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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
May 30, 2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
liquid

Method

Target gene:
his (Salmonella typhimurium strains), trp (E. coli)
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
0.316, 1.0, 3.16, 10.0, 31.6 and 100 μg
Pronounced cytotoxicity was noted starting at a concentration of 100 μg/plate in the preliminary cytotoxicity tests.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: test item was not soluble in highly purified water or dimethylsulfoxide
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
2-nitrofluorene
sodium azide
benzo(a)pyrene
other: 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: 1st experiment: in agar (plate incorporation); 2nd experiment: preincubation

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

NUMBER OF REPLICATIONS: 3


DETERMINATION OF CYTOTOXICITY
- Method: background lawn



Evaluation criteria:
A test item is considered to show a positive response if
- the number of revertants is significantly increased (p ≤ 0.05, U-test according to MANN and WHITNEY) compared to the solvent control to at least 2-fold of the solvent control for the Salmonella typhimurium test strains TA98, TA100, TA1535, TA1537 and Escherichia coli test strain WP2 uvrA in both independent experiments.
- in addition, a significant (p ≤ 0.05) concentration (log value)-related effect (Spearman’s rank correlation coefficient) is observed;
Biological relevance of the results should be considered first.
Positive results have to be reproducible and the histidine or tryptophan independence of the revertants has to be confirmed by streaking random samples on histidine or tryptophan-free agar plates.
A test item for which the results do not meet the above mentioned criteria is considered as non-mutagenic in the AMES test.

Results and discussion

Test results
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not reported
- Effects of osmolality: not reported
- Precipitation: not reported

HISTORICAL CONTROL DATA
- see attachment

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation, pronounced cytotoxicity (scarce background lawn and reduction of the number of revertants) were noted at the top concentration of 100 μg/plate, in all Salmonella typhimurium strains and in the Escherichia coli strain WP2 uvrA [pKM101].

Any other information on results incl. tables

Plate incorporation, without metabolic activation

 

 

TA98

TA100

TA1535

TA1537

E. coli WP2

0.316

mean

SD

27.3

1.2

117.0

2.6

15.3

1.5

6.3

1.2

30.0

0.0

1.0

mean

SD

36.3

0.6

118.7

4.6

17.0

1.0

5.7

2.1

29.3

2.9

3.16

mean

SD

36.3

1.5

127.0

13.1

17.3

1.5

7.7

0.6

37.7

8.7

10

mean

SD

39.0

6.1

128.3

7.6

16.7

1.5

5.7

1.2

45.3

6.4

31.6

mean

SD

29.0

15.5

106.3

2.1

18.0

1.7

9.0

1.0

53.7

6.8

100

mean

SD

12.0 #

1.0

66.7 #

0.6

8.7 #

1.2

2.0 #

0.0

14.3 #

0.6

Negative control

mean

SD

29.0

6.2

131.3

15.4

25.0

0.0

6.7

0.6

51.3

3.1

Positive control

mean

SD

143.3

1.5

990.3

4.9

149.3

0.6

97.3

9.5

205.3

4.9

Plate incorporation, with metabolic activation

0.316

mean

SD

33.3

1.2

143.3

17.0

17.3

2.1

5.3

2.3

42.7

2.1

1.0

mean

SD

26.7

5.5

131.0

15.6

20.7

7.4

7.0

1.7

45.7

6.4

3.16

mean

SD

33.0

4.6

128.0

22.3

17.7

0.6

7.0

1.7

47.0

4.6

10

mean

SD

25.7

2.5

131.0

2.6

17.7

0.6

7.3

1.2

41.3

4.0

31.6

mean

SD

31.0

7.8

129.3

3.2

16.3

0.6

7.0

0.0

39.0

14.4

100

mean

SD

12.7 #

1.2

68.0 #

3.6

8.3 #

0.6

2.0 #

0.0

14.3 #

1.5

Negative control

mean

SD

36.7

0.6

120.7

2.1

17.0

1.0

6.7

1.2

48.3

12.1

Positive control

mean

SD

145.7

4.0

980.0

30.8

152.3

10.4

85.0

17.3

184.7

35.9

Preincubation, without metabolic activation

0.316

mean

SD

30.7

4.7

152.7

7.6

19.0

1.0

6.3

0.6

45.0

1.7

1.0

mean

SD

26.0

9.5

164.3

18.5

22.0

2.6

7.0

1.0

53.3

5.7

3.16

mean

SD

30.7

9.3

160.7

22.9

21.7

1.5

7.3

1.2

52.7

2.1

10

mean

SD

41.7

6.4

167.3

12.4

24.7

4.0

7.3

0.6

36.0

10.4

31.6

mean

SD

31.0

1.7

108.7

5.9

24.3

3.1

6.3

0.6

43.0

1.0

100

mean

SD

14.7 #

0.6

51.0 #

1.0

7.7 #

0.6

2.0 #

0.0

15.7 #

0.6

Negative control

mean

SD

28.3

3.2

174.0

3.6

27.7

0.6

5.3

0.6

41.7

18.9

Positive control

mean

SD

146.0

3.6

845.3

4.0

193.0

1.0

86.3

2.3

143.0

28.2

Preincubation, with metabolic activation

0.316

mean

SD

30.7

3.5

139.3

24.6

25.0

4.6

6.3

2.1

50.3

1.5

1.0

mean

SD

26.3

4.7

138.7

28.5

23.7

4.6

6.3

2.1

54.3

4.2

3.16

mean

SD

29.7

5.5

111.7

1.2

20.0

0.0

6.3

1.2

57.7

1.2

10

mean

SD

29.0

4.6

122.0

4.6

22.0

3.6

4.7

0.6

54.0

7.0

31.6

mean

SD

23.3

3.1

109.7

2.1

21.0

2.0

8.0

1.7

44.7

 

0.6

100

mean

SD

13.7 #

1.5

59.3 #

8.3

7.3 #

0.6

2.0 #

0.0

15.3 #

1.2

Negative control

mean

SD

31.7

9.0

177.3

33.2

22.7

1.5

7.7

1.5

49.7

1.5

Positive control

mean

SD

137.3

5.7

830.3

16.2

153.0

13.0

80.3

5.5

159.7

3.1

Applicant's summary and conclusion

Conclusions:
Di-C12-18 alkyldimethyl ammonium chloride was not mutagenic in this bacterial reverse mutation assay in the presence and absence of metabolic activation.
Executive summary:

In a reverse gene mutation assay in bacteria according to OECD guideline 471 (1997) and EU method B.13/14 (2008), Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and E. coli WP2 uvr A were exposed to Di-C12-18 alkyldimethyl ammonium chloride in ethanol in concentrations of 0 (control), 0316, 1.0, 3.16, 10.0, 31.6 and 100 µg/plate in all strains in the absence and presence of mammalian metabolic activation (rat liver S9 mix). The assay was performed using the plate incorporation method (1st experiment) and pre-incubation method (2nd experiment; 20 min pre-incubation).

The test substance was tested up to cytotoxic concentrations. Pronounced cytotoxicity was noted at 100 μg/plate in in all strains.

The positive control items showed a significant increase in the number of revertant colonies of the respective test strain and confirmed the validity of the test conditions and the sensitivity of the test system. The results of the negative and positive control cultures were within the range of the historical data. Hence, all acceptance criteria are met.

No increase in revertant colony numbers as compared with control counts was observed for the test item in the Salmonella typhimurium and in the Escherichia coli test strains in two independent experiments without and with metabolic activation, respectively (plate incorporation and preincubation test).

Under the conditions of the study, the test substance was negative for mutagenic potential.