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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:
16 Feb - 09 Mar 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:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted in 1997
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Slovenska Narodna Akreditacna Sluzba, Bratislava, Slovenska Republika
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
his operon (for S. typhimurium strains), trp operon (for E. coli strain)
Species / strain
Species / strain / cell type:
other: S. typhimurium TA 1535, TA 97, TA 98 and TA 100 and E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
cofactor-supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of male rats, treated with Aroclor
Test concentrations with justification for top dose:
Range-finding study: 50, 150, 300, 500, 1500 and 5000 µg/plate in TA 97 and TA 100 without metabolic activation

First main experiment: 50, 150, 500, 1500 and 5000 µg/plate with and without metabolic activation
Second main experiment: 15, 50, 150, 500, 1500 and 5000 µg/plate without metabolic activation
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent: DMSO was chosen due to the insolubility of the test substance in water. The vehicle used did not affect the spontaneous mutation level and it is recommended for use in this test.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
2-nitrofluorene
sodium azide
other: 2-aminoanthracene (2AA)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) (Range Finding Test and first main experiment); preincubation (second main experiment)

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 - 72 h

NUMBER OF REPLICATIONS: 3 replications each in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: The bacterial growth was controlled in order to determine toxicity of the test substance.
Evaluation criteria:
Acceptance criteria
The study is considered valid if all of the following criteria are met:
- tester strains fulfil the criteria for sensitivity to UV light
- tester strains exhibit sensitivity to crystal violet
- tester Salmonella strains TA97, TA98, TA100 strain exhibit resistance to ampicillin
- all tester Salmonella strains and E.coli WP 2 uvrA exhibit a characteristic number of spontaneous revertant colonies when plated. The mean should be within the range of historical control values or within the published historical range
- all tester strains exhibit at least a three-fold increase in mutagen-induced revertant colonies when plated with positive control chemicals

Evaluation criteria
Considering biological relevance the test substance is considered positive if the assay is valid and the following conditions are met:
- concentration-related increase over the tested range and reproducible increase at one or more concentrations in the number of revertant colonies per plate in at least one strain with or without metabolic activation
- Mutation factor >2

The positive result indicates that the test substance induces mutations in Salmonella typhimurium or E.coli cells.

The test item for which results do not meet the above criteria is considered non-mutagenic in this test.
Negative results indicate that under the test conditions, the test substance does not produce mutations in Salmonella typhimurium or E.coli cells.
Statistics:
The mutation frequency at each dose concentration level of the test item was compared to the one observed in negative and positive controls. The statistical analysis was carried out using unpaired T-test.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 97
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium 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
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
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
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: No inhibition of the bacterial growth was observed in the two tested Salmonella strains TA 97 and TA 100 in the concentration range 50 - 5000 µg/plate without metabolic activation. Therefore the same doses (except 300 µg/plate) were tested in all Salmonella strains (TA 97, TA 98, TA 100 and TA 1535) and in E. coli WP2 uvr A in the main tests, with and without metabolic activation.

HISTORICAL CONTROL DATA
The mean revertants for controls for Salmonella tester strains and E.coli WP2 uvr were within the range of historical control values (please refer to Table 3 in "Any other information on results incl. tables").

Any other information on results incl. tables

Table 1:Summary of test results (experiment 1; Plate Incorporation Method)

With or without S9-Mix

Test substance concentration (μg/plate)

Mean number of revertant colonies per plate
(average of 3 plates ± SD)

Frameshift type

Base-pair substitution type

TA 97

TA98

TA100

TA1535

WP2 uvr A

Solvent control (DMSO)

124 ± 3

18 ± 2

145 ± 12

17 ± 3

26 ± 7

50

116 ± 3 *

17 ± 3

127 ± 3

16 ± 3

24 ± 5

150

119 ± 2

19 ± 3

130 ± 2

14 ± 1

32 ± 3

500

105 ± 5 *

18 ± 7

122 ± 18

15 ± 3

28 ± 9

1500

128 ± 9

17 ± 3

148 ± 19

17 ± 3

25 ± 4

5000

115 ± 8

18 ± 4

144 ± 7

18 ± 3

33 ± 5

Positive
controls (µg/plate)

9AA
(75)

2NF
(3)

SA
(1.5)

SA
(1.5)

4NQO
(5)

Mean No. of colonies/plate (average of 3 plates)

577 ± 38 *

432 ± 78 *

531 ± 28 *

448 ± 71 *

145 ± 8 *

+

Solvent control (DMSO)

152 ± 16

28 ± 9

133 ± 13

18 ± 5

39 ± 4

50

136 ± 16

28 ± 2

134 ± 6

22 ± 7

44 ± 3

150

149 ± 9

27 ± 1

124 ± 15

19 ± 3

43 ± 2

500

153 ± 11

26 ± 8

113 ± 21

15 ± 2

38 ± 6

1500

148 ± 4

26 ± 1

128 ± 7

17 ± 1

39 ± 8

5000

159 ± 9

28 ± 2

118 ± 7

17 ± 2

43 ± 2

Positive
controls
 

(µg/plate)

2AA
(5)

Mean No. of colonies/plate (average of 3 plates)

549 ± 57 *

159 ± 12 *

772 ± 25 *

211 ± 8 *

328 ± 17 *

2AA = 2-aminoanthracene

2NF = 2-nitro-fluorene

4NQO = 4-nitroquinoline-1-oxide

9AA = 9-aminoacridine

SA = sodium azide

SD = standard deviation

* = p < 0.05

Table 2: Summary of test results (experiment 2; Preincubation Method)

 

Test substance concentration (μg/plate)

Mean number of revertant colonies per plate
(average of 3 plates ± SD)

Frameshift type

Base-pair substitution type

TA 97

TA98

TA100

TA1535

WP2 uvr A

Without S9 Mix

Solvent control (DMSO)

159 ± 10

18 ± 5

130 ± 17

14 ± 3

34 ± 8

15

145 ± 8

17 ± 7

129 ± 8

13 ± 3

28 ± 6

50

151 ± 2

13 ± 4

119 ± 12

16 ± 1

28 ± 1

150

151 ± 2

17 ± 5

116 ± 4

15 ± 2

30 ± 4

500

153 ± 10

16 ± 2

117 ± 12

15 ± 3

34 ± 4

1500

153 ± 12

13 ± 4

116 ± 17

14 ± 2

31 ± 4

5000

155 ± 4

15 ± 1

127 ± 7

12 ± 4

33 ± 6

Positive
controls (µg/plate)

9AA
(75)

2NF
(3)

SA
(1.5)

SA
(1.5)

4NQO
(5)

Mean No. of colonies/plate (average of 3 plates)

657 ± 60 *

400 ± 17

531 ± 28 *

448 ± 71 *

145 ± 8 *

2AA = 2-aminoanthracene

2NF = 2-nitro-fluorene

4NQO = 4-nitroquinoline-1-oxide

9AA = 9-aminoacridine

SA = sodium azide

SD = standard deviation

* = p < 0.05

Table 3: Historical negative and positive control values (2015 -2018)

S9 Mix   Revertants per plate
TA 100 TA 1535 TA 97 TA 98 WP2 uvr A
Neg Pos Neg Pos Neg Pos Neg Pos Neg Pos
- Mean 194 698 23 624 163 1135 25 246 38 423
SD 35 170 8 170 30 527 7 141 12 270
Min 115 440 6 336 114 400 12 117 25 82
Max 272 1148 43 964 227 2776 43 680 68 912
+ Mean 180 1041 23 192 195 893 36 779 46 397
SD 29 443 8 130 36 369 12 438 10 145
Min 123 398 11 34 127 370 19 256 31 194
Max 231 2888 43 604 286 1616 85 1644 70 648

SD = standard deviation

Min = minimum value

Max = maximum value

Neg = negative control

Pos = positive control

Applicant's summary and conclusion

Conclusions:
Based on the results of the conducted study the test substance did not exhibit mutagenic properties in bacterial cells tested up to limit concentration.