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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:
2017-02-01 to 2017-02-23
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Specific details on test material used for the study:
- Characteristics: (Off-) white powder
- Storage conditions: at room temperature (+10 °C to +25 °C)

Method

Target gene:
histidine operon
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 98
Additional strain / cell type characteristics:
other: uvrB-, rfa-, pKM 101, resistance to Ampicillin
Species / strain / cell type:
S. typhimurium TA 100
Additional strain / cell type characteristics:
other: uvrB-, rfa-, pKM 101, resistance to Ampicillin
Species / strain / cell type:
S. typhimurium TA 102
Additional strain / cell type characteristics:
other: rfa-, pKM 101/pAQ1, resistance to Ampicillin
Species / strain / cell type:
S. typhimurium TA 1535
Additional strain / cell type characteristics:
other: uvrB-, rfa-, non-resistance to Ampicillin
Species / strain / cell type:
S. typhimurium TA 1537
Additional strain / cell type characteristics:
other: uvrB-, rfa-, non-resistance to Ampicillin
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction, Aroclor 1254-induced, from male Sprague-Dawley rat liver (Trinova Biochem GmbH, Gießen, Germany
Test concentrations with justification for top dose:
1, 3.16, 10, 31.6, 100 and 316 µg/plate. Two preliminary cytotoxicity tests (plate incorporation test without and with metabolic activation) were carried out in test strain TA100. Ten concentrations ranging from 0.316 to 5000 µg/plate were tested. Concentrations of 1000 µg/plate and higher were not evaluable due to pronounced test item precipitation. Hence, 316 µg/plate were chosen as top concentration for the main study in the plate incorporation test and in the preincubation test.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol (Batch no. F2540; Honeywell Specialty Chemicals)
- Justification for choice of solvent/vehicle: The test item was not soluble in water or dimethylsulfoxide (DMSO).
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
benzo(a)pyrene
mitomycin C
other: 2-amino-anthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation
- Cell density at seeding: 0.1 mL of approximately 1.00E+08 viable cells in the late exponential or early stationary phase
DURATION
- Preincubation period: 20 minutes at 37°C
- Exposure duration: 37°C for 48 -72 h
NUMBER OF REPLICATIONS:
- Plates: 3 per concentration and experiment
- Experiments: 2 independent experiments, each with and without metabolic activation
DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity is evidenced by a reduction in the number of spontaneous revertants by at least 50%, a clearing or diminution of the background lawn or by the degree of survival of the treated cultures. Insolubility could have been assessed as precipitation in the final mixture under the actual test conditions and evident to the unaided eye.
Evaluation criteria:
Acceptance Criteria:
The results of the negative and positive control cultures should be within the range of the historical data generated by the testing facility. The range of spontaneous reversion frequencies per plate is based on Kirkland (1990):
TA98: 20-60
TA100: 100-200
TA102: 240-320
TA1535: 10-35
TA1537: 3-20
The numbers may be slightly different on plates with S9 and may vary slightly from experiment to experiment. Cytotoxicity is defined as a reduction in the number of colonies by more than 50 % compared with the vehicle control and/or a scarce background lawn.
Historical background data of revertant colony numbers of the negative and positive controls without and with metabolic activation for the experiments of the years 2015 to 2017 (most recent background data, not audited by the QAU-department) are given in Table 1 in section “any other details on results incl. tables”.
Statistics:
Interpretation of results:
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 TA98, TA100, TA1535 and TA1537 and 1.5-fold of the solvent control for TA102 in both independent experiments.
or
- a concentration-related increase over the range tested in the number of the revertants per plate is observed. The Spearman's rank correlation coefficient may be applied.
- positive results have to be reproducible and the histidine independence of the revertants has to be confirmed by streaking random samples on histidine-free agar plates.
Biological relevance of the results should be considered first. 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 resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
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
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
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
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
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
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not reported
- Effects of osmolarity: not reported
- Evaporation from medium: not reported
- Water solubility: the test item is insoluble in water
- Precipitation: see section “any other information on results incl. tables”.
RANGE-FINDING/SCREENING STUDIES: see section “any other information on results incl. tables”.
HISTORICAL CONTROL DATA see tables 1 and 2 in section “any other information on results incl. tables”.

Any other information on results incl. tables

Preliminary test

The test substance was examined in two preliminary cytotoxicity tests (plate incorporation test without and with metabolic activation) in test strain TA100. Ten concentrations ranging from 0.316 to 5000 µg/plate were tested. Test item precipitation was noted starting at the concentration of 316 µg/plate. Concentrations of 1000 µg/plate and higher were not evaluable due to pronounced test item precipitation (see tables 2 and 3). Hence, 316 µg/plate were chosen as top concentration for the main study in the plate incorporation test and in the preincubation test.

Main study

Six concentrations ranging from 1.0 to 316 µg of the test item per plate were employed in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation.

Cytotoxicity 

Test item precipitation was noted in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation at the top concentration of 316 µg/plate in all test strains. Reduction of the number of revertants by more than 50% was noted in both experiments at the top concentration of 316 µg/plate in the following test strains:  

Plate incorporation test:

- S9: TA1535 and TA1537;

+S9: TA1537;

Preincubation test:

- S9: TA98, TA102, TA1535 and TA1537;

+S9: TA1535 and TA1537.  

Mutagenicity

No increase in revertant colony numbers as compared with control counts was observed up to a concentration of 316 µg/plate, that led to test item precipitation, in any of the 5 test strains in two independent experiments without and with metabolic activation, respectively (plate incorporation test and preincubation test).

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 are within the range of the historical data generated by the test facility (see table 1). A summary of the results is given in tables 4 to 7.

Table 1: history profile of negative and positive control values of the years 2015 to 2017 (n= 80 studies). Data obtained from plate incorporation and preincubation tests.

Negative reference item

Strain

TA 98

TA 100

TA 102

TA 1535

TA 1537

S9-mix

-S9

+ S9

-S9

+ S9

-S9

+ S9

-S9

+ S9

-S9

+ S9

Mean

30.3

32.1

145.1

145.2

277.3

279.0

19.7

19.9

6.7

6.7

SD

5.6

5.9

18.4

19.2

16.5

17.2

4.4

4.6

1.7

1.8

Min

20

20

107

101

245

203

10

10

2

3

Max

49

49

195

198

323

324

34

36

10

10

Positive reference item

Strain

TA 98

TA 100

TA 102

TA 1535

TA 1537

S9-mix

-S9

+ S9

-S9

+ S9

-S9

+ S9

-S9

+ S9

-S9

+ S9

 

2-nitro-fluorene

Benzo[a]-pyrene

Sodium azide

2-amino-anthracene

Mitomycin C

Benzo[a]-pyrene

Sodium azide

2-amino-anthracene

9-amino-acridine

Benzo[a]-pyrene

Mean

151.2

150.4

952.9

948.8

1029.7

1024.3

135.6

135.4

76.7

77.6

SD

27.9

28.8

99.7

103.7

102.3

97.1

28.8

28.4

26.5

26.4

Min

91

96

677

703

756

781

51

49

28

31

Max

293

291

1213

1195

1637

1366

266

270

185

184

 Table 2: Preliminary cytotoxicity test without metabolic activation. Plate incorporation test with TA 100.

Test item (µg/plate)

Background lawn

Revertants plate 1

Revertants plate 2

0.316

normal

121

127

1

normal

144

142

3.16

normal

116

149

10

normal

144

134

31.6

normal

142

149

100

normal

146

146

316

test item precipitation

110

100

1000

test item precipitation

0

0

3160

test item precipitation

0

0

5000

test item precipitation

0

0

Solvent control

100 µL/plate

normal

147

149

 Table 3: Preliminary cytotoxicity test with metabolic activation. Plate incorporation test with TA 100.

Test item (µg/plate)

Background lawn

Revertants plate 1

Revertants plate 2

0.316

normal

130

120

1

normal

163

127

3.16

normal

120

153

10

normal

131

131

31.6

normal

158

161

100

normal

144

143

316

test item precipitation

114

97

1000

test item precipitation

0

0

3160

test item precipitation

0

0

5000

test item precipitation

0

0

Solvent control 100 µL/plate

normal

153

157

Table 4: Plate incorporation test without metabolic activation

Test item (µg/plate)

Number of reverted colonies (mean values, n=3)

 

TA98

TA100

TA102

TA1535

TA1537

1.0

22.7

112.3

281.7

24.3

6.3

3.16

24.0

119.3

292.0

15.0

6.0

10.0

28.3

118.0

287.7

18.7

6.0

31.6

26.0

120.3

284.0

17.3

5.3

100

25.7

128.0

251.0

16.3

6.7

316

22.7

test item precipitation

109.0

test item precipitation

260.7

test item precipitation

11.0

test item precipitation

2.0

test item precipitation

Vehicle control

100 µL/plate

32.7

128.0

271.3

26.7

6.7

Positive reference item

2-nitro-fluorene

Sodium azide

Mitomycin C

Sodium azide

9-amino-acridine

Concentration

µg/plate

10

10

10

10

100

 

193.7

993.0

1093.3

136.0

73.3

 Table 5: Plate incorporation test with metabolic activation

Test item (µg/plate)

Number of reverted colonies (mean values, n=3)

 

TA98

TA100

TA102

TA1535

TA1537

1.0

29.7

119.3

255.3

23.0

4.3

3.16

29.3

132.0

293.0

19.0

6.0

10.0

24.7

119.0

264.3

22.7

4.0

31.6

24.3

132.0

277.7

16.7

5.7

100

30.7

118.7

258.3

19.0

5.0

316

22.7

test item precipitation

108.3

test item precipitation

245.0

test item precipitation

10.0

test item precipitation

2.0

test item precipitation

Vehicle control

100 µL/plate

30.7

120.0

274.0

17.0

5.3

Positive reference item

Benzo[a]pyrene

2-amino-anthracene

Benzo[a]pyrene

2-amino-anthracene

Benzo[a]pyrene

Concentration

µg/plate

10

2

10

2

10

 

184.0

992.7

1088.3

137.7

77.3

 Table 6: Preincubation test without metabolic activation

Test item (µg/plate)

Number of reverted colonies (mean values, n=3)

1.0

31.7

117.3

281.7

16.7

8.0

3.16

29.3

113.0

276.0

17.0

8.3

10.0

28.7

118.7

258.7

18.0

6.3

31.6

28.3

129.0

267.0

16.0

6.7

100

29.7

120.3

288.7

20.7

5.7

316

12.0

test item precipitation

79.7

test item precipitation

126.7

test item precipitation

9.0

test item precipitation

2.3

test item precipitation

Vehicle control

100 µL/plate

30.7

125.0

286.7

18.3

6.0

Positive reference item

2-nitro-fluorene

Sodium azide

Mitomycin C

Sodium azide

9-amino-acridine

Concentration

µg/plate

10

10

10

10

100

 

165.7

998.3

1036.7

139.7

32.3

 Table 7: Preincubation test with metabolic activation

Test item (µg/plate)

Number of reverted colonies (mean values, n=3)

 

TA98

TA100

TA102

TA1535

TA1537

1.0

32.0

116.3

267.3

22.7

9.7

3.16

32.0

118.3

273.7

23.3

9.3

10.0

33.3

109.3

276.3

26.3

6.7

31.6

32.3

118.7

271.0

20.7

4.3

100

29.0

111.3

252.3

18.3

7.0

316

15.3

test item precipitation

75.3

test item precipitation

202.7

test item precipitation

9.0

test item precipitation

2.0

test item precipitation

Vehicle control

100 µL/plate

22.7

131.0

302.0

25.0

7.3

Positive reference item

Benzo[a]pyrene

2-amino-anthracene

Benzo[a]pyrene

2-amino-anthracene

Benzo[a]pyrene

Concentration

µg/plate

10

2

10

2

10

 

163.3

1029.7

997.3

137.7

65.0

 


Applicant's summary and conclusion

Conclusions:
Under the described conditions and tested up to a concentration of 316 µg/plate, that led to test item precipitation, the test substance caused no mutagenic effect in the Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 neither in the plate incorporation test nor in the preincubation test each carried out without and with metabolic activation. Therefore, the test substance is considered non-mutagenic in the bacterial reverse mutation assay.
Executive summary:

The potential of Ceramide IIIB to induce gene mutations was examined in 5 Salmonella typhimurium strains in two independent experiments without and with metabolic activation (plate incorporation test and preincubation test).

The test item was completely dissolved in ethanol for concentrations lower than 1000 µg/plate. The vehicle ethanol was employed as the negative control.

In two preliminary cytotoxicity tests, test item precipitation was noted starting at the concentration of 316 µg/plate. Concentrations of 1000 µg/plate and higher were not evaluable due to pronounced test item precipitation. Thus, six concentrations ranging from 1.0 to 316 µg/plate were employed in the main experiment.

Cytotoxicity as determined by the reduction of the number of revertants by more than 50% was noted at the top concentration of 316 µg/plate in some of the test strains with and without metabolic activation.

No increase in revertant colony numbers as compared with control up to a concentration of 316 µg/plate was observed in any of the 5 test strains in two independent experiments without and with metabolic activation, respectively (plate incorporation test and preincubation test).

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.

In conclusion, the test substance was tested up to a concentration of 316 µg/plate, which led to precipitation but caused no mutagenic effect in the Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 neither in the plate incorporation test nor in the preincubation test each carried out without and with metabolic activation.