 1000 μg test substance/mL medium in the experiments without and with metabolic activation (24-hour or 4-hour exposure, respectively). Hence, 500 μg/mL were employed as the top concentration for the genotoxicity tests without and with metabolic activation.
HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: see table 2
- Negative (solvent/vehicle) historical control data: see table 2
ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: The CBPI indicates the average number of cell cycles per cell during the period of exposure to CytoB, and is used to calculate cell proliferation.
The RI indicates the relative number of nuclei in treated cultures compared to control cultures and can be used to calculate the % cytostasis. An RI of 53% means that, compared to the numbers of cells that have divided to form binucleate and multinucleate cells in the control culture, only 53% of this number divided in the treated culture, i.e. 47% cytostasis. - Conclusions:
- Under the present test conditions, the test substance revealed no indications of chromosomal damage in the in vitro micronucleus test.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
- Version / remarks:
- 2016
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Behörde für Gesundheit und Verbraucherschutz, Hamburg, Germany
- Type of assay:
- other: forward gene mutation assay in mammalian cells
- Target gene:
- TK locus
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: American Type Culture Collection, 0801 University Blvd., Manassas, VA 20110-2209, USA.
MEDIA USED
- Type and identity of media including CO2 concentration if applicable: growth medium: RPMI 1640 with glutamax medium supplemented with Pluronic® F68, gentamycin, amphotericin B and horse serum (10% by volume); treatment medium: growth medium with a reduced horse serum content (5% by volume); Plating medium: growth medium with increased horse serum content but without Pluronic® F68; Selection medium: growth medium that contains 3 μg/mL of TFT
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically 'cleansed' against high spontaneous background: yes - Additional strain / cell type characteristics:
- other: heterzygous at TK locus (+/-)
- Metabolic activation:
- with and without
- Metabolic activation system:
- cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254
- Test concentrations with justification for top dose:
- 31.6, 100, 316, 1000 and 2000 μg /mL
Highest dose used as recommended in guideline. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: 0.05 M HCl
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 3-methylcholanthrene
- methylmethanesulfonate
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
- Cell density at seeding: 0.5 cells/mL
DURATION
- Exposure duration: 3 or 24 hours
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 11 - 14 days
SELECTION AGENT (mutation assays): 5-trifluoro-thymidine (TFT) (3 µg/mL)
NUMBER OF REPLICATIONS: 2
DETERMINATION OF CYTOTOXICITY
- Method: relative total growth - Evaluation criteria:
- For the MLA, significant work on biological relevance and criteria for a positive response has been conducted by The Mouse Lymphoma Expert Workgroup of the IWGT (M.M. Moore et al., 2006). Therefore, the interpretation of test chemical results is based on those recommendations:
To define positive and negative results and to assure that the increased mutation frequency (MF) is biologically relevant instead of a statistical analysis (generally used for other tests), the interpretation relies on the use of a predefined induced mutant frequency (i.e. increase in MF above concurrent control), designated as the Global Evaluation Factor (GEF). The GEF (126 x 10E-6) is based on the analysis of the distribution of the negative control MF data from participating laboratories (M.M. Moore et al., 2006).
Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if, in any of the experimental conditions examined, the increase in MF above the concurrent background exceeds the GEF and the increase is concentration related (e.g., using a trend test). The test chemical is then considered able to induce mutation in this test system.
Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly negative if, in all experimental conditions examined there is no concentration related response or, if there is an increase in MF, it does not exceed the GEF. The test chemical is then considered unable to induce mutations in this test system. In cases when the response is neither clearly negative nor clearly positive as described above and/or in order to assist in establishing the biological relevance of a result the data is evaluated by expert judgement and/or further investigations. - Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at 1000 and 2000 µg/mL
- 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: no relevant changes
- Effects of osmolality: no relevant changes
- Precipitation: see result tables
RANGE-FINDING/SCREENING STUDIES: A preliminary cytotoxicity study was conducted to establish the top concentration for the main study. Concentrations of 10.0, 31.6, 100, 316, 1000 and 2000 μg test substance per mL medium were employed in an experiment without and with metabolic activation. Cytotoxicity (decreased survival) and test item precipitation were noted in the absence and in the presence of metabolic activation at the top concentration of 2000 μg test substance/mL. In addition, cytotoxicity was noted at 1000 μg/mL in the presence of metabolic activation.
HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: see attached file
- Negative (solvent/vehicle) historical control data: see attached file - Conclusions:
- Under this test condtions, the test substance was not mutagenic in mammalian cells with and without metabolic activation because it did neither induce mutations nor have any chromosomal aberration potential.
Referenceopen allclose all
Table 1: Plate incorporation test – number of reverted colonies (mean±standard deviation) |
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EXPERIMENT 1 (Plate incorporation test) |
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S9-Mix |
Without
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Test item (µg/plate) |
TA98 |
TA100 |
TA102 |
TA1535 |
TA1537 |
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NC |
32.7±4.2 |
157.3±15.0 |
275.3±16.9 |
14.7±0.6 |
6.3±1.2 |
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31.6 |
36.3±3.8 |
142.3±10.8 |
275.7±11.0 |
21.3±1.5 |
4.7±0.6 |
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100 |
35.0±5.6 |
126.3±8.1 |
278.3±16.0 |
17.7±4.7 |
6.0±3.5 |
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316 |
34.7±5.9 |
132.0±20.3 |
287.0±3.6 |
18.7±6.7 |
6.0±2.6 |
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1000 |
31.7±4.9 |
152.0±25.4 |
287.7±8.3 |
19.7±8.3 |
4.7±0.6 |
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3160 |
31.7±6.8 |
141.7±11.0 |
291.3±12.7 |
17.0±2.6 |
6.3±0.6 |
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5000 |
34.2±2.1 |
131.0±9.2 |
270.0±16.7 |
16.3±7.8 |
4.3±2.1 |
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Positive reference item |
2-Nitrofluorene 10 µg/plate |
Sodium azide 10 µg/plate |
Mitomycin C 10 µg/plate |
Sodium azide 10 µg/plate |
9-Aminoacridine 100 µg/plate |
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168.3±4.7 |
979.3±2.3 |
1025.3±83.5 |
153.0±4.0 |
82.0±5.6 |
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S9-Mix |
With
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Test item (µg/plate) |
TA98 |
TA100 |
TA102 |
TA1535 |
TA1537 |
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NC |
26.0±0.0 |
156.7±9.3 |
308.7±2.1 |
19.7±2.9 |
6.7±2.3 |
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31.6 |
27.3±2.1 |
154.0±3.0 |
262.7±7.5 |
21.0±2.0 |
6.3±2.3 |
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100 |
34.0±4.6 |
147.0±17.3 |
266.0±3.6 |
18.3±4.5 |
7.3±2.3 |
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316 |
24.7±0.6 |
149.3±16.6 |
267.0±2.6 |
17.0±3.6 |
8.3±1.2 |
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1000 |
31.3±4.2 |
129.0±23.5 |
264.3±4.9 |
19.7±5.5 |
8.3±1.5 |
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3160 |
31.3±2.9 |
133.7-12.4 |
269.0±4.4 |
22.3±7.0 |
5.0±2.6 |
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5000 |
40.0±10.1 |
111.3±4.0 |
271.3±1.2 |
19.7±5.0 |
5.3±2.3 |
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Positive reference item |
Benzo(a)pyrene 10 µg/plate |
2-Aminoanthracene 2 µg/plate |
Benzo(a)pyrene 10 µg/plate |
2-Aminoanthracene 2 µg/plate |
Benzo(a)pyrene 10 µg/plate |
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|
157.0±3.0 |
975.7±10.7 |
1018.7±63.7 |
157.7±6.7 |
111.3±10.7 |
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NC = Vehicle Control
|
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Table 1: Results |
|||||
Test item |
Concentration |
CBPI |
RI |
Number of micronucleated cells per 1000 binucleated cells |
|
|
in µg/mL |
|
% |
|
|
|
Exposure period 4 h, fixation time 24 h, without S9 mix |
||||
HCl |
0.05 M |
1.28 |
100 |
4.5 |
|
Mitomycin C |
0.2 |
1.33 |
122 |
37.0* |
|
Test substance |
62.5 |
1.41 |
150 |
7.0 |
|
125 |
1.40 |
147 |
6.5 |
||
250 |
1.18 |
66 |
7.0 |
||
500 |
1.12 |
43 |
7.5 |
||
|
Exposure period 4 h, fixation time 24 h, with S9 mix |
||||
HCl |
0.05 M |
1.42 |
100 |
7.0 |
|
Cyclophosphamide |
0.2 |
1.37 |
87 |
20.0* |
|
Test substance |
62.5 |
1.41 |
97 |
6.5 |
|
125 |
1.43 |
101 |
5.0 |
||
250 |
1.29 |
69 |
6.5 |
||
|
500 |
1.20 |
48 |
6.5 |
|
|
Exposure period 24 h, fixation time 44 h, without S9 mix |
||||
HCl |
0.05 M |
1.50 |
100 |
5.0 |
|
Colchicin |
0.02 |
1.49 |
99 |
114.5* |
|
Test substance |
62.5 |
1.53 |
108 |
4.5 |
|
125 |
1.54 |
109 |
6.0 |
||
250 |
1.33 |
67 |
5.0 |
||
|
500 |
1.25 |
50 |
8.0 |
|
CBPI: Cytokinesis block proliferation index RI: Replicative Index * : significantly different from negative control (p < 0.05), CHI-SQUARE Test |
|||||
Table 2: Historical background data |
||||
|
Micronucleus frequency per 1000 cells |
|||
|
Without metabolic activation |
With metabolic activation |
||
|
Untreated control |
Vehicle control |
Untreated control |
Vehicle control |
mean |
6.6 |
6.3 |
6.4 |
6.1 |
SD |
2.9 |
3.1 |
2.6 |
4.2 |
range |
2.0 - 17 |
2.0 - 18 |
3.0 - 13 |
1 - 22 |
95% Confidence interval |
5.9 – 7.3 |
5.7 – 6.8 |
5.7 – 7.1 |
5.1 – 6.7 |
|
Mitomycin C Positive control |
Colchicine Positive control |
Cyclophosphamide Positive control |
|
mean |
46.9 |
25.9 |
44.0 |
|
SD |
35.0 |
9.5 |
37.7 |
|
range |
17 -137 |
16 -63 |
14 -158 |
|
|
|
|
|
|
Table 1: Results |
|
|
|||||
Concentration [µg/mL] |
S9 mix |
RTG |
CE % |
MF/10E6 |
Diff/10E6 |
% large colonies of total colonies |
% large colonies of total colonies |
1stexperiment (3-hour exposure) |
|||||||
0 |
- |
100 |
81.64 |
60.30 |
- |
86.1 |
13.9 |
31.6 |
- |
62 |
100.63 |
59.54 |
-0.76 |
60.0 |
40.0 |
100 |
- |
162 |
98.04 |
53.14 |
-7.16 |
68.4 |
31.6 |
316 |
- |
124 |
86.64 |
53.50 |
-6.80 |
70.6 |
29.4 |
1000 |
- |
86 |
92.08 |
53.43 |
-6.87 |
66.7 |
33.3 |
2000* |
- |
12 |
16.69 |
55.12 |
-5.18 |
71.4 |
28.6 |
MMS 0.013 |
- |
17 |
24.38 |
692.17 |
631.87 |
34.5 |
65.5 |
2ndexperiment (24-hour exposure) |
|||||||
0 |
- |
100 |
86.16 |
52.20 |
- |
66.7 |
33.3 |
31.6 |
- |
131 |
79.28 |
74.92 |
22.72 |
60.5 |
39.5 |
100 |
- |
109 |
84.09 |
68.85 |
16.65 |
66.7 |
33.3 |
316 |
- |
38 |
93.52 |
51.11 |
-1.09 |
68.6 |
31.4 |
1000 |
- |
14 |
81.64 |
56.77 |
4.57 |
64.7 |
35.3 |
2000* |
- |
1 |
11.39 |
115.89 |
63.69 |
40.0 |
60.0 |
MMS 0.013 |
- |
27 |
24.86 |
823.41 |
771.21 |
31.8 |
68.2 |
1stexperiment (3-hour exposure) |
|||||||
0 |
+ |
100 |
70.68 |
77.88 |
- |
75.0 |
25.0 |
31.6 |
+ |
108 |
74.82 |
101.24 |
23.36 |
59.3 |
40.7 |
100 |
+ |
123 |
90.68 |
60.65 |
-17.23 |
70.0 |
30.0 |
316 |
+ |
89 |
77.01 |
134.79 |
56.91 |
77.8 |
22.2 |
1000 |
+ |
94 |
86.64 |
73.64 |
-4.24 |
56.5 |
43.5 |
2000* |
+ |
10 |
15.01 |
61.29 |
-16.59 |
57.1 |
42.9 |
3-MC 1.0 |
+ |
13 |
17.55 |
731.91 |
654.03 |
43.7 |
56.3 |
2ndexperiment (3-hour exposure) |
|||||||
0 |
+ |
100 |
81.75 |
61.34 |
- |
69.9 |
30.1 |
31.6 |
+ |
114 |
72.70 |
151.65 |
90.31 |
36.8 |
63.2 |
100 |
+ |
110 |
68.66 |
110.33 |
48.99 |
59.3 |
40.7 |
316 |
+ |
99 |
65.80 |
115.12 |
53.78 |
44.4 |
55.6 |
1000 |
+ |
43 |
69.65 |
54.34 |
-7.00 |
50.0 |
50.0 |
2000 |
+ |
16 |
20.19 |
75.50 |
17.16 |
50.0 |
50.0 |
3-MC 1.0 |
+ |
17 |
22.48 |
639.90 |
578.56 |
37.5 |
62.5 |
*: test substance precipitation MMS: Methylmethansulfonate; 3-MC: 3-Methylcholanthrene RTG: Relative growth rate MF/10E6: Mutant frequency/10E6 cells CE: Cloning efficiency Diff/10E6: MF/10E6 (test substance) - MF/10E6 (control) |
|||||||
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Reliable studies regarding genetic toxicity are available for the test substance.
In vitro:
- Gene mutation in bacteria:
The potential of the test substance to induce gene mutations was assessed in a study performed according to OECD Guideline 471 and in compliance with GLP (LTP, 2016). The test substance was examined in the 5 Salmonella typhimurium strains TA 98, TA 100, TA 102, TA 1535, and TA 1537 in two independent experiments, each carried out without and with metabolic activation (a microsomal preparation derived from Aroclor 1254-induced rat liver). The first experiment was carried out as a plate incorporation test and the second as a preincubation test. 0.05 M HCl solution was used as vehicle. Six concentrations, 31.6, 100, 316, 1000, 3160, and 5000 μg test substance/plate were employed in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation with three replicates each. No signs of cytotoxicity were noted up to the top concentration of 5000 μg test substance/plate in any test strain. No increase in revertant colony numbers as compared with control counts was observed for the test substance, tested up to a concentration of 5000 μg/plate in any of the 5 test strains in two independent experiments without and with metabolic activation, respectively (plate incorporation and preincubation test). The positive control items showed a significant increase in the number of revertant colonies of the respective test strain and confirmed the validity of the test conditions and the sensitivity of the test system. Thus, under the conditions of this study, the test substance is considered to be non-mutagenic.
- Gene mutation in mammalian cells:
The potential of the test substance to induce gene mutations was assessed in a study performed according to OECD Guideline 490 and in compliance with GLP (LTP, 2016). The test substance was assayed in a gene mutation assay in cultured mammalian cells (L5178Y TK +/-) both in the presence and absence of metabolic activation by a liver post-mitochondrial fraction (S9 mix) from Aroclor 1254-induced rats. The test was carried out employing two exposure times without S9 mix: 3 and 24 hours, and one exposure time with S9 mix: 3 hours; the experiment with S9 mix was carried out in two independent assays. 0.05 M HCl solution served as vehicle. The concentrations 31.6, 100, 316, 1000 and 2000 µg/mL for the experiments without and with metabolic activation were chosen based on the results of a cytotoxicity study. The mutation frequencies of the cultures treated with the test substance ranged from 53.14 to 59.54 per 10E06 cloneable cells (3-hour exposure) and from 51.11 to 115.89 per 10E06 cloneable cells (24-hour exposure) in the experiments without metabolic activation and from 60.65 to 134.79 per 10E06 cloneable cells (3-hour exposure, first assay) and from 54.34 to 151.65 per 10E06 cloneable cells (3-hour exposure, second assay) in the experiments with metabolic activation. These results were within the range of the negative control values and the normal range of 50 to 170 mutants per 10E06 viable cells and, hence, no mutagenicity was observed according to the criteria for assay evaluation. In addition, no change was observed in the ratio of small to large mutant colonies, ranging from 0.29 to 1.71 for test substance-treated cells and from 0.13 to 0.55 for the negative controls. A ratio of 1.71, calculated for the lowest dose tested in the 2nd experiment with S9 activation (31.6 μg/mL) is slightly outside historical control data. This finding is regarded to be without any biological relevance due to lacking of dose dependence. Cytotoxicity (decreased relative total growth (RTG: 10% to 16%)) was noted at the top concentration of 2000 μg test substance/mL in the absence and presence of metabolic activation (3-hour exposure) and at 1000 and 2000 μg/mL medium in the experiment without S9 mix with a 24-hour exposure (RTG: 14% and 1%, respectively). In addition, test item precipitation was noted at the top concentration of 2000 μg/mL in all experiments.
The positive controls Methylmethanesulfonate (MMS) and 3-Methylcholanthrene (3-MC) caused pronounced increases in the mutation and the colony size ratio was moderately shifted towards an increase in small colonies, showing the validity of the test system.
Thus, under the conditions of this study, the test substance is considered to be non-mutagenic because it neither induces mutations nor has any chromosomal aberration potential.
- Cytogenicity / micronucleus test in mammalian cells:
The potential of the test substance to induce chromosome aberration in human peripheral lymphocytes was assessed in a study performed according to OECD Guideline 487 and in compliance with GLP (LPT, 2016). The test substance was assayed in an in vitro micronucleus test using human peripheral lymphocytes both in the presence and absence of metabolic activation by a rat liver post-mitochondrial fraction (S9 mix) from Aroclor 1254-induced animals. The test was carried out employing two exposure times without S9 mix: 4 and 24 hours, and one exposure time with S9 mix: 4 hours. The harvesting time was 20 hours after the end of exposure. The cytokinesis-block technique was applied. 0.05 M HCl solution served as the vehicle. The concentrations employed were chosen based on the results of a cytotoxicity study. Cytotoxicity was noted starting at concentrations of 316.2 or 1000 μg test substance/mL medium in the experiments without and with metabolic activation (24-hour or 4-hour exposure, respectively). Hence, concentrations of 62.5, 125, 250 or 500 μg/mL were used for the genotoxicity tests without and with metabolic activation. In the main study cytotoxicity was noted in the experiments without and with metabolic activation at the top concentration of 500 μg test item/mL medium. Mitomycin C (at 0.2 μg/mL) and colchicine (at 0.02 μg/mL) were used as positive controls in the absence and cyclophosphamide (at 20 μg/mL) in the presence of metabolic activation. The micronucleus frequencies of cultures treated with the test substance in the absence of metabolic activation (4- and 24-hour exposure, respectively) ranged from 4.5 to 8.0 micronucleated cells per 1000 binucleated cells. The results for the vehicle control from this study were all within the historical control data. The test substance data were within or slightly above the upper limit of the 95% confidence interval of the historical background data at LPT. However, the test substance data in this study gave reproducibly low and consistent micronuclei frequencies without extreme outliers. There was no dose-related increase in micronuclei up to the top concentration of 500 μg test substance/mL medium. The micronucleus frequencies of cultures treated with the test substance (4-h exposure) in the presence of metabolic activation ranged from 5.0 to 6.5 micronucleated cells per 1000 binucleated cells. There was no dose-related increase in micronuclei up to the top concentration of 500 μg test substance/mL medium. The frequency of micronucleated cells was within the historical control range of the untreated and vehicle controls.
In the respective positive control cultures the micronucleus frequency was increased. This demonstrated that test system is valid.
Thus, under the conditions of this study, the test substance is considered to be not clastogenic and not aneugenic.
Justification for classification or non-classification
The available data on genetic toxicity do not meet the criteria for classification according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
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