Registration Dossier
Registration Dossier
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EC number: 250-151-3 | CAS number: 30364-51-3
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Bacterial Mutagenicity (OECD 471), Ames: negative with and without metabolic activation
Mammalian Cytogenicity (OECD 473), Chromosome Aberration: negative with and without metabolic activation
Mammalian Mutagenicity (OECD 476): negative with and without metabolic activation
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:
- 26 Sept 2012 - 6 Nov 2012
- 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:
- (adopted 21 July 1997)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Version / remarks:
- (adopted 30 May 2008)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- The Department of Health of the Government of the United Kingdom
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- his operon (S. typhimurium strains)
trp operon (E. coli) - Species / strain / cell type:
- S. typhimurium, other: TA 1535, TA 1537, TA98, and TA100
- Species / strain / cell type:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Metabolic activation system:
- cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Phenobarbitone/beta-Naphthoflavone
- Test concentrations with justification for top dose:
- - Preliminary Toxicity Test:
TA100 or WP2uvrA: 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate
- Mutation Test - Experiment 1
All Salmonella strains (with and without S9-mix): 1.5, 5, 15, 50, 150, 500 and 1500 μg/plate
WP2uvrA (with and without S9-mix): 15, 50, 150, 500, 1500 and 5000 μg/plate
- Mutation Test - Experiment 2
All Salmonella strains (with and without S9-mix): 0.5, 1.5, 5, 15, 50, 150 and 500 μg/plate
WP2uvrA (with and without S9-mix): 15, 50, 150, 500, 1500 and 5000 μg/plate - Vehicle / solvent:
- - Vehicle used: sterile, distilled water
- Justification for choice of vehicle: based on solubility checks performed in-house - Untreated negative controls:
- no
- 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:
- - S9: N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG): 2 μg/plate (WP2uvrA), 3 μg/plate (TA100), 5 μg/plate (TA1535); 9-Aminoacridine (9AA): 80 μg/plate (TA1537), 4-Nitroquinoline-1-oxide (4NQO): 0.2 μg/plate (TA98)
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- benzo(a)pyrene
- other: 2-Aminoanthracene
- Remarks:
- + S9: 2-Aminoanthracene (2AA): 1 μg/plate (TA100), 2 μg/plate (TA1535 and TA1537), 10 μg/plate (WP2uvrA); Benzo(a)pyrene (BP): 5 μg/plate (TA98)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: plate incorporation (Experiment 1) and pre-incubation (Experiment 2)
DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h
NUMBER OF REPLICATIONS: triplicates each in two independent experiments
DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of revertant colonies, a clearing or diminution of the background lawn (Preliminary Toxicity Test) - Evaluation criteria:
- There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby (1979)).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al (1989)).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response).
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 judgement about test item activity. Results of this type will be reported as equivocal. - Species / strain:
- S. typhimurium, other: TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Preliminary Toxicity Test: TA100 at 500 μg/plate and above (+ and - S9); plate incorporation Experiment 1: all strains at 150 μg/plate and above (+ and - S9); pre incubation Experiment 2: all strains at 50 μg/plate and above (+ and - S9)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Species / strain:
- E. coli WP2 uvr A
- 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:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: none
- Other confounding effects: none reported
RANGE-FINDING/SCREENING STUDIES: RANGE-FINDING/SCREENING STUDIES: Range finding test: in order to select the appropriate dose levels for use in the main test, a preliminary test was carried out to determine the toxicity of the test substance. Ten concentrations of the test item formulation (0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate) and vehicle control (sterile distilled water) were tested.
The test item was toxic to TA 100 from 500 µg/plate and non-toxic to WP2 uvrA.
COMPARISON WITH HISTORICAL CONTROL DATA: The results of the vehicle and positive controls (+ and - S9) are within the historical control data of the last two years. - Conclusions:
- Interpretation of results: negative
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 11 Oct 2012 - 10 Apr 2013
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Version / remarks:
- (adopted 21 July 1997)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
- Version / remarks:
- (adopted 30 May 2008)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- The Department of Health of the Government of the United Kingdom
- Type of assay:
- other: in vitro mammalian chromosome aberration test
- Target gene:
- not applicable.
- Species / strain / cell type:
- lymphocytes: human lymphocytes of fresh heparinised whole blood
- Details on mammalian cell type (if applicable):
- - Type and identity of media:
Growth medium: Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 10% foetal bovine serum. The lymphocytes of fresh heparinised whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).
Exposure medium (for 0.75 mL heparinised whole blood): 8.05-9.05 mL MEM, 10% (FBS), 0.1 mL Li-heparin and 0.1 mL PHA
- Properly maintained: yes
- Periodically checked for karyotype stability: not applicable (the donor had not been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection) - Metabolic activation:
- with and without
- Metabolic activation system:
- cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital/beta-naphthoflavone; Experiment 1 was conducted with 2% final concentration of S9 mix, whereas Experiment 2 was conducted with 1%)
- Test concentrations with justification for top dose:
- - Preliminary Toxicity Test: 12.6, 25.1, 50.2, 100.4, 200.9, 401.8, 803.5, 1607.0 and 3214.0 µg/mL
- Main Study: 12.5, 25, 50, 75, 100 and 200 µg/mL
- Slides evaluated in Main Study:
Experiment 1 with and without S9: 50, 75 and 100 µg/mL;
Experiment 2 without S9: 12.5, 25, 50 and 75 µg/mL;
Experiment 2 with S9: 25, 50 and 75 µg/mL - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Eagle's minimal essential medium with HEPES buffer (MEM)
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- mitomycin C
- Remarks:
- without S9: mitomycin C (MMC), 0.4 and 0.2 μg/mL in MEM; with S9: cyclophosphamide (CP), 5 μg/mL in DMSO
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: Experiment 1: 4 h with and without S9; Experiment 2: 4 h with S9 and 24 h without S9
- Expression time (cells in growth medium): 20 h (only in case of 4 h exposure duration)
- Fixation time (start of exposure up to fixation or harvest of cells): 24 h
SPINDLE INHIBITOR (cytogenetic assays): Colcemid, 0.1 μg/mL, for 2 h
STAIN (for cytogenetic assays): 5% Giemsa
NUMBER OF REPLICATIONS: two replicates each in two independent experiments
NUMBER OF CELLS EVALUATED: 100 per culture
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index
OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes - Evaluation criteria:
- A positive response was recorded for a particular treatment if the % cells with aberrations, excluding gaps, markedly exceeded that seen in the concurrent control, either with or without a clear dose-relationship. For modest increases in aberration frequency a dose response relationship is generally required and appropriate statistical tests may be applied in order to record a positive response.
- Statistics:
- The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test.
- Key result
- Species / strain:
- lymphocytes: human lymphocytes of fresh heparinised whole blood
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Experiment 1 with and without S9: at 200 µg/mL; Experiment 2 with and without metabolic activation: at 100 µg/mL and above
- 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 effect
- Effects of osmolality: no effect
- Water solubility: soluble
- Precipitation: none observed in the main study
- Other confounding effects: haemolysis
Experiment 1: haemolysis was observed at and above 100 μg/mL in the absence of S9, and at 200 μg/mL in the presence of S9. When the cultures were harvested reduced cell pellets were observed in both exposure groups at 200 μg/mL. The authors concluded that this was most likely due to cell lysis occurring due to the soap impurity in the test item.
Experiment 2: haemolysis and reduced cell pellets were observed at the end of the exposure period at 200 μg/mL in the absence of S9, and 100 μg/mL in the presence of S9.
RANGE-FINDING/SCREENING STUDIES:
The dose range for the Preliminary Toxicity Test was 12.6 - 3214 μg/mL. The maximum dose was based on the maximum recommended concentration (10 mM). A precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure, at and above 200.9 μg/mL, in all three of the exposure groups. Haemolysis was also observed at and above 100.4 μg/mL in the two 4 h exposure groups, respectively, and at and above 200.9 μg/mL in the 24 h continuous exposure group. The authors stated that haemolysis is the toxic effect on the erythrocytes in the culture and does not normally represent toxicity to the lymphocytes, and was probably due to the soap impurity. Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 100.4 μg/mL in all three of the exposures groups. The test item induced clear evidence of toxicity in all of the exposure groups. The selection of the maximum dose level was therefore based on toxicity for all exposure groups in both experiments.
COMPARISON WITH HISTORICAL CONTROL DATA:
The results of this study were within the range of the historical control data of this laboratory. - Conclusions:
- Interpretation of results: negative
- Executive summary:
The test item did not induce a statistically significant increase in the frequency of cells with chromosome aberrations in either the absence or presence of a liver enzyme metabolising system in either of two separate experiments. The test item was therefore considered to be non-clastogenic to human lymphocytes in vitro.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 04 Oct 2012 - 12 Mar 2013
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- The Department of Health of the Government of the United Kingdom
- Type of assay:
- other: mammalian cell gene mutation assay
- Target gene:
- TK locus
- Species / strain / cell type:
- other: mouse lymphoma L5178Y TK+/- 3.7.2c cells
- Details on mammalian cell type (if applicable):
- - Type and identity of media: RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/mL), Streptomycin (100 µg/mL), Sodium pyruvate (1 mM), Amphotericin B (2.5 µg/mL) and 10% donor horse serum (giving R10 media) at 37 °C with 5% CO2 in air. RPMI 1640 with 20% donor horse serum (R20) and without serum (R0) are used during the course of the study.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes - Metabolic activation:
- with and without
- Metabolic activation system:
- cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Phenobarbital/beta-Naphthoflavone
- Test concentrations with justification for top dose:
- Preliminary Toxicity Test:
12.55, 25.11, 50.11, 100.44, 200.88, 401.75, 803.5, 1607 and 3214 µg/mL
Mutagenicity Test/Experiment I
without S9 mix: 1.25, 2.5, 5, 10, 20, 30, 40 and 50 µg/mL (4 h)
with S9 mix: 1.25, 2.5, 5, 10, 20, 30, 40 and 50 µg/mL (4 h)
Mutagenicity Test/Experiment II
without S9 mix: 5, 10, 20, 30, 40, 45, 50, 55 and 60 µg/mL (24 h)
with S9 mix: 10, 20, 30, 35, 40, 42.5, 45, 47.5 and 50 µg/mL (4 h) - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: RPMI 1640 Medium without serum
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- ethylmethanesulphonate
- Remarks:
- - S9 mix: 400 µg/mL (4 h); 150µg/mL (24 h)
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- Remarks:
- +S9 mix: 2 µg/mL
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration:
1st experiment: 4 h exposure with and without S9 mix.
2nd experiment: 4 h exposure with S9 mix and 24 h without S9 mix.
- Expression time (cells in growth medium): 2 days, then cells were plated for determination of the cloning efficiency and the mutation frequency in 96-well microtitre plates containing TFT selective medium.
- Selection time (if incubation with a selection agent): 10 - 14 days
- Fixation time (start of exposure up to fixation or harvest of cells): 13-17 days
SELECTION AGENT (mutation assays): 4 µg/mL 5-trifluorothymidine (TFT)
STAIN: MTT
NUMBER OF REPLICATIONS: duplicates each in two independent experiments in 96-well microtitre plates
DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency and relative total growth
OTHER EXAMINATIONS:
- Other: small and large colonies were differentiated, as small colonies are capable to indicate chromosomal mutations.
- Evaluation criteria:
- The normal range for mutant frequency per survivor is 50-170 x 10E-6 for the TK+/- locus in L5178Y cells at this laboratory. Positive control chemicals should induce at least three to five fold increases in mutant frequency greater than the corresponding vehicle control. For a test item to demonstrate a mutagenic response it must produce a statistically significant increase in the induced mutant frequency (IMF) over the concurrent vehicle mutant frequency value.
- Statistics:
- The experimental data was analysed using a dedicated computer program, Mutant 240C by York Electronic Research, which follows the statistical guidelines recommended by the UKEMS (Robinson W D et al., 1989)
- Key result
- Species / strain:
- other: mouse lymphoma L5178Y TK+/- 3.7.2c cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at ≥ 40 µg/mL
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none
- Effects of osmolality: none
- Water solubility: soluble
- Precipitation: none observed
RANGE-FINDING/SCREENING STUDIES:
In all three of the exposure groups there was evidence of marked dose-related reductions in the Relative Suspension Growth (RSG) of cells treated with the test item when compared to the concurrent vehicle controls. The toxicity was very steep in all three exposure groups. Based on the % RSG values observed, the maximum dose levels in the subsequent Mutagenicity Test were limited by test item-induced toxicity at doses of 50 µg/mL or higher.
COMPARISON WITH HISTORICAL CONTROL DATA: neither of the vehicle control mutant frequency values were outside the acceptable range of 50 to 170 x 10E-6 viable cells. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional. - Conclusions:
- Interpretation of results: negative
- Executive summary:
The test item did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells and is therefore considered to be non-mutagenic under the conditions of the test
Referenceopen allclose all
Table 1 Test Results: Experiment 1 (plate incorporation) – Without Metabolic Activation
Test group |
Number of revertants (mean) ± SD |
|||||
Base-pair substitution strains |
Frameshift strains |
|||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
||
Solvent control |
105±7.6 |
18±3.8 |
34±9.8 |
21±6.8 |
15±3.5 |
|
Test item |
1.5 µg/plate |
96±3.1 |
16±2.6 |
N/T |
19±7.4 |
9±1.5 |
5 µg/plate |
97±11.7 |
21±2.6 |
N/T |
22±5.6 |
16±2.1 |
|
15 µg/plate |
94±4.7 |
16±3.5 |
29±2.0 |
16±3.2 |
9±2.5 |
|
50 µg/plate |
107±16.5 |
13±3.2 |
28±2.9 |
22±8.5 |
10±2.3 |
|
150 µg/plate |
79±6.0 |
11±4.0 |
35±7.6 |
13±2.6 |
9±5.5 |
|
500 µg/plate |
58±4.7 |
7±4.2 |
32±8.1 |
12±3..1 |
2±1.2 |
|
1500 µg/plate |
0±0 |
2±1.0 |
25±7.0 |
0±0 |
0±0 |
|
5000 µg/plate |
N/T |
N/T |
29±5.5 |
N/T |
N/T |
|
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 |
||
514±45.2 |
453±17.9 |
859±31.6 |
159±12.7 |
1122±74.5 |
||
Table 2 Test Results: Experiment 1 (plate incorporation) – With Metabolic Activation
Test group |
Number of revertants (mean) ± SD |
|||||
Base-pair substitution strains |
Frameshift strains |
|||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
||
Solvent control |
125±10.2 |
11±1.0 |
36±6.0 |
26±4.2 |
17±7.0 |
|
Test item |
1.5 µg/plate |
122±12.5 |
11±1.2 |
N/T |
26±7.1 |
16±7.6 |
5 µg/plate |
119±7.8 |
11±2.6 |
N/T |
19±5.0 |
15±6.1 |
|
15 µg/plate |
124±26.0 |
12±3.5 |
40±8.1 |
21±4.2 |
14±1.2 |
|
50 µg/plate |
126±12.8 |
11±5.5 |
33±7.1 |
22±8.7 |
16±3.8 |
|
150 µg/plate |
103±15.5 |
11±0.6 |
39±6.7 |
24±7.1 |
10±0.6 |
|
500 µg/plate |
68±5.2 |
5±0.6 |
34±3.8 |
11±2.6 |
3±1.2 |
|
1500 µg/plate |
0±0 |
0±0 |
35±3.5 |
3±2.6 |
0±0 |
|
5000 µg/plate |
N/T |
N/T |
30±2.6 |
N/T |
N/T |
|
Positive controls S9 mix (+) |
Name Dose Level No. of revertants |
2AA |
2AA |
2AA |
BP |
2AA |
1 µg |
2 µ |
10 µg |
5 µg |
2 µg |
||
1939±74.5 |
314±31.6 |
362±29.3 |
169±4.2 |
384±9.3 |
||
Table 3 Test Results: Experiment 2 (pre-incubation) – Without Metabolic Activation
Test group |
Number of revertants (mean) ± SD |
|||||
Base-pair substitution strains |
Frameshift strains |
|||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
||
Solvent control |
109±7.6 |
21±2.3 |
29±3.0 |
19±3.1 |
12±2.6 |
|
Test item |
0.5 µg/plate |
116±2.5 |
23±4.2 |
N/T |
19±2.6 |
15±3.5 |
1.5 µg/plate |
103±8.5 |
22±4.0 |
N/T |
19±5.1 |
12±2.0 |
|
5 µg/plate |
87±13.2 |
19±4.0 |
N/T |
17±3.8 |
12±4.7 |
|
15 µg/plate |
98±8.5 |
17±3.1 |
26±8.2 |
18±4.5 |
6±1.0 |
|
50 µg/plate |
100±11.6 |
18±3.5 |
27±13.2 |
15±1.2 |
5±2.1(S) |
|
150 µg/plate |
82±2.9 |
17±1.5 |
23±2.6 |
22±5.8 |
0±0 (V) |
|
500 µg/plate |
0±0 |
0±0 |
21±6.5 |
10±2.1 |
0±0 (V) |
|
1500 µg/plate |
N/T |
N/T |
21±5.0 |
N/T |
N/T |
|
5000 µg/plate |
N/T |
N/T |
27±1.5 |
N/T |
N/T |
|
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 |
||
635±35.8 |
335±87.5 |
775±38.9 |
121±30.0 |
953±53.4 |
||
Table 4 Test Results: Experiment 2 (pre-incubation) – With Metabolic Activation
Test group |
Number of revertants (mean) ± SD |
|||||
Base-pair substitution strains |
Frameshift strains |
|||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
||
Solvent control |
105±15.0 |
11±1.2 |
33±7.6 |
28±4.0 |
13±5.9 |
|
Test item |
0.5 µg/plate |
106±7.6 |
9±0.6 |
N/T |
22±7.6 |
14±2.6 |
1.5 µg/plate |
101±4.7 |
13±3.0 |
N/T |
25±3.2 |
13±3.8 |
|
5 µg/plate |
94±6.2 |
10±0.6 |
N/T |
21±1.5 |
12±1.7 |
|
15 µg/plate |
91±11.9 |
11±1.2 |
33±2.6 |
28±4.2 |
11±6.7 |
|
50 µg/plate |
89ׅ10.6 |
12±1.7 |
31±12.4 |
18±3.2 |
10±5.2 |
|
150 µg/plate |
85±7.1 |
10±3.6 |
34±2.0 |
21±6.0 |
6±2.3 |
|
500 µg/plate |
54±8.3 |
0±0 |
29±5.8 |
18±3.5 |
0±0 |
|
1500 µg/plate |
N/T |
N/T |
30±5.5 |
N/T |
N/T |
|
5000 µg/plate |
N/T |
N/T |
32±1.5 |
N/T |
N/T |
|
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 |
||
1161±93.3 |
162±16.7 |
292±14.0 |
138±20.6 |
169±38.7 |
||
N/T Not tested at this dose level
(S) = Sparse bacterial background lawn
(V) = Very weak bacterial background lawn
solvent control:sterile, destilled water
positive controls (- S9):
-N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG): 2 μg/plate (WP2uvrA), 3 μg/plate (TA100), 5 μg/plate (TA1537)
- 9-Aminoacridine (9AA): 80 μg/plate (TA1537), 4-Nitroquinoline-1-oxide (4NQO): 0.2 μg/plate (TA98)
positive controls (+ S9):
- 2-Aminoanthracene (2AA): 1 μg/plate (TA100), 2 μg/plate (TA1535 and TA1537), 10 μg/plate (WP2uvrA)
- Benzo(a)pyrene (BP): 5 μg/plate (TA98)
Table 1: Experiment I - 4 h treatment, 24 h fixation - Without Metabolic Activation
Concentration |
Mitotic index [%] |
Polyploid cells |
Aberrant cells (total) |
|
incl. gaps |
excl. gaps |
|||
solvent control (MEM) |
100 |
0 |
3 |
0 |
50 |
102 |
0 |
1 |
0 |
75 |
138 |
0 |
2 |
0 |
100 |
81 |
0 |
0 |
0 |
Positive control # (MMC), 0.4 |
34 |
0 |
44 |
38*** |
MEM: Eagle's minimal essential medium
MMC: Mitomycin C
*** P < 0.001
# For one culture slide evaluation terminated at 50 cells because at least 30% cells with aberrations had been observed.
Table 2: Experiment I - 4 h treatment, 24 h fixation - With Metabolic Activation
Concentration |
Mitotic index [%] |
Polyploid cells |
Aberrant cells (total) |
|
incl. gaps |
excl. gaps |
|||
solvent control (MEM) |
100 |
0 |
0 |
0 |
50 |
96 |
0 |
2 |
1 |
75 |
73 |
0 |
2 |
0 |
100 |
66 |
0 |
0 |
0 |
Positive control # (CP), 5 |
20 |
0 |
46 |
40*** |
MEM: Eagle's minimal essential medium
CP: Cyclophosphamide
*** P < 0.001
# For one culture slide evaluation terminated at 50 cells because at least 30% cells with aberrations had been observed.
Table 3: Experiment II - 24 h treatment, 24 h fixation - Without Metabolic Activation
Concentration |
Mitotic index [%] |
Polyploid cells |
Aberrant cells (total) |
|
incl. gaps |
excl. gaps |
|||
solvent control (MEM) |
100 |
0 |
3 |
1 |
12.5 |
120 |
0 |
1 |
0 |
25 |
85 |
0 |
2 |
2 |
50 |
82 |
0 |
5 |
2 |
75 |
37 |
0 |
0 |
0 |
Positive control # (MMC), 0.2 |
21 |
0 |
46 |
43*** |
MEM: Eagle's minimal essential medium
MMC: Mitomycin C
*** P < 0.001
# For one culture slide evaluation terminated at 50 cells because at least 30% cells with aberrations had been observed.
Table 4: Experiment II - 4 h treatment, 24 h fixation - With Metabolic Activation
Concentration |
Mitotic index [%] |
Polyploid cells |
Aberrant cells |
|
incl. gaps |
excl. gaps |
|||
solvent control (MEM) |
100 |
0 |
1 |
1 |
25 |
113 |
0 |
4 |
4 |
50 |
92 |
0 |
3 |
3 |
75 |
84 |
0 |
1 |
1 |
Positive control # (CP), 5 |
33 |
0 |
40 |
34*** |
MEM: Eagle's minimal essential medium
CP: Cyclophosphamide
*** P < 0.001
# For one culture slide evaluation terminated at 50 cells because at least 30% cells with aberrations had been observed.
Table 1: Experiment I - 4 h exposure - With Metabolic Activation |
|||
Concentration |
Cloning efficiency [%] |
Relative Total Growth [%] |
Mutants per 1E+06 surviving cells |
[µg/mL] |
|||
0 (RPMI) |
100 |
100 |
98.11 |
1.25 |
113 |
- |
- |
2.5 |
105 |
- |
- |
5 |
104 |
90 |
145.32 |
10 |
105 |
100 |
126.27 |
20 |
100 |
91 |
120.9 |
30 |
94 |
105 |
118.11 |
40 |
70 |
70 |
110.73 |
50 |
70 |
9 |
120.72 |
CP, |
72 |
57 |
1223.95 |
CP = Cyclophosphamide |
|||
Table 2: Experiment I - 4 h exposure - Without Metabolic Activation |
|||
Concentration |
Cloning efficiency [%] |
Relative Total Growth [%] |
Mutants per 1E+06 surviving cells |
[µg/mL] |
|||
0 (RPMI) |
100 |
100 |
106.7 |
1.25 |
93 |
- |
- |
2.5 |
82 |
83 |
116.42 |
5 |
91 |
88 |
121.19 |
10 |
89 |
80 |
111.6 |
20 |
88 |
100 |
88.9 |
30 |
82 |
85 |
98.83 |
40 |
60 |
58 |
129.32 |
50 |
1 |
- |
- |
EMS, |
72 |
57 |
835.68 |
EMS = Ethyl methane sulphonate |
|||
Table 3: Experiment II - 4 h Exposure - With Metabolic Activation |
|||
Concentration |
Cloning efficiency [%] |
Relative Total Growth [%] |
Mutants per 1E+06 surviving cells |
[µg/mL] |
|||
0 (RPMI) |
100 |
100 |
96.35 |
10 |
92 |
- |
- |
20 |
92 |
101 |
100.65 |
30 |
86 |
85 |
82.01 |
35 |
71 |
72 |
72.56 |
40 |
43 |
48 |
86.53 |
42.5 |
37 |
32 |
131.03 |
45 |
15 |
10 |
114.82 |
47.5 |
6 |
- |
- |
50 |
2 |
- |
- |
CP, |
36 |
22 |
1160.44 |
CP = Cyclophosphamide |
|||
Table 4: Experiment II - 24 h exposure - Without Metabolic Activation |
|||
Concentration |
Cloning efficiency [%] |
Relative Total Growth [%] |
Mutants per 1E+06 surviving cells |
[µg/mL] |
|||
0 (RPMI) |
100 |
100 |
115.26 |
5 |
91 |
87 |
110.97 |
10 |
74 |
79 |
100.71 |
20 |
55 |
74 |
104.46 |
30 |
37 |
60 |
116.99 |
40 |
18 |
36 |
116.23 |
45 |
10 |
24 |
138.37 |
50 |
5 |
- |
- |
55 |
2 |
- |
- |
60 |
1 |
- |
- |
EMS, |
43 |
28 |
1552.76 |
EMS = Ethyl methane sulphonate |
|||
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
In vitro gene mutation in bacteria and mammalian cells as well as chromosome aberration in mammalian cells were tested with Sodium N-methyl-N-(1-oxotetradecyl)aminoacetate (CAS 30364-51-3).
Genetic toxicity (mutagenicity) in bacteria in vitro
A bacterial gene mutation assay (Ames test) was performed with Sodium N-methyl-N-(1-oxotetradecyl)aminoacetate (CAS 30364-51-3) according to OECD TG 471 (Harlan, 2013e).
The S. typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvr A were tested conducting both the plate incorporation and the pre-incubation method in the absence and presence of a metabolic activation system (Phenobarbitone/beta-Naphthoflavone-induced rat liver S9-mix). The experiment was conducted in 3 replicates each up to the limit concentration of 5000 µg/plate (vehicle: water). No increase in the number of revertant colonies was noted in any of the bacterial strains, with and without metabolic activation system. Cytotoxicity was observed for all S. typhimurium strains at 150 µg/plate and above (with and without metabolic activation) in the plate incorporation test and for all S. typhimurium strains at 50 µg/plate and above (with and without metabolic activation) in the pre-incubation experiment. However, no cytotoxicity was observed for E. coli WP2 uvr A up to the limit concentration of 5000 µg/plate. Appropriate positive and solvent controls were included in the test and showed the expected results. Under the conditions of the study, Sodium N-methyl-N-(1-oxotetradecyl)aminoacetate did not induce mutations in the bacterial mutation assay in the absence and presence of a metabolic activation system in any of the strains tested.
Genetic toxicity (cytogenicity) in mammalian cells in vitro
An in vitro mammalian chromosome aberration test was conducted with Sodium N-methyl-N-(1-oxotetradecyl)aminoacetate (CAS 30364-51-3) in accordance with OECD TG 473 under GLP conditions (Harlan, 2013f). The induction of structural chromosome aberrations was evaluated in vitro in lymphocytes of freshly heparised human whole blood cultures, incubated for 4 and 24 h with and without a metabolic activation system (S9-mix from rats treated with phenobarbitone and beta-naphthoflavone). Concentrations of 12 - 200 µg/mL (4 h and 24 h incubation) of the test substance in Minimal Essential Media (MEM) with and without metabolic activation were applied. The negative as well as the positive controls showed the expected results and were within the historical control data. Haemolysis was also observed at and above 100.4 µg/mL in the two 4(20)-hour exposure groups and at and above 200.9 µg/mL in the 24-hour continuous exposure group. In the first experiment, haemolysis was seen at and above 100 µg/mL in the exposure groups in absence of S9, and at 200 µg/mL in presence of S9. The mitotic index data show that 19% mitotic inhibition and 34% mitotic inhibition was achieved at 100 µg/mL in the absence and presence of S9, respectively. In the second experiment haemolysis and reduced cell pellets were observed at the end of the exposure period at 200 µg/mL in the absence of metabolic activation, and at 100 µg/mL in the presence of metabolic activation. The mitotic index data show that 63% mitotic inhibition was achieved at 75 µg/mL in the absence of S9. In the presence of S9 the response was not as marked with a modest 16% mitotic inhibition being observed. The test material did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in any of the exposure groups. No statistically or biologically significant increase in the incidence of chromosome aberrations was observed. Therefore, under the conditions of the study, the test substance did not show clastogenic activity in this chromosomal aberration test with and without metabolic activation performed in peripheral human lymphocytes in vitro.
Genetic toxicity (mutagenicity) in mammalian cells in vitro
The in vitro mammalian cell gene mutation study of Sodium N-methyl-N-(1-oxotetradecyl)aminoacetate (CAS 30364-51-3) was carried out according to OECD TG 476 under GLP conditions (Harlan, 2013g). Gene mutations in the thymidine kinase locus were investigated in L5178Y mouse lymphoma cells in the presence and absence of a metabolic activation system (Phenobarbital/beta-naphtoflavone-induced rat liver, S9). In the first experiment, cells were exposed for 4 h to the test substance at eight concentrations of 1.25 - 50 µg/mL without and with metabolic activation. Nine concentrations of the second experiment without metabolic activation for an exposure time of 24 h ranged from 5 - 60 µg/mL. Nine concentrations of the second experiment for an exposure of 4 h ranged from 10 - 50 µg/mL in presence of metabolic activation. The vehicle and positive controls in the study showed the expected results and were within the range of historical control data of the laboratory. In the first and second experiment there was evidence of marked toxicity following exposure to the test item in both the absence and presence of metabolic activation, as indicated by the RTG and % RSG values. There was no significant increase in the number of forward mutations at the thymidine kinase locus of L5178Y mouse lymphoma cells treated with the test material, neither in the presence nor in the absence of a metabolic activation system. Under the conditions of the study, Sodium N-methyl-N-(1-oxotetradecyl)aminoacetate did not show gene mutation activity in this test performed in L5178Y mouse lymphoma cells in vitro.
Conclusion
Taken together, the available data on genetic toxicity from the registered substance do not indicate any mutagenic and clastogenic potential in vitro. Therefore, according to EU classification criteria, the registered substance is not to be classified.
Justification for classification or non-classification
The available genotixicity/mutagenicity data are negative and thus conclusive but not sufficient for classification. Therefore, the registered substance Sodium N-methyl-N-(1-oxotetradecyl)aminoacetate (CAS 30364-51-3) does not warrant any classification according to Regulation (EC) No. 1272/2008 (CLP) with respect to genotoxicity/mutagenicity.
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