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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:
December 16, 2017 - February 6, 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
2-hydroxy-4-(trifluoromethyl)benzoic acid
EC Number:
700-368-9
Cas Number:
328-90-5
Molecular formula:
C8H5F3O3
IUPAC Name:
2-hydroxy-4-(trifluoromethyl)benzoic acid
Test material form:
solid: particulate/powder
Details on test material:
Beige to brownish solid

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Additional strain / cell type characteristics:
other: rfa mutation (histidine dependence), uvrB mutation (biotin dependence)
Metabolic activation:
with and without
Test concentrations with justification for top dose:
Test 1: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate (all strains)
Test 2: 156.25, 512.5, 625, 1250, 2500 and 5000 µg/plate (all strains)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: A preliminary solubility and precipitation test was used. The substance was insoluble in distilled water.
Controls
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
mitomycin C
other: 2-Aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)
Tubes containing 2 mL of molten top agar with 0.5 mM histidine/biotin were maintained at 45 ± 2 °C. A volume of 500 µL of 0.2 M phosphate buffer was added in the absence of metabolic activation system and 500 µL of 5% v/v S9 mix (Test 1) or 10% v/v S9 (Test 2) was added in the presence of metabolic activation system. Volume of 100 µL of the relevant stock solution of test item, DMSO and relevant positive control were used for treatment, as a negative control and as a positive control, respectively. Finally, 100 µL of bacterial culture (1 - 2 x 10E9 bacteria/mL) was added to the tubes and mixed. Two sets were maintained for each concentration of dihydromyrcenyl formate, positive control and negative control. The petriplates were incubated at 37 ± 1 °C for 48 hours and then examined to assess the state of background bacterial lawn inhibition and reduction in number of colonies.

DURATION
- Expression time (cells in growth medium): 48 h

NUMBER OF REPLICATIONS: 2 (Test 1) and 3 (Test 2)

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity was detectable as decrease in the number of revertant colonies per plate and/or by a thinning of the bacterial background lawn.

OTHER EXAMINATIONS:
Cell viability test: Fresh cultures for the test were prepared by inoculating frozen permanent cultures to a flask containing 10 mL of sterile nutrient broth N° 2 (Oxoid). The flasks were then incubated at 37 ± 1 °C in an orbital shaking incubator (120 rpm) for 15 h up to early stationary or late exponential phase. After the incubation period, the culture flasks were removed from the orbital shaking incubator. Aseptically, the cultures were diluted with oxoid nutrient broth (ONB) and the optical density was measured at 660 nm using a Spectrophotometer (Model visiscan 167, manufactured by Systronics). Oxoid nutrient broth was used as the control blank. Cell viability of the tester strains was determined prior to treatment. The optical density of the cultures was found to be in the acceptable range and so they were used for the study.

Genotype confirmation test: The genotype of the tester strain was confirmed for all the strains regularly (once a month). The tester strains of Salmonella typhimurium were tested for histidine dependence, biotin dependence, histidine and biotin dependence, rfa mutation, uvrB mutation through sensitivity to ultraviolet light and the R-factor resistance for ampicillin and tetracycline.

Sterility Check for the Operating System: Top agar, S9 mix, Solvent, Test item, ONB solution, 0.2 M Sodium Phosphate Buffer, MGA Plate.
Rationale for test conditions:
Hence DMSO was selected as the vehicle for treatment. Precipitation was not observed at the tested concentration of 5 µL/plate. Hence 5 µL/plate was selected as the highest concentration to be tested for initial toxicity-mutation test both in the absence and presence (5% v/v S9 mix) of metabolic activation.
Evaluation criteria:
The conditions necessary for determining a positive result were: there should be a dose related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing doses of the test item either in the absence or presence of the metabolic activation system.
Biological relevance of the results was considered first:
Strains TA1535, TA1537:
Data sets were judged positive, when the increase in mean revertants at the peak of the dose response was equal to or greater than 3.0 times the mean negative control value.
Strain TA98, TA100 and TA102:
Data sets were judged positive, when the increase in mean revertants at the peak of the dose response was equal to or greater than 2.0 times the mean negative control value.
A response that did not meet all three of the above criteria (magnitude, concentration-responsiveness, reproducibility) was not evaluated as positive.
Negative results obtained in the initial toxicity-mutation test were confirmed by a second trial, using the same method as specified above, with an alteration in concentration spacing.
Statistics:
Statistical analysis was used as an aid in the evaluation of dose response.

Results and discussion

Test results
Key result
Species / strain:
S. typhimurium, other: TA1537, TA1535, TA98, TA100 and TA102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
(at 5000 µg/plate)
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
No increase in the number of revertant colonies was observed both in the absence and presence of metabolic activation at any of the tested concentration in all the tester strains.

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No observed.
- Definition of acceptable cells for analysis:
- Other confounding effects: Cell viability and genotype confirmatory tests were performed (see above). The cells were determined to be suitable for the test.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: Revertants per plate (mean ± SD)
Without S9 mix
TA1537: 274.27 ± 94.89
TA 1535: 376.70 ± 113.61
TA98: 542.14 ± 148.27
TA100: 880.92 ± 175.36
TA102: 1058.59 ± 152.48

With S9 mix
TA1537: 300.52 ± 98.11
TA 1535: 419.02 ± 122.05
TA98: 671.47 ± 171.10
TA100: 968.94 ± 158.04
TA102: 1088.70 ± 154.86

Positive controls in the absence of metabolic activation:
TA1537 = 9-Aminoacridine Hydrochloride Monohydrate (75 µg/plate), TA1535 = Sodium Azide (0.5 µg/plate), TA98 = 2-Nitrofluorene (7.5 µg/plate), TA100 = Sodium Azide (5 µg/plate), TA102 = Mitomycin-C (0.5 µg/plate).
Positive controls in the presence of metabolic activation
2Aa = 2-Aminoanthracene (10 µg/plate for TA1537, TA1535, TA102 and 5 µg/plate for TA98 and TA100)

- Negative (DMSO) historical control data: Revertants per plate (mean ± SD)
Without S9 mix
TA1537: 8.40 ± 2.73
TA 1535: 18.37 ± 4.44
TA98: 22.79 ± 4.92
TA100: 131.37 ± 10.65
TA102: 228.89 ± 12.48

With S9 mix
TA1537: 8.96 ± 3.02
TA 1535: 20.03 ± 4.97
TA98: 24.24 ± 5.43
TA100: 134.50 ± 10.53
TA102: 232.55 ± 13.12

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: Normal growth in bacterial background lawn pattern was observed up to the tested concentration of 5000 µg/plate. Cytotoxicity was characterised by the reduction in the number of revertant colonies:
Test 1: Significant reduction in number of revertant colonies was observed (i.e. 92.2% to 100%) at the tested concentration of 5000 µg/plate both in the absence and presence of the metabolic activation (5% v/v S9 mix) in all the tester strains.
Test 2: Significant reduction in number of revertant colonies was observed (i.e. 84.43% to 100%) at the tested concentration of 5000 µg/plate both in the absence and presence of the metabolic activation (10% v/v S9 mix) in all the tester strains. Reduction in number of revertant colonies was observed (i.e. 36.61% to 45.88%) in both presence and absence of metabolic activation in all tester strains up to the tested concentration of 2500 µg/plate.
- Other observations when applicable: No.

Any other information on results incl. tables

Test 1: Without metabolic activation (-S9)

Concentration

(µg/plate)

His+Revertant Colonies/Plate (Absence of Metabolic Activation)

TA1537

TA1535

TA98

TA100

TA102

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

NC (DMSO)

6.00

1.41

14.50

0.71

16.00

4.24

141.00

5.66

237.50

6.36

1.5

7.00

2.83

14.00

5.66

20.50

3.54

134.00

5.66

244.50

9.19

5

4.50

0.71

14.00

1.41

19.50

7.78

125.00

5.66

230.50

3.54

15

5.50

0.71

16.50

3.54

18.50

2.12

138.00

14.14

230.50

2.12

50

6.00

0.00

16.00

1.41

18.00

7.07

136.50

3.54

231.50

13.44

150

6.00

4.24

14.50

6.36

17.00

4.24

138.00

8.49

232.50

6.36

500

2.50

0.71

15.00

4.24

17.00

5.66

140.50

7.78

237.00

22.63

1500

5.00

2.83

17.00

2.83

16.00

4.24

131.50

12.02

231.50

16.26

5000

0.00

0.00

0.00

0.00

0.00

0.00

10.50

4.95

17.50

4.95

PC

239.50

37.48

324.00

39.60

495.00

87.68

961.00

8.49

1106.00

289.91

2Aa

-

-

-

-

-

-

140.50

12.02

-

-

Test 1: With metabolic activation (+S9)

Concentration

(µg/plate)

His+Revertant Colonies/Plate

[Presence of Metabolic Activation (5% v/v S9 mix)]

TA1537

TA1535

TA98

TA100

TA102

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

NC (DMSO)

6.50

0.71

15.50

0.71

19.00

5.66

135.00

4.24

243.50

10.61

1.5

6.50

3.54

17.50

0.71

19.00

7.07

136.00

19.80

242.00

4.24

5

8.50

0.71

15.50

4.95

21.00

2.83

134.50

12.02

240.50

13.44

15

5.50

0.71

17.00

4.24

20.00

2.83

134.50

13.44

236.50

21.92

50

6.50

4.95

16.50

3.54

21.50

2.12

132.50

0.71

245.00

2.83

150

6.00

1.41

18.50

4.95

17.50

2.12

134.00

15.56

232.50

0.71

500

5.50

2.12

15.50

2.12

20.00

4.24

135.00

5.66

249.00

15.56

1500

6.50

2.12

16.50

3.54

19.50

2.12

131.50

6.36

247.00

14.14

5000

0.00

0.00

0.00

0.00

0.00

0.00

7.50

2.12

19.00

12.73

PC-2Aa

295.00

84.85

322.00

16.97

720.00

46.67

904.00

19.80

910.50

36.06

Test 2: Without metabolic activation (-S9)

Concentration

(µg/plate)

His+Revertant Colonies/Plate

[Absence of Metabolic Activation]

TA1537

TA1535

TA98

TA100

TA102

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

NC (DMSO)

6.00

2.00

14.00

3.00

23.67

6.51

133.00

6.56

222.00

11.53

156.25

8.00

2.65

15.33

3.51

20.67

2.52

133.33

8.62

230.00

15.39

312.5

6.33

0.58

12.33

1.53

22.00

5.57

132.00

8.19

232.00

19.31

625

7.00

3.00

14.00

5.29

21.33

3.06

132.00

5.29

229.00

3.00

1250

7.00

3.61

13.33

2.31

21.00

5.57

132.00

8.00

232.00

15.52

2500

3.33

0.58

7.67

2.08

14.00

2.65

78.33

13.65

134.00

26.51

5000

0.00

0.00

0.00

0.00

0.00

0.00

14.67

3.51

30.67

14.57

PC

209.67

27.30

360.00

56.31

437.33

113.07

878.67

130.37

951.67

134.24

2Aa

-

-

-

-

-

-

132.00

8.19

-

-

Test 2: With metabolic activation (+S9)

Concentration

(µg/plate)

His+Revertant Colonies/Plate

[Presence of Metabolic Activation (10% v/v S9 mix)]

TA1537

TA1535

TA98

TA100

TA102

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

NC (DMSO)

8.00

3.61

15.67

2.52

23.33

7.77

133.33

7.37

244.00

8.54

156.25

8.00

2.00

16.33

4.51

23.00

4.58

130.00

2.65

236.00

16.64

312.5

9.33

1.53

15.33

2.52

19.00

4.58

133.67

8.08

237.67

16.04

625

8.33

0.58

14.33

4.04

22.67

7.02

134.00

10.82

234.00

9.85

1250

8.33

1.53

13.67

6.43

23.33

7.09

130.33

10.02

233.67

14.01

2500

4.33

1.53

9.33

2.08

14.00

2.65

82.33

11.50

154.67

27.68

5000

0.00

0.00

0.00

0.00

0.00

0.00

10.00

3.61

38.00

19.67

PC-2Aa

223.67

34.49

322.67

28.01

642.00

170.71

969.33

147.77

972.33

44.97

Applicant's summary and conclusion

Conclusions:
The test item was determined to be non-mutagenic to all of the five strains of Salmonella typhimurium viz., TA1537, TA1535, TA98, TA100 and TA102 when tested with and without metabolic activation up to 5000 µg/plate.


Executive summary:

A bacterial reverse mutation assay was performed according to OECD Guideline 471 (GLP study) using five histidine deficient mutant tester strains of Salmonella typhimurium (i.e., TA1537, TA1535, TA98, TA100, and TA102). Two independent experiments, in the absence and presence of metabolic activation, were performed. In the initial test, bacterial cultures were exposed to the test item at 8 concentrations (two plates/concentration) between 1.5 and 5000 µg/plate. No increase in the number of revertant colonies (no mutagenic effect) were observed both in the absence and presence of the metabolic activation system (5% v/v S9 mix) in all the tester strains. To confirm the negative results obtained in the initial toxicity mutation test, confirmatory mutation test was conducted with an increased S9 concentration i.e., 10% v/v S9 mix and modified concentration spacing. In the confirmatory test, bacterial cultures were exposed to the test item at 6 concentrations (three plates/concentration) between 156.25 and 5000 µg/plate both in the absence and presence (10 % v/v S9 mix) of metabolic activation system. After 48 hours of incubation at 37 ± 1°C, the revertant colonies were scored. Test item did not induce any significant increase in the number of revertants, in both trials, with and without S9 mix, in any of the five tester strains. All the values for the negative control were within historical control ranges of the laboratory and positive controls showed an increase in the number of revertant colonies, demonstrating the efficiency of the test system. All criteria for a valid study were met. From the results of this study, under the specified experimental conditions, test item was concluded to be non-mutagenic in the Bacterial Reverse Mutation Test using Salmonella typhimurium.