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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant, guideline study, available as an unpublished report.
Justification for type of information:
See IUCLID section 13 for category and read across justification

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
impurity
Test material form:
solid
Details on test material:
- Batch number: A194-99
- Expiry date: 30 June 2013

Method

Target gene:
- Salmonella: +Histidine
- E.Coli: Tryptophan
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Not applicable.
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
Not applicable.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbitone/beta­naphthoflavone induced rat liver, S9
Test concentrations with justification for top dose:
- Preliminary Toxicity Test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
- Main test experiment one: 50, 150, 500, 1500 and 5000 µg/plate
- Main test experiment two: 50, 150, 500, 1500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used:
- Justification for choice of solvent/vehicle:
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA100
Negative solvent / vehicle controls:
yes
Remarks:
Sterile distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene: 1 µg/plate
Remarks:
With S9 mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA1535
Negative solvent / vehicle controls:
yes
Remarks:
Sterile distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene: 2 µg/plate
Remarks:
With S9 mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA1537
Negative solvent / vehicle controls:
yes
Remarks:
Sterile distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene: 2 µg/plate
Remarks:
With S9 mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of WP2uvrA
Negative solvent / vehicle controls:
yes
Remarks:
Sterile distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene: 10 µg/plate
Remarks:
With S9 mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA98
Negative solvent / vehicle controls:
yes
Remarks:
Sterile distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
With S9 mix. Benzo(a)pyrene: 5 µg/plate
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA98
Negative solvent / vehicle controls:
yes
Remarks:
Sterile distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-Nitroquinoline-1-oxide: 0.2 µg/plate
Remarks:
Without S9 mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA1537
Negative solvent / vehicle controls:
yes
Remarks:
Sterile distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Without S9 mix. 9-Aminoacridine: 80 µg/plate
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA100
Negative solvent / vehicle controls:
yes
Remarks:
Sterile distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Without S9 mix. N-ethyl-N'-nitro-N-nitrosoguanidine: 3 µg/plate
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA1535
Negative solvent / vehicle controls:
yes
Remarks:
Sterile distilled water
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Without S9 mix. N-ethyl-N'-nitro-N-nitrosoguanidine: 5 µg/plate
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of WP2uvrA
Negative solvent / vehicle controls:
yes
Remarks:
Sterile distilled water
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Without S9 mix. N-ethyl-N'-nitro-N-nitrosoguanidine: 2 µg/plate
Details on test system and experimental conditions:
METHODS OF APPLICATION
- Experiment 1: In agar (plate incorporation)
- Experiment 2: Pre-incubation

DURATION
- Preincubation period for bacterial strains: 10h
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): Not applicable
- Selection time (if incubation with a selection agent): Not applicable

NUMBER OF REPLICATIONS: Triplicate plating.

DETERMINATION OF CYTOTOXICITY
- Method: Plates were assessed for numbers of revertant colonies using a colony counter and examined for effects on the growth of the bacterial background lawn. Manual counts were performed at 5000 µg/plate because of excessive test item precipitation.
Evaluation criteria:
ACCEPTANCE CRITERIA
The reverse mutation assay may be considered valid if the following criteria are met:
- All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls.
- The appropriate characteristics for each tester strain have been confirmed, e.g. rfa cell-wall mutation and pKM101 plasmid R-factor etc.
- All tester strain cultures should be in the approximate range of 1 to 9.0 x 10(+09) bacteria per ml.
- Diagnostic mutagens (positive control chemicals) must be included to demonstrate both the intrinsic sensitivity of the tester strains to mutagen exposure and the integrity of the S9-mix. All of the positive control chemicals used in the study should induce marked increases in the frequency of revertant colonies, both with or without metabolic activation.
- There should be a minimum of four non-toxic test material dose levels.
- There should not be an excessive loss of plates due to contamination.

EVALUATION CRITERIA
There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results will be considered first, statistical methods, as recommended by the UKEMS can also be used as an aid to evaluation, however, statistical significance will not be the only determining factor for a positive response. A test material 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 a definitive judgement about the test material activity. Results of this type will be reported as equivocal.
Statistics:
Standard deviation

Results and discussion

Test resultsopen allclose all
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
Remarks:
Tested up to maximum recommended dose of 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
Tested up to maximum recommended dose of 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Solubility: The test item was insoluble in sterile distilled water (25 and 50 mg/mL), dimethyl sulphoxide, dimethyl formamide and acetonitrile (50 mg/mL), acetone (100 mg/mL) and tetrahydrofuran (200 mg/mL) in solubility checks performed in-house. The test item formed the best doseable suspension in sterile distilled water at 25 mg/mL; therefore, this solvent was selected as the vehicle.
- Precipitation: A test item precipitate (particulate in appearance) was noted at and above 1500 µg/plate, this observation did not prevent the scoring of revertant colonies.

RANGE-FINDING/SCREENING STUDIES:
- Preliminary Toxicity Test:The test item was non-toxic to the strains of bacteria used (TA100 and WP2uvrA). The test item formulation and S9-mix used in this experiment were both shown to be sterile.

COMPARISON WITH HISTORICAL CONTROL DATA:
- Master strains: Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory).
- Negative control: Results for the negative controls (spontaneous mutation rates) were considered to be acceptable.
- Positive control: All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

ADDITIONAL INFORMATION ON CYTOTOXICITY: None
Remarks on result:
other: all strains/cell types tested

Any other information on results incl. tables

RESULTS

Preliminary ToxicityTest

The test item was non-toxic to the strains of bacteria used (TA100 and WP2uvrA). The test item formulation and S9-mix used in this experiment were both shown to be sterile. The numbers of revertant colonies for the toxicity assay were:

With (+) or without (-) S9-mix Strain Dose (µg/plate)
0 0.15 0.5 1.5 5 15 50 150 500 1500 5000
- TA100 83 91 92 87 101 91 97 85 86P 67SP 45SP
+ TA100 87 102 79 101 67 75 74 71 74P 82P 39SP
- WP2uvrA 12 11 15 13 12 17 15 15 18P 15P 10SP
+ WP2uvrA 22 16 13 18 16 21 22 15 24P 12P 5P

P: Precipitate

S: Sparse bacterial background lawn

      

MutationTest

Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). These data are not given in the report. The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile.

Results for the negative controls (spontaneous mutation rates) are presented in Table1(see below) and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.

The individual plate counts, the mean number of revertant colonies and the standard deviations, for the test item, positive and vehicle controls, both with and without metabolic activation, are presented in Table 2 and Table 3 for Experiment 1 and Table 4 and Table 5 for Experiment 2.

No significant increases in the frequency of revertant colonies were recorded for any of the strains of bacteria, at any dose level either with or without metabolic activation or exposure method.


All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.


Table1               Spontaneous Mutation Rates (Concurrent Negative Controls)

Experiment 1

Number of revertants (mean number of colonies per plate)
Base-pair substitution type Frameshift type
TA100 TA1535 WP2uvrA TA98 TA1537
106 114 9 16 27 23 20 17 3 10
118 28 20 15 17
119 11 21 16 11

Experiment 2

Number of revertants (mean number of colonies per plate)
Base-pair substitution type Frameshift type
TA100 TA1535 WP2uvrA TA98 TA1537
103 119 17 14 21 21 19 18 7 7
124 13 21 17 7
130 13 20 17 7
25 20* 12 10*
13 8
23 9

* Experimental procedure repeated at a later date (without S9-mix) due to excessive toxicity in the original test

Table 2: Test Results: Experiment 1– Without Metabolic Activation

With or without S9-Mix Dose Level Per Plate Number of revertants (mean) ± SD
Base-pair substitution strains Frameshift strains
TA100 TA1535 WP2uvrA TA98 TA1537
    Value Mean (s.d.) Value Mean (s.d.) Value Mean (s.d.) Value Mean (s.d.) Value Mean (s.d.)
S9-Mix (-) Solvent Control (Acetone) 104 112 (10.8) 19 19 (2.0) 20 20 (0.6) 13 13 (6.0) 1 6 (6.4)
107 17 20 7 3
124 21 19 19 13
5 μg 136 125 (11.0) 16 14 (7.8) 25 25 (8.0) 17 19 (3.5) 5 6 (1.2)
126 5 33 23 5
114 20 17 17 7
15 μg 102 116 (11.8) 28 29 (3.2) 24 20 (9.6) 12 14 (1.7) 7 5 (2.0)
123 33 27 15 5
122 27 9 15 3
50 μg 83 91 (10.6) 17 17 (1.5) 32 20 (10.2) 8 13 (5.7) 1 4 (3.5)
103 19 13 11 8
87 16 16 19 4
150 μg 104 98 (9.8) 13 13 (0.6) 24 24 (0.6) 4 10 (9.0) 5 5 (2.0)
87 13 24 20 7
104 12 25 5 3
500 μg 116P 98 (15.7) 5P 9 (3.5) 17P 18 (1.7) 13P 13 (1.5) 8P 5 (3.5)
91P 11P 17P 15P 5P
87P 11P 20P 12P 1P
1500 μg 115P 107 (18.0) 13SP 20 (7.0) 28P 23 (4.4) 15SP 14 (1.2) 7SP 7 (2.0)
86P 27SP 21P 13SP 9SP
119P 19SP 20P 15SP 5SP
5000 μg 144SP 97 (42.2) 11SP 9 (1.5) 24SP 17 (6.1) 7SP 6 (1.5) 0SP 0 (0.0)
83SP 8SP 13SP 6SP 0SP
63SP 9SP 14SP 4SP 0SP
Positive controls S9-Mix (-) Name ENNG ENNG ENNG 4NQO 9AA
Dose level 3 μg 5 μg 2 μg 0.2 μg 80 μg
  Value Mean (s.d.) Value Mean (s.d.) Value Mean (s.d.) Value Mean (s.d.) Value Mean (s.d.)
No. revertants 529 435 (181.6) 160 161 (6.1) 521 504 (20.5) 186 185 (11.0) 668 605 (82.2)
226 156 481 196 635
551 168 509 174 512

ENNG: N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO: 4-Nitroquinoline-1-oxide

9AA: 9-Aminoacridine

S: Sparse bacterial background lawn

P: Precipitate

Table 3: Test Results: Experiment 1– With Metabolic Activation

With or without S9-Mix Dose Level Per Plate Number of revertants (mean) ± SD
Base-pair substitution strains Frameshift strains
TA100 TA1535 WP2uvrA TA98 TA1537
    Value Mean (s.d.) Value Mean (s.d.) Value Mean (s.d.) Value Mean (s.d.) Value Mean (s.d.)
S9-Mix (+) Solvent Control (Acetone) 90 104 (17.1) 13 13 (3.5) 33 30 (2.6) 16 16 (0.6) 7 9 (2.1)
123 16 28 17 11
99 9 29 16 8
5 μg 103 117 (19.5) 8 13 (6.2) 19 25 (6.0) 29 19 (8.5) 8 5 (3.5)
108 20 24 13 5
139 11 31 16 1
15 μg 115 112 (2.6) 9 8 (3.1) 29 23 (6.0) 15 19 (4.0) 11 11 (0.0)
110 11 23 19 11
111 5 17 23 11
50 μg 95 102 (9.1) 20 16 (4.5) 23 22 (1.7) 16 20 (6.4) 4 3 (0.6)
98 16 23 16 3
112 11 20 27 3
150 μg 88 96 (10.6) 7 12 (4.6) 13 15 (3.8) 23 18 (5.0) 7 8 (3.1)
108 15 19 13 5
92 15 12 19 11
500 μg 115P 108 (6.4) 15P 10 (4.2) 33P 29 (4.0) 19P 18 (6.0) 1P 4 (3.1)
104P 7P 25P 12P 7P
104P 9P 29P 24P 3P
1500 μg 116P 112 (10.6) 21SP 20 (1.0) 16P 17 (0.6) 21SP 21 (2.5) 4SP 4 (2.5)
100P 19SP 17P 24SP 6SP
120P 20SP 17P 19SP 1SP
5000 μg 112SP 118 (8.1) 1SP 7 (5.3) 12SP 16 (7.2) 16SP 14 (2.6) 0SP 0 (0.0)
114SP 11SP 24SP 11SP 0SP
127SP 9SP 11SP 15SP 0SP
Positive controls S9-Mix (+) Name 2AA 2AA 2AA BP 2AA
Dose level 1 μg 2 μg 10 μg 5 μg 2 μg
  Value Mean (s.d.) Value Mean (s.d.) Value Mean (s.d.) Value Mean (s.d.) Value Mean (s.d.)
No. revertants 1070 1029 (92.2) 191 190 (2.3) 255 305 (47.3) 289 302 (11.0) 406 426 (25.0)
1093 187 311 307 454
923 191 349 309 418

2AA 2-Aminoanthracene

BP Benzo(a)pyrene

P Precipitate

S: Sparse bacterial background lawn

Tables 4 and 5 below

Applicant's summary and conclusion

Conclusions:
Interpretation of results: Negative
Lithium myristate was considered to be non-mutagenic under the conditions of this test.
Executive summary:

The in vitro mutagenicity of lithium myristate was assessed in a GLP-compliant Bacterial Reverse Mutation Test, following OECD guideline 471 (Harlan 2013). S. typhimurium and E. coli strains were treated with suspensions of lithium myristate using both the Ames plate incorporation and pre-incubation methods at five dose levels in triplicate, both with and without the addition of a rat liver homogenate metabolising system. The vehicle, positive and negative controls confirmed the sensitivity of the assay and the efficacy of the S9 -mix.