Registration Dossier
Registration Dossier
Data platform availability banner - registered substances factsheets
Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.
The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.
Diss Factsheets
Use of this information is subject to copyright laws and may require the permission of the owner of the information, as described in the ECHA Legal Notice.
EC number: 264-935-8 | CAS number: 64519-44-4
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Ames test: non mutagenic (OECD 471, GLP, K, rel. 1).
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:
- 19 July - 25 August 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- GLP study conducted according to OECD Guideline 471 without any deviation
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- dated 21 July 1997
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Version / remarks:
- dated 30 May 2008
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
- Version / remarks:
- dated August 1998
- Deviations:
- no
- Principles of method if other than guideline:
- Not applicable
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- 28 October 2016
- Type of assay:
- bacterial reverse mutation assay
- Specific details on test material used for the study:
- TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- The test item was accurately weighed and approximate half-log dilutions prepared in dimethyl sulphoxide by mixing on a vortex mixer and sonication for 10 minutes at approximately 40 °C on the day of each experiment. No correction was required for purity. Prior to use, the solvent was dried to remove water using molecular sieves i.e. 2 mm sodium alumino silicate pellets with a nominal pore diameter of 4 x 10^-4 microns. All formulations were used within four hours of preparation and were assumed to be stable for this period. - Target gene:
- histidine locus for Salmonella strains and tryptophan for E. coli strain
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
- Details on mammalian cell type (if applicable):
- Not applicable
- Additional strain / cell type characteristics:
- not applicable
- Cytokinesis block (if used):
- Not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- rat liver homogenate metabolizing system (10% liver S9 in standard co-factors)
- Test concentrations with justification for top dose:
- - Experiment 1 - Plate Incorporation Method: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate, with and without S9-mix
- Experiment 2 - Pre-Incubation Method: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate, with and without S9-mix - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: The test item was immiscible in sterile distilled water at 50 mg/mL but was fully miscible in dimethyl sulphoxide at the same concentration in solubility checks performed in house. Dimethyl sulphoxide was therefore selected as the vehicle. - Untreated negative controls:
- yes
- Remarks:
- untreated
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- 9-aminoacridine
- N-ethyl-N-nitro-N-nitrosoguanidine
- Remarks:
- without metabolic activation
- Untreated negative controls:
- yes
- Remarks:
- untreated
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- benzo(a)pyrene
- other: 2-Aminoanthracene
- Remarks:
- with metabolic activation
- Details on test system and experimental conditions:
- SOURCE OF TEST SYSTEM: The bacteria used in the test were obtained from:
- University of California, Berkeley, on culture discs, on 04 August 1995
- British Industrial Biological Research Association, on a nutrient agar plate, on 17 August 1987
METHOD OF APPLICATION: in agar (plate incorporation) and preincubation
DURATION
- Preincubation period: 20 minutes in Experiment 2.
- Exposure duration: ca. 48 hours
CONTROLS:
- Vehicle/solvent control: DMSO
- Negative (untreated) controls were performed to assess the spontaneous revertant colony rate.
- Positive control items used demonstrated a direct and indirect acting mutagenic effect depending on the presence or absence of metabolic activation.
- Sterility controls were performed in triplicate as follows:
Top agar and histidine/biotin or tryptophan in the absence of S9-mix;
Top agar and histidine/biotin or tryptophan in the presence of S9-mix; and
The maximum dosing solution of the test item in the absence of S9-mix only (test in singular only).
NUMBER OF REPLICATIONS: Triplicate
- OTHER: All of the plates were incubated at 37 ± 3 °C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). Several manual counts were required due to revertant colonies spreading slightly, thus distorting the actual plate count. - Rationale for test conditions:
- The dose range for Experiment 1 was predetermined and was 1.5 to 5000 µg/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was the same as Experiment 1 (15 to 5000 µg/plate). Eight test item concentrations were selected in Experiment 2 in order to achieve both four non toxic dose levels and the potential toxic limit of the test item following the change in test methodology.
- Evaluation criteria:
- Criteria for determining a positive result:
- A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
- A reproducible increase at one or more concentrations.
- Biological relevance against in-house historical control ranges.
- Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out of historical range response (Cariello and Piegorsch, 1996)).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal. - Statistics:
- None
- Key result
- Species / strain:
- other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- refer tables 7.6.1/2 to 7.6.1/5
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.
MUTAGENICITY
- The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
- In the first experiment (plate incorporation method), the test item caused a visible reduction in the growth of the bacterial background lawn in all of the tester strains without metabolic activation (S9), initially from 1500 µg/plate (TA100, TA98 and TA1537) and at 5000 µg/plate (TA1535 and WP2uvrA). In the presence of S9, toxicity was observed at and above 1500 µg/plate to TA98 and TA1537 and at 5000 µg/plate to strains TA100, TA1535 and WP2uvrA. Additionally, a substantial decrease in the frequency of revertant colonies was observed to TA100 at 1500 µg/plate.
In the second experiment (pre-incubation method), the test item caused a slightly stronger toxic response with visible reductions in the growth of the bacterial background lawn noted in the absence of S9 from 500 µg/plate (TA100, TA1535, WP2uvrA, and TA1537) and from 1500 µg/plate (TA98). In the presence of S9-mix, toxicity to the bacterial background lawns of all the tester strains was observed at 1500 µg/plate. Again, a substantial decrease in the frequency of revertant colonies was observed to TA100 at 500 µg/plate (presence of S9).
- No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method) and Experiment 2 (pre incubation method).
- Refer Tables 7.6.1/1 to 7.6.1/5 for more details.
HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: Refer Table 7.6.1/6
- Negative (solvent/vehicle) historical control data: Refer Table 7.6.1/6
OTHERS:
- Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile. The test item formulation was also shown to be sterile.
- Results for the negative controls (spontaneous mutation rates) are presented in 7.6.1/1 and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test. - Conclusions:
- Under the test conditions, the test item is not considered as mutagenic in Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100, and Escherichia coli strain WP2uvrA.
- Executive summary:
In a reverse gene mutation assay in bacteria, performed according to the OECD Guideline 471 and in compliance with GLP, Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100, and Escherichia coli strain WP2uvrA were exposed to the test item at the following concentrations:
- Experiment 1 - Plate Incorporation Method: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate, with and without S9-mix
- Experiment 2 - Pre-Incubation Method: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate, with and without S9-mix
Rat liver homogenate (10% liver S9 in standard co-factors) was used as a metabolizing system. Vehicle control, negative (untreated) and positive control groups were also included in mutagenicity tests.
The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
In the first experiment (plate incorporation), the test item caused a visible reduction in the growth of the bacterial background lawn in all of the tester strains both with and without metabolic activation (S9), initially from 1500 µg/plate.
In the second experiment (pre-incubation), the test item caused a visible reduction in the growth of the bacterial background lawn in all of the tester strains, initially from 500 and 1500 µg/plate in the absence and presence of S9-mix, respectively.
No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.
No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method) and Experiment 2 (pre incubation method).
Under the test conditions, the test item is not considered as mutagenic in these bacterial systems.
Reference
Table 7.6.1/1:Spontaneous Mutation Rates (Concurrent Negative Controls)
Number of revertants (mean number of colonies per plate) |
|||||||||
Base-pair substitution type |
Frameshift type |
||||||||
Experiment 1 |
|||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
|||||
74 |
|
14 |
|
15 |
|
25 |
|
12 |
|
71 |
(71) |
21 |
(16) |
12 |
(13) |
22 |
(23) |
6 |
(9) |
69 |
|
13 |
|
13 |
|
23 |
|
8 |
|
Experiment 2 |
|||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
|||||
75 |
|
20 |
|
16 |
|
28 |
|
14 |
|
66 |
(73) |
19 |
(20) |
21 |
(19) |
16 |
(20) |
12 |
(11) |
78 |
|
21 |
|
20 |
|
17 |
|
8 |
|
Table 7.6.1/2:Test Results: Experiment 1 – Without Metabolic Activation
Test Period |
From: 08 August 2017 |
To: 11 August 2017 |
||||||||||
S9-Mix (-) |
Dose Level Per Plate |
Number of revertants (mean) +/- SD |
||||||||||
Base-pair substitution strains |
Frameshift strains |
|||||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
||||||||
Solvent Control (DMSO) |
63 68 80 |
(70) 8.7# |
16 18 18 |
(17) 1.2 |
22 22 19 |
(21) 1.7 |
35 29 35 |
(33) 3.5 |
8 13 12 |
(11) 2.6 |
||
1.5 µg |
60 68 70 |
(66) 5.3 |
16 22 23 |
(20) 3.8 |
17 19 20 |
(19) 1.5 |
27 26 31 |
(28) 2.6 |
9 16 9 |
(11) 4.0 |
||
5 µg |
72 73 94 |
(80) 12.4 |
24 22 18 |
(21) 3.1 |
20 13 16 |
(16) 3.5 |
27 27 25 |
(26) 1.2 |
23 14 8 |
(15) 7.5 |
||
15 µg |
83 61 80 |
(75) 11.9 |
19 21 17 |
(19) 2.0 |
20 21 15 |
(19) 3.2 |
25 22 22 |
(23) 1.7 |
12 19 8 |
(13) 5.6 |
||
50 µg |
76 80 75 |
(77) 2.6 |
12 8 14 |
(11) 3.1 |
13 14 17 |
(15) 2.1 |
25 25 17 |
(22) 4.6 |
13 13 10 |
(12) 1.7 |
||
150 µg |
84 69 88 |
(80) 10.0 |
14 25 14 |
(18) 6.4 |
11 16 15 |
(14) 2.6 |
18 19 17 |
(18) 1.0 |
10 12 18 |
(13) 4.2 |
||
500 µg |
72 73 62 |
(69) 6.1 |
21 14 14 |
(16) 4.0 |
11 15 19 |
(15) 4.0 |
20 22 14 |
(19) 4.2 |
7 22 10 |
(13) 7.9 |
||
1500 µg |
54 S 61 S 47 S |
(54) 7.0 |
20 18 22 |
(20) 2.0 |
17 16 17 |
(17) 0.6 |
12 S 12 S 21 S |
(15) 5.2 |
0 V 0 V 0 V |
(0) 0.0 |
||
5000 µg |
0 V 0 V 0 V |
(0) 0.0 |
0 V 0 V 0 V |
(0) 0.0 |
7 S 5 S 5 S |
(6) 1.2 |
0 V 0 V 0 V |
(0) 0.0 |
0 T 0 T 0 T |
(0) 0.0 |
||
Positive controls S9-Mix (-) |
Name Dose Level No. of Revertants |
ENNG |
ENNG |
ENNG |
4NQO |
9AA |
||||||
3 µg |
5 µg |
2 µg |
0.2 µg |
80 µg |
||||||||
448 547 472 |
(489) 51.6 |
317 374 477 |
(389) 81.1 |
531 543 570 |
(548) 20.0 |
161 176 178 |
(172) 9.3 |
158 273 236 |
(222) 58.7 |
|||
ENNG:N-ethyl-N'-nitro-N-nitrosoguanidine
4NQO:4-Nitroquinoline-1-oxide
9AA: 9-Aminoacridine
S: Sparse bacterial background lawn
V: Very weak bacterial background lawn
T: Toxic, no bacterial background lawn
#: Standard deviation
Table 7.6.1/3:Test Results: Experiment 1 – With Metabolic Activation
Test Period |
From: 08 August 2017 |
To: 11 August 2017 |
||||||||||
S9-Mix (+) |
Dose Level Per Plate |
Number of revertants (mean) +/- SD |
||||||||||
Base-pair substitution strains |
Frameshift strains |
|||||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
||||||||
Solvent Control (DMSO) |
65 90 88 |
(81) 13.9# |
11 24 13 |
(16) 7.0 |
27 27 15 |
(23) 6.9 |
26 28 27 |
(27) 1.0 |
10 6 15 |
(10) 4.5 |
||
1.5 µg |
82 97 77 |
(85) 10.4 |
27 26 10 |
(21) 9.5 |
25 19 19 |
(21) 3.5 |
29 26 29 |
(28) 1.7 |
11 17 12 |
(13) 3.2 |
||
5 µg |
68 81 72 |
(74) 6.7 |
22 20 16 |
(19) 3.1 |
12 19 28 |
(20) 8.0 |
26 27 28 |
(27) 1.0 |
14 15 2 |
(10) 7.2 |
||
15 µg |
84 109 70 |
(88) 19.8 |
12 17 28 |
(19) 8.2 |
25 22 19 |
(22) 3.0 |
21 27 27 |
(25) 3.5 |
4 12 15 |
(10) 5.7 |
||
50 µg |
74 82 84 |
(80) 5.3 |
23 15 20 |
(19) 4.0 |
20 22 13 |
(18) 4.7 |
34 12 18 |
(21) 11.4 |
16 20 6 |
(14) 7.2 |
||
150 µg |
66 72 66 |
(68) 3.5 |
12 19 19 |
(17) 4.0 |
17 18 20 |
(18) 1.5 |
24 25 13 |
(21) 6.7 |
11 8 10 |
(10) 1.5 |
||
500 µg |
62 61 64 |
(62) 1.5 |
14 11 14 |
(13) 1.7 |
18 22 21 |
(20) 2.1 |
24 24 23 |
(24) 0.6 |
16 8 10 |
(11) 4.2 |
||
1500 µg |
45 59 47 |
(50) 7.6 |
16 16 19 |
(17) 1.7 |
25 16 23 |
(21) 4.7 |
18 S 11 S 12 S |
(14) 3.8 |
8 S 8 S 8 S |
(8) 0.0 |
||
5000 µg |
0 V 0 V 0 V |
(0) 0.0 |
0 V 0 V 0 V |
(0) 0.0 |
17 S 22 S 16 S |
(18) 3.2 |
0 V 0 V 0 V |
(0) 0.0 |
0 T 0 T 0 T |
(0) 0.0 |
||
Positive controls S9-Mix (+) |
Name Dose Level No. of Revertants |
2AA |
2AA |
2AA |
BP |
2AA |
||||||
1 µg |
2 µg |
10 µg |
5 µg |
2 µg |
||||||||
1129 1130 1390 |
(1216) 150.4 |
234 244 202 |
(227) 21.9 |
111 81 87 |
(93) 15.9 |
188 155 175 |
(173) 16.6 |
338 308 265 |
(304) 36.7 |
|||
2AA: 2-Aminoanthracene
BP: Benzo(a)pyrene
S: Sparse bacterial background lawn
V: Very weak bacterial background lawn
T: Toxic, no bacterial background lawn
#: Standard deviation
Table 7.6.1/4:Test Results: Experiment 2 – Without Metabolic Activation
Test Period |
From: 22 August 2017 |
To: 25 August 2017 |
||||||||||
S9-Mix (-) |
Dose Level Per Plate |
Number of revertants (mean) +/- SD |
||||||||||
Base-pair substitution strains |
Frameshift strains |
|||||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
||||||||
Solvent Control (DMSO) |
77 70 66 |
(71) 5.6# |
23 15 21 |
(20) 4.2 |
20 19 21 |
(20) 1.0 |
15 13 20 |
(16) 3.6 |
11 14 16 |
(14) 2.5 |
||
1.5 µg |
79 68 67 |
(71) 6.7 |
23 18 16 |
(19) 3.6 |
12 18 25 |
(18) 6.5 |
12 26 20 |
(19) 7.0 |
13 8 9 |
(10) 2.6 |
||
5 µg |
64 71 76 |
(70) 6.0 |
18 18 20 |
(19) 1.2 |
14 13 18 |
(15) 2.6 |
22 22 17 |
(20) 2.9 |
13 12 9 |
(11) 2.1 |
||
15 µg |
78 64 64 |
(69) 8.1 |
14 22 24 |
(20) 5.3 |
21 28 21 |
(23) 4.0 |
17 21 14 |
(17) 3.5 |
13 8 19 |
(13) 5.5 |
||
50 µg |
76 61 68 |
(68) 7.5 |
26 24 25 |
(25) 1.0 |
17 21 17 |
(18) 2.3 |
24 17 23 |
(21) 3.8 |
13 10 15 |
(13) 2.5 |
||
150 µg |
61 72 69 |
(67) 5.7 |
11 22 17 |
(17) 5.5 |
26 20 19 |
(22) 3.8 |
24 21 22 |
(22) 1.5 |
10 6 18 |
(11) 6.1 |
||
500 µg |
61 S 50 S 47 S |
(53) 7.4 |
16 S 17 S 17 S |
(17) 0.6 |
20 S 9 S 11 S |
(13) 5.9 |
14 17 17 |
(16) 1.7 |
4 S 5 S 3 S |
(4) 1.0 |
||
1500 µg |
0 V 0 V 0 V |
(0) 0.0 |
0 V 0 V 0 V |
(0) 0.0 |
11 S 14 S 25 S |
(17) 7.4 |
0 V 0 V 0 V |
(0) 0.0 |
0 V 0 V 0 V |
(0) 0.0 |
||
5000 µg |
0 T 0 T 0 T |
(0) 0.0 |
0 T 0 T 0 T |
(0) 0.0 |
0 T 0 T 0 T |
(0) 0.0 |
0 T 0 T 0 T |
(0) 0.0 |
0 T 0 T 0 T |
(0) 0.0 |
||
Positive controls S9-Mix (-) |
Name Dose Level No. of Revertants |
ENNG |
ENNG |
ENNG |
4NQO |
9AA |
||||||
3 µg |
5 µg |
2 µg |
0.2 µg |
80 µg |
||||||||
686 770 706 |
(721) 43.9 |
353 416 455 |
(408) 51.5 |
760 695 863 |
(773) 84.7 |
235 280 250 |
(255) 22.9 |
246 308 332 |
(295) 44.4 |
|||
ENNG:N-ethyl-N'-nitro-N-nitrosoguanidine
4NQO:4-Nitroquinoline-1-oxide
9AA: 9-Aminoacridine
S: Sparse bacterial background lawn
V: Very weak bacterial background lawn
T: Toxic, no bacterial background lawn
#: Standard deviation
Table 7.6.1/5:Test Results: Experiment 2 – With Metabolic Activation
Test Period |
From: 22 August 2017 |
To: 25 August 2017 |
||||||||||
S9-Mix (+) |
Dose Level Per Plate |
Number of revertants (mean) +/- SD |
||||||||||
Base-pair substitution strains |
Frameshift strains |
|||||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
||||||||
Solvent Control (DMSO) |
71 70 69 |
(70) 1.0# |
12 17 17 |
(15) 2.9 |
25 24 28 |
(26) 2.1 |
29 22 25 |
(25) 3.5 |
17 14 14 |
(15) 1.7 |
||
1.5 µg |
67 66 63 |
(65) 2.1 |
21 17 18 |
(19) 2.1 |
22 38 22 |
(27) 9.2 |
20 24 25 |
(23) 2.6 |
17 9 11 |
(12) 4.2 |
||
5 µg |
64 74 64 |
(67) 5.8 |
25 23 17 |
(22) 4.2 |
28 37 28 |
(31) 5.2 |
18 15 27 |
(20) 6.2 |
17 5 9 |
(10) 6.1 |
||
15 µg |
63 69 68 |
(67) 3.2 |
22 22 22 |
(22) 0.0 |
28 21 36 |
(28) 7.5 |
18 12 25 |
(18) 6.5 |
10 18 19 |
(16) 4.9 |
||
50 µg |
68 68 64 |
(67) 2.3 |
22 24 12 |
(19) 6.4 |
29 18 22 |
(23) 5.6 |
25 32 29 |
(29) 3.5 |
10 8 6 |
(8) 2.0 |
||
150 µg |
73 64 70 |
(69) 4.6 |
13 18 24 |
(18) 5.5 |
20 21 20 |
(20) 0.6 |
24 20 24 |
(23) 2.3 |
13 25 13 |
(17) 6.9 |
||
500 µg |
73 50 38 |
(54) 17.8 |
26 19 26 |
(24) 4.0 |
23 21 23 |
(22) 1.2 |
19 23 16 |
(19) 3.5 |
16 8 12 |
(12) 4.0 |
||
1500 µg |
50 V 0 V 0 V |
(17) 28.9 |
11 S 12 S 11 S |
(11) 0.6 |
16 S 10 S 18 S |
(15) 4.2 |
0 V 0 V 0 V |
(0) 0.0 |
0 V 0 V 0 V |
(0) 0.0 |
||
5000 µg |
0 T 0 T 0 T |
(0) 0.0 |
0 V 0 V 0 V |
(0) 0.0 |
0 V 0 V 0 V |
(0) 0.0 |
0 T 0 T 0 T |
(0) 0.0 |
0 T 0 T 0 T |
(0) 0.0 |
||
Positive controls S9-Mix (+) |
Name Dose Level No. of Revertants |
2AA |
2AA |
2AA |
BP |
2AA |
||||||
1 µg |
2 µg |
10 µg |
5 µg |
2 µg |
||||||||
970 923 1076 |
(990) 78.4 |
231 279 241 |
(250) 25.3 |
197 140 246 |
(194) 53.1 |
137 128 163 |
(143) 18.2 |
470 395 425 |
(430) 37.7 |
|||
2AA: 2-Aminoanthracene
BP: Benzo(a)pyrene
S: Sparse bacterial background lawn
V: Very weak bacterial background lawn
T: Toxic, no bacterial background lawn
#: Standard deviation
Table 7.6.1/6:History Profile of Vehicle and Positive Control Values
COMBINED VEHICLE AND UNTREATED CONTROL VALUES 2015 |
|||||||||||||||||||||||||||||||||
Strain S9-Mix |
TA100 |
TA1535 |
TA102 |
WP2uvrA |
TA98 |
TA1537 |
WP2uvrA pKM101 |
WP2pKM101 |
|||||||||||||||||||||||||
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
||||||||||||||||||
Values† |
274 |
278 |
504 |
285 |
26 |
13 |
461 |
229 |
526 |
299 |
506 |
282 |
42 |
51 |
39 |
49 |
|||||||||||||||||
Min |
60 |
61 |
7 |
7 |
222 |
278 |
10 |
12 |
11 |
10 |
4 |
6 |
87 |
98 |
89 |
93 |
|||||||||||||||||
Max |
166 |
175 |
31 |
29 |
376 |
388 |
58 |
43 |
45 |
46 |
27 |
27 |
237 |
254 |
174 |
177 |
|||||||||||||||||
Mean |
91 |
95 |
16 |
14 |
286 |
333 |
24 |
27 |
21 |
24 |
12 |
13 |
156 |
164 |
123 |
137 |
|||||||||||||||||
SD |
19.3 |
19.1 |
4.5 |
4.0 |
48.7 |
37.6 |
5.6 |
5.9 |
6.2 |
6.1 |
3.8 |
3.4 |
42.2 |
35.6 |
23.1 |
21.2 |
|||||||||||||||||
POSITIVE CONTROL VALUES 2015 |
|
||||||||||||||||||||||||||||||||
Strain S9-Mix |
TA100 |
TA1535 |
TA102 |
WP2uvrA |
TA98 |
TA1537 |
WP2uvrA pKM101 |
WP2pKM101 |
|
||||||||||||||||||||||||
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
|
|||||||||||||||||
Values |
276 |
280 |
252 |
264 |
13 |
13 |
231 |
227 |
262 |
276 |
253 |
261 |
20 |
35 |
20 |
35 |
|
||||||||||||||||
Min |
222 |
250 |
79 |
118 |
953 |
673 |
116 |
103 |
100 |
78 |
164 |
97 |
430 |
494 |
745 |
325 |
|
||||||||||||||||
Max |
2266 |
2402 |
2779 |
457 |
3140 |
1655 |
2769 |
550 |
502 |
705 |
2318 |
823 |
1696 |
2264 |
3662 |
1174 |
|
||||||||||||||||
Mean |
614 |
927 |
472 |
246 |
2303 |
1093 |
792 |
266 |
222 |
218 |
911 |
336 |
761 |
1461 |
2257 |
569 |
|
||||||||||||||||
SD |
260.6 |
452.5 |
434.8 |
55.7 |
815.2 |
376.5 |
342.1 |
97.7 |
70.2 |
107.6 |
412.4 |
135.7 |
350.0 |
382.0 |
790.7 |
220.3 |
|
||||||||||||||||
COMBINED VEHICLE AND UNTREATED CONTROL VALUES 2016 |
|||||||||||||||||||||||||||||||||
Strain S9-Mix |
TA100 |
TA1535 |
TA102 |
WP2uvrA |
TA98 |
TA1537 |
WP2uvrA pKM101 |
WP2pKM101 |
|||||||||||||||||||||||||
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
||||||||||||||||||
Values |
399 |
401 |
758 |
393 |
60 |
30 |
690 |
345 |
788 |
415 |
762 |
398 |
32 |
32 |
16 |
24 |
|||||||||||||||||
Min |
63 |
66 |
8 |
8 |
216 |
221 |
10 |
13 |
8 |
12 |
3 |
4 |
97 |
104 |
78 |
52 |
|||||||||||||||||
Max |
154 |
156 |
34 |
39 |
340 |
375 |
53 |
53 |
49 |
51 |
24 |
23 |
268 |
243 |
148 |
166 |
|||||||||||||||||
Mean |
90 |
93 |
15 |
15 |
268 |
310 |
22 |
27 |
21 |
25 |
12 |
13 |
161 |
159 |
118 |
110 |
|||||||||||||||||
SD |
14.5 |
14.3 |
4.5 |
5.2 |
26.4 |
31.1 |
5.8 |
6.3 |
4.8 |
5.7 |
3.5 |
3.5 |
39.2 |
32.3 |
17.0 |
29.3 |
|||||||||||||||||
POSITIVE CONTROL VALUES 2016 |
|
||||||||||||||||||||||||||||||||
Strain S9-Mix |
TA100 |
TA1535 |
TA102 |
WP2uvrA |
TA98 |
TA1537 |
WP2uvrA pKM101 |
WP2pKM101 |
|
||||||||||||||||||||||||
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
|
|||||||||||||||||
Values |
409 |
406 |
381 |
386 |
30 |
28 |
341 |
335 |
388 |
385 |
379 |
381 |
14 |
24 |
8 |
16 |
|
||||||||||||||||
Min |
221 |
284 |
84 |
92 |
897 |
629 |
107 |
102 |
100 |
96 |
95 |
101 |
445 |
574 |
1674 |
372 |
|
||||||||||||||||
Max |
2222 |
2863 |
2994 |
879 |
2326 |
2140 |
1611 |
637 |
449 |
4357 |
1413 |
639 |
1117 |
1855 |
2823 |
945 |
|
||||||||||||||||
Mean |
724 |
1264 |
854 |
240 |
1633 |
950 |
718 |
240 |
186 |
188 |
406 |
290 |
743 |
1271 |
2379 |
535 |
|
||||||||||||||||
SD |
320.4 |
562.9 |
664.9 |
62.1 |
564.5 |
382.7 |
338.6 |
98.2 |
49.8 |
230.8 |
227.0 |
92.7 |
214.6 |
326.5 |
426.2 |
143.3 |
|
||||||||||||||||
SD: Standard deviation
Min: Minimum value
Max: Maximum value
†: Number of mean values used to create dataset
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
Table 7.6/1: Summary of genotoxicity test
Test n° |
Test / Guideline Reliability |
Focus |
Strains tested |
Metabolic activation |
Test concentration |
Statement |
1
Thompson, 2017 |
Ames Test (OECD 471) K, rel. 1 |
Gene mutation |
TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA |
-S9 +S9 |
Up to cytotoxic or highest recommended concentration |
-S9 : non mutagenic +S9 : non mutagenic |
In a reverse gene mutation assay in bacteria, performed according to the OECD Guideline 471 and in compliance with GLP, Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100, and Escherichia coli strain WP2uvrA were exposed to the test item at the following concentrations:
- Experiment 1 - Plate Incorporation Method: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate, with and without S9-mix
- Experiment 2 - Pre-Incubation Method: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate, with and without S9-mix
Rat liver homogenate (10% liver S9 in standard co-factors) was used as a metabolizing system. Vehicle control, negative (untreated) and positive control groups were also included in mutagenicity tests.
The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
In the first experiment (plate incorporation), the test item caused a visible reduction in the growth of the bacterial background lawn in all of the tester strains both with and without metabolic activation (S9), initially from 1500 µg/plate.
In the second experiment (pre-incubation), the test item caused a visible reduction in the growth of the bacterial background lawn in all of the tester strains, initially from 500 and 1500 µg/plate in the absence and presence of S9-mix, respectively.
No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.
No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method) and Experiment 2 (pre incubation method).
Under the test conditions, the test item is not considered as mutagenic in these bacterial systems.
Justification for classification or non-classification
Harmonized classification:
The registered substance has no harmonized classification according to the Regulation (EC) No. 1272/2008.
Self-classification:
Based on the available data, the registered substance does not require classification for mutagenicity according to the Regulation (EC) No. 1272/2008 (CLP) and GHS.
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.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.