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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
March - April 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP compliant

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2019

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
from July 21, 1997
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Certificate of Compliance with Good Laboratory Practices according to Directives 2004/9/CE and 2004/10/CE, Groupe Interministeriel des Produits Chimiques, Republique Francaise, Certificat n°: 2016/48, dated 2 Nov 2016
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: delivered from Sponsor, BATCH: 05297/MA
- Expiration date of the lot/batch: October 2021
- Purity test date: 27.10.2017


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature (15 - 25°C), non hygroscopic
- Stability: stable under storage conditions
- Solubility and stability of the test substance in the solvent/vehicle: soluble in DMSO, test solutions used immediately after preparation, no further details mentioned
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: no data

Method

Target gene:
his D, his C, his G, tryp E
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Remarks:
E. coli uvrA-
Metabolic activation:
with and without
Metabolic activation system:
S9 microsome fraction prepared from Sprague Dawley rat liver homogenate, was provided by MOLTOXTM (POB Box 1189 - 157 Industrial Park Dr - Boone, NC 28607 - USA) (S9 Moltox-11101-5-3919 validated on 28.06.2018 – expiry date: 07.02.2020).
Test concentrations with justification for top dose:
50, 150, 500, 1500 and 5000 µg/plate with and without S9 mix, top dose chosen according to guideline
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: test item is soluble in DMSO
Controls
Untreated negative controls:
yes
Remarks:
absolute negative control containing no test item; negative controls with solvents used to solubilize positive controls (Acetone, DMSO, NaCl 0.15 M)
Negative solvent / vehicle controls:
yes
Remarks:
vehicle used to solubilize test item (DMSO)
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
9-aminoacridine
2-nitrofluorene
sodium azide
other: cis-Platinum (II) Diammine Dichloride, CAS 15663-27-1: 1 µg/plate, E. coli without S9; 2-anthramine, CAS 613-13-8: 2 µg/plate, TA 98, 100, 1535, 1537 with S9 (plate incorporation); 1 µg/plate, TA 98, 100, 1535, 1537, with S9 (pre-incubation)
Details on test system and experimental conditions:
METHOD OF APPLICATION:
Salmonella Typhimurium strains: for each strain, 0.1 mL of the bacterial suspension containing 1-9 x10exp9 bacteria/mL) and 0.1 mL (in -aqueous or -oily vehicle / 50 µL (in non-aqueous or non-oily vehicle as ethanol ….) of each dilution of the original solution and 0.5 mL of sterile phosphate buffer were successively added to 2 mL of overlay agar, maintained supercooled at 45° C, containing 10 % (v/v) of a L-Histidine-D-Biotine solution (0.5 mM).

Escherichia coli strain : in a test tube 0.1 mL of the bacterial suspension containing 1-9 x 10exp9 bacteria/mL and ) and 0.1 mL (in -aqueous or -oily vehicle / 50 µL (in non-aqueous or non-oily vehicle as ethanol ….) of each dilution of the original solution and 0.5 mL of phosphate buffer were successively added to 2 mL of overlay agar maintained super cooled in 45° C containing 5% (v/v) of nutrient broth n° 2 to which are added 5 µL of a L-Tryptophane solution at 2 mg/mL.

Plates were incubated at 37° C over a 48-72-hour period. The number of revertant colonies per plate was counted.

Moreover the following controls were carried out:
Negative controls :
o absolute negative control containing no test item corresponding to the spontaneous reversion rate,
o solvent used to solubilize positive controls : Acetone, DMSO, NaCl 0.15 M
Vehicle used to solubilize test item : DMSO
Positive control

Two methodologies can be used: - either a standard plate incorporation method where the protocol is similar to that described above, except that, 500 µL of S9-mix fraction is quickly added, before pouring the mixture onto the plates ; - or the pre-incubation assay where the solution of the test item solution with the test strain, and 500 µL of S9-mix fraction are preincubated with shaking for 30 min., at 37° C prior to mixing with the overlay agar and pouring onto the minimal agar plate.

1st assay:
- without metabolic activation: in agar (plate incorporation),
- with metabolic activation: in agar (plate incorporation)
2nd assay (this method used because the first assay was negative):
- without metbolic activation: in agar (plate incorporation),
- with metabolic activation: pre-incubation

DURATION
- Preincubation period: 30 min (at 37°C, with shaking)
- Exposure duration: 48-72 h

NUMBER OF REPLICATIONS: 3 plates per experimental point

DETERMINATION OF CYTOTOXICITY
- Method: Bacteriostatic activity was tested in all concentrations in strain TA 100 in a preliminary cytotoxicity experiment: percent of survival in the plates treated with test item concentrations compared to negative control colonies was measured. In a test tube, 0.1 mL of the bacterial suspension (1-9 x 10exp3 bacteria/mL) and 0.1 mL (in -aqueous or -oily vehicle / 50 µL (in non-aqueous or non-oily vehicle as ethanol ….) of the stock solution and dilutions, were successively added to 2 mL of top agar at 45°C, containing 10 % (v/v) of a solution of L-Histidine-D-Biotine (2.5 mM). After homogenization, the content of the tube was poured onto a Petri plate (90 mm in diameter) containing minimal agar (20 mL). 3 plates per concentration were incubated for 48-72 hours at 37°C, and the colonies counted. A negative control containing the blank alone was run in parallel.

In case bacteriostatic activity is detected, the highest concentration to be retained is that exhibiting a bacteriostatic activity of 75 % or less. The precipitate, if present, should not interfere with the scoring. The following four dilutions studied are distributed according to a semi-logarithmic progression.
Evaluation criteria:
Study was judged to be valid if the following criteria were met:
- the bacteriostatic activity of the highest concentration tested is equal of less than 75%,
- the spontaneous reversion rate of the absolute negative control complies with the historical values of the laboratory,
- the spontaneous reversion rate of the solvent is not statistically different from absolute negative control,
- the mean number of revertant colonies obtained for each strain and the corresponding positive control, with and/or without metabolic activation complies with the historical values of the laboratory,
- negative and positive values don't show significant difference with the historical values of the laboratory (± 2 standard deviations).

Criteria for mutagenic activity in this assay:
The result of the test is considered as negative, if the revertant number is below three fold the number of spontaneous reversions, for TA 1535 and TA 1537 strains, and below two fold the number of spontaneous reversions for TA 98, TA 100 and Escherichia coli WP2 (uvrA-)(pKM101) strains without and with metabolic activation.
The result of the test is considered positive if a dependent relationship concentration is obtained in one, or several of the 5 strains, without and/or with metabolic activation, a mutagenic effect being taken into account for a given dilution of test item if the number of revertant colonies is at least two fold that of spontaneous revertant colonies for TA 98, TA 100 and Escherichia coli WP2 (uvrA-)(pKM101), and three fold for TA 1535 and TA 1537.
All results must be confirmed in an independent experiment.
Statistics:
Mean values of replicates with standard deviations were calculated per plate.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
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:
no cytotoxicity
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:
no cytotoxicity
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:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: Results of the bacteriostatic activity control testing show that neither original solution nor dilutions have bacteriostatic effect.

HISTORICAL CONTROL DATA (with number of experiments, ranges, means and standard deviation) (2009-2018)

POSITIVE HISTORICAL CONTROL DATA
TA 1535:
- without metabolic activation, plate incorporation, Sodium Azide: n=1017, range: 190 - 1487, mean: 757.7 ± 217.3
- with metabolic activation, plate incorporation, 2-Anthramine: n=540, range: 26 - 269, mean: 1112.9 ± 56.3
- with metabolic activation, pre-incubation, 2-Anthramine: n=534, range: 25 - 997, mean: 83.0 ± 77.7
TA 1537:
- without metabolic activation, plate incorporation, 9-Aminoacridine: n=1017, range: 224 - 1979, mean: 917.5 ± 465.2
- with metabolic activation, plate incorporation, 2-Anthramine: n=540, range: 24 - 170, mean: 55.4 ± 25.0
- with metabolic activation, pre-incubation, 2-Anthramine: n=534, range: 20 - 182, mean: 47.9 ± 25.3
TA 98:
- without metabolic activation, plate incorporation, 2-Nitrofluorene: n=1017, range: 187 - 1667, mean: 501.5 ± 219.9
- with metabolic activation, plate incorporation, 2-Anthramine: n=540, range: 220 - 1499, mean: 585.0 ± 225.6
- with metabolic activation, pre-incubation, 2-Anthramine: n=534, range: 174 - 1368, mean: 468.0 ± 207.4
TA 100:
- without metabolic activation, plate incorporation, Sodium Azide: n=1017, range: 381 - 1690, mean: 1038.8 ± 331.9
- with metabolic activation, plate incorporation, 2-Anthramine: n=537, range: 361 - 2163, mean: 850.7 ± 373.0
- with metabolic activation, pre-incubation, 2-Anthramine: n=537, range: 291 - 1720, mean: 640.5 ± 279.9
Escherichia coli WP2 (pKM101)(uvr A-)
- without metabolic activation, plate incorporation, cis-Platinium (II) Diamine Dichloride: n=819, range: 248 - 1089, mean: 485.4 ± 171.6
- with metabolic activation, plate incorporation, Dimethyl Benzanthracene: n=402, range: 365 - 1680, mean: 680.9 ± 246.8
- with metabolic activation, pre-incubation, Dimethyl Benzanthracene: n=420, range: 281 - 1680, mean: 668.0 ± 225.4

NEGATIVE (SOLVENT/VEHICLE) HISTORICAL CONTROL DATA
TA 1535:
- without metabolic activation, plate incorporation: n=1017, range: 4 - 23, mean: 11.0 ± 3.7
- with metabolic activation, plate incorporation: n=540, range: 3 - 23, mean: 12.3 ± 4.1
- with metabolic activation, pre-incubation: n=534, range: 5 - 25, mean: 12.9 ± 4.2
TA 1537:
- without metabolic activation, plate incorporation: n=1017, range: 1 - 20, mean: 6.1 ± 2.5
- with metabolic activation, plate incorporation: n=540, range: 1 - 24, mean: 8.0 ± 3.6
- with metabolic activation, pre-incubation: n=534, range: 1 - 21, mean: 8.2 ± 3.5
TA 98:
- without metabolic activation, plate incorporation: n=1017, range: 6 - 29, mean: 15.9 ± 3.9
- with metabolic activation, plate incorporation: n=540, range: 11 - 38, mean: 23.1 ± 5.2
- with metabolic activation, pre-incubation: n=534, range: 10 - 36, mean: 23.1 ± 5.4
TA 100:
- without metabolic activation, plate incorporation: n=1017, range: 40 - 121, mean: 60.1 ± 12.2
- with metabolic activation, plate incorporation: n=537, range: 55 - 152, mean: 90.8 ± 17.9
- with metabolic activation, pre-incubation: n=534, range: 51 - 156, mean: 90.5 ± 19.5
Escherichia coli WP2 (pKM101)(uvr A-)
- without metabolic activation, plate incorporation: n=819, range: 41 - 188, mean: 88.8 ± 36.3
- with metabolic activation, plate incorporation: n=420, range: 80 - 264.0, mean: 156.5 ± 34.0
- with metabolic activation, pre-incubation: n=420, range: 69 - 250, mean: 159.2 ± 36.1

Any other information on results incl. tables

Table #1: Assay n° 1, without metabolic activation, plate incorporation

Concentration [µg/plate] Revertant colonies / plate
TA 98 TA 100 TA 1535 TA 1537 E. coli WP2 uvrA
mean±SD R mean±SD R mean±SD R mean±SD R mean±SD R
Negative control 16.00 ± 1.73 - 69.67 ± 4.51 - 9.33 ± 2.52 - 7.67 ± 2.31 - 159.00 ± 12.12 -
Vehicle (DMSO) 17.33 ± 4.62 - 64.67 ± 5.51 - 13.00± 1.73 - 7.67 ± 3.51 - 169.00 ± 12.12 -
50 19.00 ± 0.00 1.10 59.33 ± 5.03 0.92 12.67 ± 2.52 0.97 7.00 ± 2.65 0.91 107.00 ± 8.72 0.70
150 15.33 ± 3.79 0.88 58.00 ± 2.00 0.90 11.67 ± 1.53 0.90 5.33 ± 2.52 0.70 125.00 ± 30.81 0.74
500 18.00 ± 2.00 1.04 54.67 ± 11.93 0.85

13.33 ± 2.08

1.03

4.43 ± 0.58

0.57

135.33 ± 12.50

0.80

1500

18.33 ± 4.16

1.06

58.00 ± 5.57

0.90

7.67 ± 2.89

0.59

6.00 ± 2.00

0.78

148.00 ± 24.58

0.88

5000

14.00 ± 3.61

0.81

57.00 ± 7.55

0.88

6.67 ± 2.08

0.51

5.33 ± 3.21

1.25

128.33 ± 20.03

0.76

Positive control solvent

17.00 ± 5.20

-

60.67 ± 2.08

-

13.33 ± 2.08

-

8.33 ± 0.58

-

136.33 ± 5.51

-

Positive control

833.00 ± 38.00

49.00

1606.33 ± 47.44

26.48

950.67 ± 114.99

71.3

909.00 ± 94.17

109.08

303.33 ± 18.99

2.22

Table #2: Assay n° 1, with metabolic activation (10% S9 mix), plate incorporation

Concentration [µg/plate]

Revertant colonies / plate

TA 98

TA 100

TA 1535

TA 1537

E. coli WP2 uvrA

mean±SD

R

mean±SD

R

mean±SD

R

mean±SD

R

mean±SD

R

Negative control

19.33 ± 4.51

-

75.00 ± 8.72

-

14.67 ± 1.51

-

11.00 ± 3.61

-

247.00 ± 9.00

-

Vehicle (DMSO)

19.67 ± 5.13

-

81.67 ± 5.86

-

15.67 ± 2.31

- 10.00 ± 2.00 - 224.67 ± 6.11 -
50 17.33 ± 1.15 0.88 81.33 ± 2.08 1.00 13.33 ± 2.52 0.85 7.33 ± 3.79 0.73 237.00 ± 12.00

1.05

150

28.00 ± 2.65

1.42

79.33 ± 5.51

0.97

9.33 ± 1.53

0.60

9.33 ± 3.06

0.93

226.00 ± 24.58

1.01

500

27.00 ± 5.29

1.37

72.00 ± 5.57

0.88

11.00 ± 5.29

0.70

8.00 ± 2.65

0.80

220.00 ± 16.09

0.98

1500

27.00 ± 5.29

1.37

66.67 ± 13.65

0.82

9.67 ± 3.06

0.62

8.67 ± 4.51

0.87

232.00 ± 27.62

1.03

5000

24.67 ± 4.62

1.25

71.00 ± 6.56

0.87

9.33 ± 6.81

0.60

9.67 ± 3.51

0.97

268.33 ± 17.56

1.19

Positive control solvent

21.00 ± 5.29

-

76.67 ± 11.59

-

17.00 ± 2.00

-

9.00 ± 2.65

-

214.00 ± 1.73

-

Positive control

430.33 ± 90.14

23.45

625.00 ± 118.65

8.15

106.33 ± 27.54

6.25

47.00 ± 12.53

5.22

477.33 ± 21.83

2.23

Table #3: Assay n° 2, without metabolic activation, plate incorporation

Concentration [µg/plate]

Revertant colonies / plate

TA 98

TA 100

TA 1535

TA 1537

E. coli WP2 uvrA

mean±SD

R

mean±SD

R

mean±SD

R

mean±SD

R

mean±SD

R

Negative control

12.67 ± 3.79

-

56.67 ± 7.64

-

8.67 ± 4.73

-

4.33 ± 0.58

-

95.00 ± 14.42

-

Vehicle (DMSO)

14.67 ± 5.77

-

67.67 ± 1.53

-

15.00 ± 5.20

-

3.00 ± 2.00

-

93.33 ± 11.93

-

50

16.67 ± 0.58

1.14

49.00 ± 2.65

0.72

11.67 ± 4.04

0.78

3.67 ± 2.31

1.22

78.67 ± 3.21

0.84

150

17.00 ± 1.73

1.73

53.00 ± 7.81

0.78

10.00 ± 1.00

0.67

5.00 ± 4.36

1.67

90.33 ± 5.69

0.97

500

12.33 ± 4.93

0.84

48.33 ± 2.31

0.71

8.33 ± 1.53

0.56

4.33 ± 1.15

1.44

80.00 ± 2.65

0.86

1500

12.00 ± 3.46

0.82

54.67 ± 4.73

0.81

6.67 ± 1.15

0.44

4.67 ± 2.08

1.56

74.00 ± 9.85

0.79

5000

13.00 ± 5.20

0.89

47.00 ± 4.58

0.69

7.67 ± 2.08

0.51

3.67 ± 2.89

1.22

73.00 ± 5.29

0.78

Positive control solvent

10.00 ± 6.08

-

61.00 ± 3.00

-

12.33 ± 4.04

-

2.00 ± 1.73

-

72.33 ± 10.07

-

Positive control

363.67 ± 37.17

36.17

1363.00 ± 145.32

22.34

1265.00 ± 63.93

102.57

631.33 ± 99.73

118.22

302.33 ± 54.45

4.18

Table #4: Assay n° 2, with metabolic activation, pre-incubation

Concentration [µg/plate]

Revertant colonies / plate

TA 98

TA 100

TA 1535

TA 1537

E. coli WP2 uvrA

mean±SD

R

mean±SD

R

mean±SD

R

mean±SD

R

mean±SD

R

Negative control

16.67 ± 1.15

-

79.67 ± 6.43

-

17 -33 ± 1.53

-

5.33 ± 3.51

-

134.67 ± 8.33

-

Vehicle (DMSO)

22.67 ± 7.51

-

100.67 ± 10.02

-

11.67 ± 4.16

-

5.33 ± 4.16

-

113.67 ± 12.50

-

50

22.33 ± 9.81

0.99

65.33 ± 4.93

0.65

11.00 ± 2.00

0.94

7.00 ± 1.00

1.31

121.33 ± 6.66

1.07

150

14.00 ± 1.73

0.62

69.67 ± 13.58

0.69

9.00 ± 7.00

0.77

5.00 ± 1.73

0.94

141.33 ± 3.21

1.24

500

23.00 ± 10.39

1.01

78.00 ± 10.82

0.77

7.00 ± 3.00

0.60

7.67 ± 2.08

1.44

137.00 ± 8.72

1.21

1500

23.00 ± 8.66

1.01

79.33 ± 4.93

0.79

10.00 ± 3.61

0.86

4.33 ± 0.58

0.81

135.33 ± 3.06

1.19

5000

12.67 ± 2.89

0.56

57.33 ± 3.79

0.57

5.67 ± 1.53

0.49

4.00 ± 1.00

0.75

142.67 ± 10.41

1.26

Positive control solvent

17.67 ± 8.33

-

73.00 ± 7.81

-

14.33 ± 5.51

-

5.67 ± 2.08

-

142.67 ± 3.06

-

Positive control

247.67 ± 17.90

14.02

329.33 ± 37.81

4.51

41.67 ± 11.59

2.91

28.33 ± 3.51

5.00

340.00 ± 70.00

2.38

SD = Standard deviation; R = Number of revertant colonies in the presence of the test item/ Number of revertant colonies in the absence of the test item

Applicant's summary and conclusion

Conclusions:
Doses (5 000, 1 500, 500, 150 and 50 µg/plate) prepared from solutions of the test item 2-Hexyldecanoic acid do not induce any mutagenic change in Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and in Escherichia coli WP2(uvrA¯) (pKM 101) without, or with metabolic activation, according to the OECD Guideline n° 471.

Executive summary:

A bacterial reverse mutation test using Salmonella typhimurium his- and Escherichia coli WP2 (uvrA-)(pKM101) was performed according to OECD Guideline n°471 in compliance with Good Laboratory Practices (GLP).

Solutions obtained from the test item have been tested for their capacity to induce reverse mutation in four Salmonella typhimurium strains and one Escherichia coli WP2 (uvrA-)(pKM101) strain. This study was performed in the absence and presence of metabolic activation. Two independent assays were carried out. For assay n°1, various concentrations were put in contact with the strains in the absence and presence of a metabolic activation system (S9 -mix 10% (v/v)). For assay n°2, various concentrations were put in contact with test strains in absence and presence of a metabolic activation system (S9 -mix 10%(v/v)). For the two assays, negative and postive controls were carried out in parallel. Positive controls induced a significant increase in the number of revertant colonies compared to negative controls. There was no siginificant difference between the number of spontaneous reversions, the number of reversions obtained in the prositive controls (without and with metabolic activation), and the mean of corresponding experimental "historical" values obtained in the laboratory. These results validated the two tests. There was no evidence of any increase in the number of revertant colonies in the presence of the various concentrations of the test item (50, 150, 500, 1500, and 5000 µg/plate), without and with metabolic activation in Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100, and in Escherichia coli WP2 (uvrA-)(pKM101). The test item was judged to be non-mutagenic in this test system under the described conditions.