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- Life Cycle description
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Genetic toxicity in vitro
Description of key information
Gene mutation in bacteria (Ames test, OECD 471): 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:
- 28 Oct - 16 Nov 2020
- 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 in 2020
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Version / remarks:
- adopted in 2008
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
- Version / remarks:
- adopted in 1998
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: The Japanese Ministry of Health, Labour and Welfare (MHLW), Ministry of Economy, Trade and Industry (METI), and Ministry of the Environment (MOE) Guidelines of 31 March 2011
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: ICH S2(R1) guideline adopted June 2012 (ICH S2(R1) Federal Register)
- Version / remarks:
- Adopted in 2012; 77:33748-33749
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- his operon (for S. typhimurium strains) and trp operon (for E. coli strain)
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells and date obtained: British Industrial Biological Research Association, obtained 17 August 1987, and Trinova Biochem GmbH obtained 27 June 2017
MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature: Top agar was prepared using 0.6% Bacto agar (lot number 9294156 expiry date 08/2024) and 0.5% sodium chloride with 5 mL of 1.0 mM histidine and 1.0 mM biotin solution added to each 100 mL of top agar. Vogel-Bonner Minimal agar plates were purchased from SGL Ltd (lot numbers 56249 expiry date 11/2020 and 56310 expiry date 12/2020). - Species / strain / cell type:
- E. coli WP2 uvr A
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells and date obtained:
British Industrial Biological Research Association, obtained 17 August 1987, and Trinova Biochem GmbH obtained 27 June 2017
MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature: Top agar was prepared using 0.6% Bacto agar (lot number 9294156 expiry date 08/2024) and 0.5% sodium chloride with 5 mL of 1.0 mM tryptophan solution added to each 100 mL of top agar. Vogel-Bonner Minimal agar plates were purchased from SGL Ltd (lot numbers 56249 expiry date 11/2020 and 56310 expiry date 12/2020). - Metabolic activation:
- with and without
- Metabolic activation system:
- Type and composition of metabolic activation system: cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of Sprague-Dawley rats (male, 5 - 6 weeks, 175 - 199 g) treated with phenobarbitone / β-naphthoflavone
- source of S9 : purchased from Moltox/Trinova Biochem GmbH, Giessen, Germany (Lot No. 4222)
- Protein level in S9: 20 mg/mL
- Method of preparation of S9 mix: S9-mix was prepared before use using sterilised co-factors and maintained on ice for the duration of the test
- Composition of S9 mix:
S9: 5.0 mL
1.65 M KCl/0.4 M MgCl2: 1.0 mL
0.1 M Glucose-6-phosphate: 2.5 mL
0.1 M NADP: 2.0 mL
0.2 M Sodium phosphate buffer (pH 7.4): 25.0 mL
Sterile distilled water: 14.5 mL
- Concentration or volume of S9 mix in the final culture medium: 0.5 mL (10%)
- Quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): checked at producer and specified in quality control certificate - Test concentrations with justification for top dose:
- Experiment 1 (plate incorporation method): 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate (5000 μg/plate being the maximum recommended dose level)
Experiment 2 (pre-incubation method): 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate (determined by the results of Experiment 1) - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: acetone
- Purity: > 99.75%
- Supplier: Acros Organics
- Batch number: 1911510
- Expiry: Nov 2021
- Justification for choice of solvent/vehicle: the test substance was not soluble/miscible in sterile distilled water and dimethyl sulphoxide at 50 mg/mL but fully miscible in acetone at 100 mg/mL
- Justification for percentage of solvent in the final culture medium: acetone is toxic to the bacterial cells at 0.1 mL (100 μL) after employing the pre-incubation modification; therefore all of the formulations for Experiment 2 were prepared at concentrations two times greater than required on Vogel-Bonner agar plates; to compensate, each formulation was dosed using 0.05 mL (50 μL) aliquots. - Untreated negative controls:
- yes
- 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
- benzo(a)pyrene
- other: 2-Aminoanthracene
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate) : triplicate
- Number of independent experiments: two independent experiments
METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation, Experiment 1) and preincubation (Experiment 2)
TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 20 min at 37 ± 3 °C (Experiment 2)
- Exposure duration/duration of treatment: 48 - 72 h at 37 ± 3 °C
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: inspection of the bacterial background lawn
METHODS FOR MEASUREMENT OF GENOTOXICIY
- Method: count of revertant colonies - Evaluation criteria:
- 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.
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. A fold increase greater than two times the concurrent solvent control for TA100, TA98 and WP2uvrA or a three-fold increase for TA1535 and TA1537 (especially if accompanied by an out-of-historical range response.
5. Statistical analysis of data as determined by UKEMS.
A test item is considered non-mutagenic (negative) in the test system if the above criteria are not met. - Statistics:
- Mean values and standard deviation were calculated. Statistical significance was confirmed using Dunnett’s Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies.
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Experiment 1 (plate incorporation): 1500 μg/plate +/- S9-mix; Experiment 2 (pre-incubation): 1500 µg/plate -S9, 5000 µg/plate +S9
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Experiment 1 (plate incorporation): 1500 μg/plate +/- S9-mix; Experiment 2 (pre-incubation): 500 µg/plate -S9, 1500 µg/plate +S9
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Experiment 1 (plate incorporation): 1500 μg/plate +/- S9-mix; Experiment 2 (pre-incubation): 500 µg/plate -S9, 1500 µg/plate +S9
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Experiment 1 (plate incorporation): 1500 μg/plate +/- S9-mix; Experiment 2 (pre-incubation): 500 µg/plate -S9, 1500 µg/plate +S9
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Experiment 1 (plate incorporation): 1500 μg/plate +/- S9-mix; Experiment 2 (pre-incubation): 1500 µg/plate -S9, 5000 µg/plate +S9
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: A test item precipitate (globular in appearance) was noted at and above 1500 μg/plate in both the presence and absence of metabolic activation (S9-mix) in both experiments. This observation did not prevent the scoring of revertant colonies.
STUDY RESULTS
- Concurrent vehicle negative and positive control data provided as pdf file under 'Attached background material'.
- Study results fall within the negative historical control data range.
Ames test:
- Detailed results are provided in tabular form under 'Any other information on results incl. tables'. - Conclusions:
- Interpretation of results: negative
Reference
Table 1: 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 |
|||||
162 |
|
34 |
|
13 |
|
20 |
|
12 |
|
149 |
(153) |
36 |
(34) |
26 |
(21) |
17 |
(19) |
12 |
(11) |
148 |
|
32 |
|
25 |
|
20 |
|
9 |
|
Viability – Bacterial cells 109 per mL |
|||||||||
2.6 |
2.4 |
2.5 |
1.4 |
2.1 |
Experiment 2
Number of revertants (mean number of colonies per plate) |
|||||||||
Base-pair substitution type |
Frameshift type |
||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
|||||
100 |
|
15 |
|
20 |
|
22 |
|
16 |
|
93 |
(97) |
24 |
(21) |
19 |
(20) |
17 |
(20) |
12 |
(14) |
98 |
|
24 |
|
22 |
|
20 |
|
14 |
|
Viability – Bacterial cells 109 per mL |
|||||||||
2.3 |
1.1 |
5.3 |
1.2 |
2.3 |
Table 2: Test Results: Experiment 1 – Without Metabolic Activation (Plate Incorporation)
Test Period |
From: 02 November 2020 |
To: 05 November 2020 |
|||||||||||
S9-Mix (-) |
Dose Level Per Plate |
Number of revertants (mean) +/- SD |
|||||||||||
Base-pair substitution strains |
Frameshift strains |
||||||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
|||||||||
Solvent Control (Acetone) |
164 167 170 |
(167) 3.0# |
36 33 33 |
(34) 1.7 |
25 21 21 |
(22) 2.3 |
20 19 25 |
(21) 3.2 |
7 17 16 |
(13) 5.5 |
|||
1.5 µg |
154 183 172 |
(170) 14.6 |
34 29 39 |
(34) 5.0 |
25 28 17 |
(23) 5.7 |
18 14 14 |
(15) 2.3 |
15 9 8 |
(11) 3.8 |
|||
5 µg |
155 171 183 |
(170) 14.0 |
29 33 35 |
(32) 3.1 |
21 13 18 |
(17) 4.0 |
20 25 11 |
(19) 7.1 |
12 7 16 |
(12) 4.5 |
|||
15 µg |
165 158 167 |
(163) 4.7 |
28 31 33 |
(31) 2.5 |
16 18 17 |
(17) 1.0 |
15 15 23 |
(18) 4.6 |
7 5 13 |
(8) 4.2 |
|||
50 µg |
182 129 130 |
(147) 30.3 |
34 30 26 |
(30) 4.0 |
14 25 18 |
(19) 5.6 |
17 18 27 |
(21) 5.5 |
11 6 6 |
(8) 2.9 |
|||
150 µg |
169 153 157 |
(160) 8.3 |
37 29 38 |
(35) 4.9 |
21 17 17 |
(18) 2.3 |
14 26 22 |
(21) 6.1 |
9 7 12 |
(9) 2.5 |
|||
500 µg |
169 140 156 |
(155) 14.5 |
26 30 31 |
(29) 2.6 |
30 21 13 |
(21) 8.5 |
17 13 17 |
(16) 2.3 |
8 6 9 |
(8) 1.5 |
|||
1500 µg |
127 SP 129 SP 129 SP |
(128) 1.2 |
25 SP 27 SP 23 SP |
(25) 2.0 |
21 SP 16 SP 15 SP |
(17) 3.2 |
16 SP 19 SP 9 SP |
(15) 5.1 |
6 SP 7 SP 11 SP |
(8) 2.6 |
|||
5000 µg |
87 SP 99 SP 90 SP |
(92) 6.2 |
14 SP 17 SP 13 SP |
(15) 2.1 |
22 SP 13 SP 24 SP |
(20) 5.9 |
16 SP 11 SP 20 SP |
(16) 4.5 |
0 SP 3 SP 5 SP |
(3) 2.5 |
|||
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 |
|||||||||
405 466 454 |
(442) 32.3 |
347 452 365 |
(388) 56.2 |
355 378 319 |
(351) 29.7 |
129 136 142 |
(136) 6.5 |
389 120 416 |
(308) 163.7 |
||||
ENNG: N-ethyl-N'-nitro-N-nitrosoguanidine
4NQO: 4-Nitroquinoline-1-oxide
9AA: 9-Aminoacridine
P: Test item precipitate
S: Sparse bacterial background lawn
#: Standard deviation
Table 3: Test Results: Experiment 1 – With Metabolic Activation (Plate Incorporation)
Test Period |
From: 02 November 2020 |
To: 05 November 2020 |
||||||||||||
S9-Mix (+) |
Dose Level Per Plate |
Number of revertants (mean) +/- SD |
||||||||||||
Base-pair substitution strains |
Frameshift strains |
|||||||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
||||||||||
Solvent Control (Acetone) |
146 145 161 |
(151) 9.0# |
18 11 15 |
(15) 3.5 |
27 26 18 |
(24) 4.9 |
24 33 27 |
(28) 4.6 |
7 10 8 |
(8) 1.5 |
||||
1.5 µg |
150 167 180 |
(166) 15.0 |
13 16 14 |
(14) 1.5 |
25 20 21 |
(22) 2.6 |
15 29 25 |
(23) 7.2 |
9 8 12 |
(10) 2.1 |
||||
5 µg |
148 161 180 |
(163) 16.1 |
11 12 7 |
(10) 2.6 |
18 30 15 |
(21) 7.9 |
35 24 16 |
(25) 9.5 |
9 13 15 |
(12) 3.1 |
||||
15 µg |
156 166 150 |
(157) 8.1 |
23 12 10 |
(15) 7.0 |
24 23 21 |
(23) 1.5 |
19 14 16 |
(16) 2.5 |
13 13 12 |
(13) 0.6 |
||||
50 µg |
150 146 168 |
(155) 11.7 |
11 9 19 |
(13) 5.3 |
23 23 24 |
(23) 0.6 |
24 16 21 |
(20) 4.0 |
10 9 13 |
(11) 2.1 |
||||
150 µg |
148 142 141 |
(144) 3.8 |
13 16 15 |
(15) 1.5 |
26 18 13 |
(19) 6.6 |
18 23 25 |
(22) 3.6 |
8 12 10 |
(10) 2.0 |
||||
500 µg |
116 134 114 |
(121) 11.0 |
14 13 7 |
(11) 3.8 |
24 23 19 |
(22) 2.6 |
21 15 19 |
(18) 3.1 |
11 8 12 |
(10) 2.1 |
||||
1500 µg |
97 SP 96 SP 78 SP |
(90) 10.7 |
6 SP 7 SP 9 SP |
(7) 1.5 |
20 SP 20 SP 18 SP |
(19) 1.2 |
12 SP 24 SP 14 SP |
(17) 6.4 |
5 SP 3 SP 7 SP |
(5) 2.0 |
||||
5000 µg |
64 SP 76 SP 58 SP |
(66) 9.2 |
5 SP 3 SP 9 SP |
(6) 3.1 |
23 SP 19 SP 21 SP |
(21) 2.0 |
24 SP 15 SP 16 SP |
(18) 4.9 |
0 SP 0 SP 0 SP |
(0) 0.0 |
||||
Positive controls S9-Mix (+) |
Name |
2AA |
2AA |
2AA |
BP |
2AA |
||||||||
Dose Level |
1 µg |
2 µg |
10 µg |
5 µg |
2 µg |
|||||||||
No. of Revertants |
1493 1595 1753 |
(1614) 131.0 |
293 295 310 |
(299) 9.3 |
118 142 145 |
(135) 14.8 |
137 174 157 |
(156) 18.5 |
224 234 251 |
(236) 13.7 |
||||
BP: Benzo(a)pyrene
2AA: 2-Aminoanthracene
P: Test item precipitate
S: Sparse bacterial background lawn
#: Standard deviation
Table 4: Test Results: Experiment 2 – Without Metabolic Activation (Pre-Incubation)
Test Period |
From: 12 November 2020 |
To: 15 November 2020 |
|||||||||||
S9-Mix (-) |
Dose Level Per Plate |
Number of revertants (mean) +/- SD |
|||||||||||
Base-pair substitution strains |
Frameshift strains |
||||||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
|||||||||
Solvent Control (Acetone) |
115 118 117 |
(117) 1.5# |
28 22 27 |
(26) 3.2 |
29 25 20 |
(25) 4.5 |
23 25 18 |
(22) 3.6 |
10 14 13 |
(12) 2.1 |
|||
1.5 µg |
130 112 118 |
(120) 9.2 |
19 21 20 |
(20) 1.0 |
20 25 21 |
(22) 2.6 |
28 14 19 |
(20) 7.1 |
16 12 14 |
(14) 2.0 |
|||
5 µg |
102 117 111 |
(110) 7.5 |
15 26 16 |
(19) 6.1 |
22 18 25 |
(22) 3.5 |
12 19 22 |
(18) 5.1 |
5 21 15 |
(14) 8.1 |
|||
15 µg |
136 119 117 |
(124) 10.4 |
28 18 19 |
(22) 5.5 |
15 12 21 |
(16) 4.6 |
19 33 28 |
(27) 7.1 |
22 17 13 |
(17) 4.5 |
|||
50 µg |
106 111 115 |
(111) 4.5 |
26 17 13 |
(19) 6.7 |
29 19 24 |
(24) 5.0 |
26 19 23 |
(23) 3.5 |
15 14 13 |
(14) 1.0 |
|||
150 µg |
129 134 92 |
(118) 22.9 |
13 16 13 |
(14) 1.7 |
31 26 21 |
(26) 5.0 |
18 14 35 |
(22) 11.2 |
11 12 9 |
(11) 1.5 |
|||
500 µg |
0 V 0 V 0 V |
(0) 0.0 |
17 10 13 |
(13) 3.5 |
18 16 19 |
(18) 1.5 |
0 V 0 V 0 V |
(0) 0.0 |
9 S 11 S 12 S |
(11) 1.5 |
|||
1500 µg |
0 T P 0 T P 0 T P |
(0) 0.0 |
10 S P 13 S P 8 S P |
(10) 2.5 |
0 V P 0 V P 0 V P |
(0) 0.0 |
0 T P 0 T P 0 T P |
(0) 0.0 |
5 S P 6 S P 7 S P |
(6) 1.0 |
|||
5000 µg |
0 T P 0 T P 0 T P |
(0) 0.0 |
0 V P 0 V P 0 V P |
(0) 0.0 |
0 V P 0 V P 0 V P |
(0) 0.0 |
0 T P 0 T P 0 T P |
(0) 0.0 |
0 V P 0 V P 0 V P |
(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 |
|||||||||
303 418 414 |
(378) 65.3 |
519 371 650 |
(513) 139.6 |
186 220 194 |
(200) 17.8 |
178 204 187 |
(190) 13.2 |
48 57 65 |
(57) 8.5 |
||||
ENNG: N-ethyl-N'-nitro-N-nitrosoguanidine
4NQO: 4-Nitroquinoline-1-oxide
9AA: 9-Aminoacridine
P: Test item precipitate
S: Sparse bacterial background lawn
T: Toxic, no bacterial background lawn
V: Very weak bacterial background lawn
#: Standard deviation
Table 5: Test Results: Experiment 2 – With Metabolic Activation (Pre-Incubation)
Test Period |
From: 12 November 2020 |
To: 15 November 2020 |
||||||||||||
S9-Mix (+) |
Dose Level Per Plate |
Number of revertants (mean) +/- SD |
||||||||||||
Base-pair substitution strains |
Frameshift strains |
|||||||||||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
||||||||||
Solvent Control (Acetone) |
143 138 153 |
(145) 7.6# |
11 9 10 |
(10) 1.0 |
32 24 27 |
(28) 4.0 |
19 22 21 |
(21) 1.5 |
20 8 12 |
(13) 6.1 |
||||
1.5 µg |
135 128 146 |
(136) 9.1 |
12 13 12 |
(12) 0.6 |
24 36 29 |
(30) 6.0 |
19 16 18 |
(18) 1.5 |
10 8 9 |
(9) 1.0 |
||||
5 µg |
155 136 124 |
(138) 15.6 |
14 8 6 |
(9) 4.2 |
30 24 40 |
(31) 8.1 |
19 23 24 |
(22) 2.6 |
20 13 14 |
(16) 3.8 |
||||
15 µg |
125 145 151 |
(140) 13.6 |
14 20 12 |
(15) 4.2 |
30 28 32 |
(30) 2.0 |
17 36 20 |
(24) 10.2 |
14 17 10 |
(14) 3.5 |
||||
50 µg |
143 131 132 |
(135) 6.7 |
21 9 15 |
(15) 6.0 |
37 20 25 |
(27) 8.7 |
29 13 22 |
(21) 8.0 |
11 18 13 |
(14) 3.6 |
||||
150 µg |
127 154 139 |
(140) 13.5 |
14 14 21 |
(16) 4.0 |
27 38 35 |
(33) 5.7 |
21 27 12 |
(20) 7.5 |
9 5 16 |
(10) 5.6 |
||||
500 µg |
114 115 97 |
(109) 10.1 |
10 11 8 |
(10) 1.5 |
30 32 33 |
(32) 1.5 |
20 29 19 |
(23) 5.5 |
4 15 15 |
(11) 6.4 |
||||
1500 µg |
104 S P 104 S P 98 S P |
(102) 3.5 |
14 P 11 P 12 P |
(12) 1.5 |
23 P 37 P 39 P |
(33) 8.7 |
16 S P 27 S P 20 S P |
(21) 5.6 |
16 S P 7 S P 12 S P |
(12) 4.5 |
||||
5000 µg |
78 S P 104 S P 79 S P |
(87) 14.7 |
11 S P 16 S P 12 S P |
(13) 2.6 |
32 S P 27 S P 31 S P |
(30) 2.6 |
25 S P 27 S P 21 S P |
(24) 3.1 |
0 V P 0 V P 0 V P |
(0) 0.0 |
||||
Positive controls Rat (+) |
Name |
2AA |
2AA |
2AA |
BP |
2AA |
||||||||
Dose Level |
1 µg |
2 µg |
10 µg |
5 µg |
2 µg |
|||||||||
No. of Revertants |
1155 1050 1209 |
(1138) 80.9 |
158 235 257 |
(217) 52.0 |
196 161 160 |
(172) 20.5 |
129 138 110 |
(126) 14.3 |
213 184 197 |
(198) 14.5 |
||||
BP: Benzo(a)pyrene
2AA: 2-Aminoanthracene
P: Test item precipitate
S: Sparse bacterial background lawn
V: Very weak bacterial background lawn
#: Standard deviation
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Gene mutation in bacteria (Ames test)
The potential of fatty acids, C6-18 (branched and linear) alkyl and hydrocarbons, C10-18, n-alkanes, branched alkanes, cycloalkanes, aromatics (EC No. 942-654-8) to induce gene mutation in bacteria was assessed in a reverse mutation assay according to OECD guideline 471 under GLP conditions (Covance, 2021d). Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using both the plate incorporation and pre-incubation methods at eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range for Experiment 1 (plate incorporation) was based on the recommendation given in the test guideline 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 in Experiment 1 (1.5 to 5000 µg/plate). Eight test item concentrations were selected in Experiment 2 in order to ensure the study achieved at least five analysable dose levels as required by the test guideline, and were selected based on the cytotoxicity noted in Experiment 1, and the potential for a change in the cytotoxicity of the test item following the change in test methodology from plate incorporation to pre-incubation. The vehicle (acetone) 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 and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The test item induced a visible reduction in the growth of the bacterial background lawn of all of the tester strains at and above 1500 µg/plate in both the presence and absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method). The test item induced a visible reduction in the growth of the bacterial background lawn of all of the tester strains dosed in the absence of metabolic activation (S9-mix) from 500 µg/plate to TA100, TA98 and TA1537 and 1500 µg/plate to TA1535 and WP2uvrA. In the presence of metabolic activation (S9-mix), toxicity was noted from 1500 µg/plate to TA100, TA98 and TA1537 and at 5000 µg/plate to TA1535 and WP2uvrA. A test item precipitate (globular in appearance) was noted at and above 1500 µg/plate in both the presence and absence of metabolic activation (S9-mix) in Experiments 1 and 2. This observation did not prevent the scoring of revertant colonies. There were no biologically relevant increases in the frequency of revertant colonies 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). A small, statistically significant increase in TA1537 revertant colony frequency was observed in the presence of S9-mix at 15 µg/plate. This increase was considered to be of no biological relevance because there was no evidence of a dose response relationship and the fold increase was only 1.5 times the concurrent vehicle control. Similarly, 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 2 (pre-incubation method). In this reverse mutation assay (Ames test) fatty acids, C6-18 (branched and linear) alkyl and hydrocarbons, C10-18, n-alkanes, branched alkanes, cycloalkanes, aromatics (EC No. 942-654-8) did not induce an increase in the frequency of revertant colonies that met the criteria for a positive result, either with or without metabolic activation (S9-mix). Under the conditions of this test the substance was considered to be non-mutagenic.
Justification for classification or non-classification
The available data on in vitro genetic toxicity of fatty acids, C6-18 (branched and linear) alkyl and hydrocarbons, C10-18, n-alkanes, branched alkanes, cycloalkanes, aromatics (EC No. 942-654-8) do not meet the criteria for classification according to Regulation (EC) No. 1272/2008 (CLP) and are therefore conclusive but not sufficient for classification.
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