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Genetic toxicity in vitro

Description of key information

Ames test (OECD 471): negative in S. typhimurium TA1535, TA1537, TA98, TA100 and TA102 with and without metabolic activation

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
31 Jul - 18 Aug 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 Jul 1997
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Bayrisches Landesamt für Arbeitsschutz, Arbeitsmedizin und Sicherheitstechnik, München, Germany
Type of assay:
bacterial reverse mutation assay
Target gene:
his operon
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of male rats treated with phenobarbital (80 mg/kg bw) and β-naphtoflavone (100 mg/kg bw)
Test concentrations with justification for top dose:
Pre-experiment: 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate with and without metabolic activation (TA98 and TA100)
Experiment I: 1.0, 3.16, 10.0, 31.6, 100, 316 and 1000 µg/plate with and without metabolic activation
Experiment II: 0.5, 1.58, 5.0, 15.8, 50, 158 and 500 µg/plate with and without metabolic activation
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
other: -S9: sodium azide (10 µg/plate) for TA100 and TA1535; methyl methane sulfonate (1 µL/plate) for TA102; 4-nitro-o-phenylendiamine (10 or 40 µg/plate) for TA98 and TA1537, respectively; +S9: 2 aminoanthracene (2.5 or 10 µg/plate) for all strains
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: triplicates each in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: diminution of the background lawn, reduction in the number of revertants (reduction in revertants smaller or equal to 50%)
Rationale for test conditions:
A test is considered acceptable if for each strain:
- the bacteria demonstrate their typical response to ampicillin (TA98, TA100, TA102)
- the control plates with and without S9 mix are within the following ranges (mean values of the spontaneous reversion frequency are within the historical control data range):
without metabolic activation: TA98 (18 - 54), TA100 (75 - 167), TA1535 (5 - 29), TA1537 (5 - 30) and TA102 (165 - 391)
with metabolic activation: TA98 (18 - 71), TA100 (81 - 168), TA1535 (6 - 31), TA1537 (6 - 36) and TA102 (163 - 594)
- corresponding background growth on negative control, solvent control and test plates is observed.
- the positive controls show a distinct enhancement of revertant rates over the control plate.
Evaluation criteria:
A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs
in at least one tester strain with or without metabolic activation.

A biologically relevant increase is described as follows:
- if in tester strains TA100 and TA102 the number of reversions is at least twice as high
- if in tester strains TA98, TA1535 and TA 1537 the number of reversions is at least three times higher than the reversion rate of the solvent control.
According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.

A test item producing neither a dose related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.
Statistics:
Mean values and standard deviations were calculated.
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
starting at ≥ 100 µg/plate (without S9 mix) and at 316 µg/plate (with S9 mix)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
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:
starting at 316 µg/plate (with and without S9 mix)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
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:
starting at 316 µg/plate (with and without S9 mix)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
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:
starting at 316 µg/plate (with and without S9 mix)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
starting at ≥ 100 µg/plate (without S9 mix) and at 316 µg/plate (with S9 mix)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation of the test substance was observed in any tester strain used in Experiment I and II with and without metabolic activation.

RANGE-FINDING/SCREENING STUDIES:
The toxicity of the test substance was determined in tester strains TA98 and TA100 at concentrations of 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/mL with and without metabolic activation. A reduced or no background lawn was observed starting at a concentration of 316 µg/ml in both strains with and without metabolic activation. Furthermore, a reduced number of revertant colonies was observed starting at 100 µg/plate. Based on these results, 1000 and 500 µg/plate were selected for Experiment I and II, respectively.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: The determined values of the positive controls fall within the range of the historical control data, please refer to Table 3.
- Negative (solvent) historical control data: The determined values of the solvent controls fall within the range of the historical control data, except tester strain TA 102 in experiment I, in which the number of mutants (149 +/-12.3) was slightly below the mean value of the historical control data (163 - 594). Considering the standard deviation, the reduced number of colonies in the vehicle control is not considered to affect the validity of the study, please refer to Table 3.

Table 1: Test results of experiment I

With or without S9-Mix Test substance concentration Mean number of revertant colonies per plate 
(μg/plate) (average of 3 plates ± Standard deviation)
  Base-pair substitution type Frameshift type
  TA 100 TA1535 TA102 TA98 TA1537
DMSO 114 ± 13.0 22 ± 2.6 188 ± 16.9 28 ± 4.0 10 ± 2.9
Aqua dest. 112 ± 8.7 16 ± 5.5 221 ± 11.1 32 ± 3.2 11 ± 3.0
1.00 112 ± 5.3 20 ± 3.5 237 ± 7.8 36 ± 5.3 9 ± 1.7
3.16 106 ± 15.3 21 ± 2.9 202 ± 9.3 34 ± 6.6 10 ± 3.6
10.0 118 ± 6.7 26 ± 8.1 212 ± 5.7 28 ± 2.3 9 ± 2.6
31.6 115 ± 4.7 21 ± 8.2 172 ± 24.5 29 ± 4.2 11 ± 3.2

100.0

114 ± 31.2

9 ± 3.8

39 ± 7.9

24 ± 4.2

11 ± 3.8

 – 

316

0 ± 0.0 B/N

0 ± 0.0 N

0 ± 0.0 N

0 ± 0.0 B/N

0 ± 0.0 N

1000

0 ± 0.0 N

0 ± 0.0 N

0 ± 0.0 N

0 ± 0.0 N

0 ± 0.0 N

Positive controls, –S9

Name 

NaN3

NaN3

MMS

4-NOPD

4-NOPD

Concentrations (μg/plate)

10

10

1

10

40

Mean No. of colonies/plate (average of 3 ± SD)

1192 ± 68.9

1546 ± 129.7

2191 ± 85.6

646 ± 89.3

183 ± 39.9

+

DMSO

108 ± 5.3

13 ± 2.1

149 ± 12.3

37 ± 9.5

6 ± 2.0

+

Aqua dest.

109 ± 19.2

13 ± 3.1

276 ± 15.7

38 ± 4.4

8 ± 1.5

+

1.00

109 ± 10.0

9 ± 1.0

255 ± 4.7

44 ± 6.7

11 ± 3.8

+

3.16

110 ± 15.9

11 ± 4.0

233 ± 3.8

38 ± 4.7

12 ± 4.0

+

10.0

121 ± 5.3

13 ± 3.5

257 ± 2.1

27 ± 4.6

10 ± 2.9

+

31.6

131 ± 16.2

14 ± 7.0 200 ± 67.4 31 ± 8.4 13 ± 7.1
+ 100.0 98 ± 6.5 9 ± 1.5 193 ± 49.1 30 ± 10.0 10 ± 3.6
+ 316 0 ± 0.0 B 0 ± 0.0  0 ± 0.0 N 0 ± 0.0  0 ± 0.0
+ 1000 0 ± 0.0 N 0 ± 0.0 N 0 ± 0.0 N 0 ± 0.0 N 0 ± 0.0 N
Positive controls, +S9 Name  2AA 2AA 2AA 2AA 2AA
Concentrations (μg/plate) 2.5 2.5 10 2.5 2.5
Mean No. of colonies/plate (average of 3 ± SD) 3103 ± 388.4 227 ± 12.2 874 ± 189.7 2824 ± 495 371 ± 71.3

NaN3 = Sodium azide

4NOPD = 4-nitro-o-phenylene-diamine

2AA = 2-Aminoanthracene

B = Background lawn reduced

N = No background lawn

Table 2: Test results of experiment II

With or without S9-Mix Test substance concentration Mean number of revertant colonies per plate 
(μg/plate) (average of 3 plates ± Standard deviation)
  Base-pair substitution type Frameshift type
  TA 100 TA1535 TA102 TA98 TA1537
DMSO 109 ± 8.0 16 ± 5.5 199 ± 12.7 19 ± 2.1 8 ± 1.0
  Aqua dest. 114 ± 15.4 14 ± 3.6 188 ± 17.0 28 ± 5.3 8 ± 3.2
0.50 100 ± 6.1 16 ± 3.5 154 ± 21.4 20 ± 3.8 11 ± 3.2
1.58 101 ± 4.6 20 ± 1.7 166 ± 7.0 26 ± 4.7  10 ± 3.1
5.0 105 ± 18.9 22 ± 5.3 201 ± 2.1 24 ± 1.5 13 ± 4.6
15.8 108 ± 18.5 26 ± 8.4 193 ± 16.3 23 ± 3.2 13 ± 2.1
  50 126 ± 11.4 28 7.2 213 ± 34.8 18 ± 5.3 12 ± 1.7
  158 0 ± 0.0 0 ± 0.0 0 ± 0.0 B 0 ± 0.0 9 ± 2.5
500 0 ± 0.0 N 0 ± 0.0 N 0 ± 0.0 N 0 ± 0.0 N 0 ± 0.0 N
Positive controls, –S9 Name  NaN3 NaN3 MMS 4-NOPD 4-NOPD
Concentrations (μg/plate) 10 10 1 10 40
Mean No. of colonies/plate (average of 3 ± SD) 1278 ± 24.6 1364 100.7 1068 ± 111.8 1038 ± 238.3 208 ± 25.2
+ DMSO 110 ± 6.9 12 ± 2.0 223 ± 5.0 29 ± 4.7 11 ± 2.1
+ Aqua dest. 110 ± 5.5 15 ± 1.0 217 ± 8.9 35 ± 3.6 10 ± 0.0
+ 0.50 103 ± 7.0 9 ± 1.5 198 ± 6.0 27 ± 5.7 8 ± 2.0
+ 1.58 102 ± 15.0 10 ± 2.6 187 ± 25.0 26 ± 3.2 10 ± 1.2
+ 5.0 107 ± 18.9 10 ± 5.5 232 ± 23.2 28 ± 4.4 12 ± 5.5
  15.8 113 ± 7.0 10 ± 5.1 244 ± 19.9 27 ± 5.7 12 ± 2.5
  50 132 ± 11.0 14 ± 3.1 291 ± 20.6 26 ± 2.0 10 ± 7.1
  158 0 ± 0.0 1 ± 1.10 44 ± 13.44 7 ± 6.2 8 ± 4.7
+ 500 0 ± 0.0 B 0 ± 0.0 B 0 ± 0.0 0 ± 0.0 N 0 ± 0.0 B
Positive controls, +S9 Name  2AA 2AA 2AA 2AA 2AA
Concentrations (μg/plate) 2.5 2.5 10 2.5 2.5
Mean No. of colonies/plate (average of 3 ± SD) 3103 ± 388.4 224 ± 21.1 1117 ± 97.4 2824 ± 495 392 ± 16.7

NaN3 = Sodium azide

4NOPD = 4-nitro-o-phenylene-diamine

2AA = 2-Aminoanthracene

B = Background lawn reduced

N = No background lawn

Table 3: Historical laboratory control data

Historical Laboratory Contral Data of the Negative Control without S9
  TA 100 TA1535 TA102 TA98 TA1537
Mean 109.3 15.1 250.4 26.8 12.0
SD 14.4 4.4 50.7 6.0 3.7
Min 75 5 165 18 5
Max 167 29 391 54 30
RSD (%) 13.1 32.4 20.3 22.2 31.0
n 751 738 547 751 723
Historical Laboratory Contral Data of the Positive Control without S9
  TA 100 TA1535 TA102 TA98 TA1537
Mean 906.1 933.3 1879.5 704.6 169.1
SD 304.6 215.6 437.1 316.6 56.1
Min 235.0 79 710 269 48
Max 66552 1630 3357 2420 1132
RSD (%) 33.6 23.1 23.3 44.9 33.2
n 748 737 548 749 721
Historical Laboratory Contral Data of the Negative Control with S9
  TA 100 TA1535 TA102 TA98 TA1537
Mean 113.6 11.8 309.6 37.2 13.1
SD 14.6 3.2 67.6 8.1 4.0
Min 81 6 163 18 6
Max 168 31 594 71 36
RSD (%) 12.9 27.0 21.8 21.9 30.6
n 759 735 548 748 729
Historical Laboratory Contral Data of the Positive Control with S9
  TA 100 TA1535 TA102 TA98 TA1537
Mean 1938.7 115.7 1040.7 2058.1 260.3
SD 585.1 58.0 320.0 703.7 96.4
Min 253 26 117 121 41
Max 3167 872 2102 3430 509
RSD (%) 30.2 50.2 546 34.2 37.0
n 757 733 427 746 726
Conclusions:
It can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the Salmonella strains used.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Genetic toxicity

The mutagenic properties of quinolin-8-ol (CAS 148-24-3) were assessed in a bacterial reverse mutation assay (Ames test) according to GLP criteria and OECD TG 471 (reference 7.6.1-1). The mutagenic potential of the test substance was assessed in S. typhimurium tester strains TA 98, 100, 102, 1535 and 1537 at concentrations up to 500 and 1000 µg/plate, respectively, in 2 independent experiments. No precipitation of the test substance was observed in any experiment with and without metabolic activation. Cytotoxicity was observed with and without metabolic activation at least at the highest concentration tested. Quinolin-8-ol did not induce an increase in reversions in any of the S. typhimurium strains tested with or without metabolic activation. The vehicle and positive controls were valid and lay within the range of historical control data. Based on the results in the conducted study, quinolin-8-ol (CAS 148-24-3) did not exhibit mutagenic properties in bacteria.

Justification for classification or non-classification

Based on the available data on genetic toxicity, there is no indication that quinolin-8-ol induces genetic toxicity in bacteria. However, no final decision on classification for genetic toxicity according to Regulation (EC) 1272/2008 can be made, as no information on chromosomal aberration and mutagenicity in mammalian cells/in vivo is available.