Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 March 2017 to 03 July 2017
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 guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
In accordance with the OECD guideline for testing of chemicals No. 471 “Bacterial Reverse Mutation Test”, adopted on 21st July 1997.
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
Fatty acids, C16-18 (even numbered), esters with 1,2,3-propanetriol and oligomers
IUPAC Name:
Fatty acids, C16-18 (even numbered), esters with 1,2,3-propanetriol and oligomers
Test material form:
solid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and batch No.of test material:Clariant Produkte (Deutschland)
GmbH 84504 Burgkirchen, Germany
- Expiration date of the batch:04.10.2018
- Purity test date:96,9%

STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient (21 to 29°C)
- Solubility of the test substance in the solvent/vehicle:Distilled water

Method

Target gene:
Hisitidine and tryptophan locus
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:
1 mL of S9 homogenate was mixed with 9 mL of co-factor
Test concentrations with justification for top dose:
The test item was tested at the concentrations of 0.02, 0.06, 0.20, 0.63 and 2 mg/plate based on the results of solubility, precipitation and initial cytotoxicity test.
Vehicle / solvent:
Distilled water- Vehicle(s)/solvent(s) used: Distilled water
- Justification for choice of solvent/vehicle:The test item at the given concentration of 50 mg/mL was soluble in distilled at 40 to 45º C
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
other: 2-aminoantracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation;

DURATION
- Preincubation period: 30 minutes
others: cytotoxicity by lawn evaluation and mutagenicity by evaluating revertant counts.

NUMBER OF REPLICATIONS: Triplicates

Rationale for test conditions:
not applicable
Evaluation criteria:
The test will be judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than 2 times the mean vehicle control value in Salmonella typhimurium strains TA98, TA100 and Escherichia coli WP2 uvrA (pKM101) or equal to or greater than 3 times the mean vehicle control value in tester strains TA1535 and TA1537.vehicle control, in both the trials, in the presence and absence of metabolic activation. There was no appreciable increase in number of revertant colonies at any of the tested concentrations in both the trials.
Statistics:
not applicable

Results and discussion

Test results
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:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS

- Water solubility: yes
- Precipitation: minimal precipitation at 2 mg/plate


HISTORICAL CONTROL DATA (with ranges, means and standard deviation)
Plate in corporation method
- Positive historical control data:
Tester strain TA 98 TA 100 TA 1535 TA 1537 E.COLI PKM 101 (With S9)
Mean 408.7 386.1 140.4 118.5 385.5
SD ±16.6 ±13.8 ±8.3 ±6.3 ±5.3
Min 280 306 118 100 370
Max 440 419 182 149 401
Tester strain TA 98 TA 100 TA 1535 TA 1537 E.COLI PKM 101 (Without S9)
Mean 401.5 382.2 141.4 119.8 386.0
SD ±26.3 ±16.5 ±10.6 ±6.5 ±5.8
Min 310 313 112 103 371
Max 428 446 190 158 416


- Negative (solvent/vehicle) historical control data:
Tester strain TA 98 TA 100 TA 1535 TA 1537 E.COLI PKM 101 (With S9)
Mean 20.7 102.5 21.4 9.3 171.8
SD ±1.7 ±2.2 ±2.0 ±1.5 ±5.8
Min 18 98 17 6 159
Max 26 108 25 12 184
Tester strain TA 98 TA 100 TA 1535 TA 1537 E.COLI PKM 101 (Without S9)
Mean 20.9 102.2 21.6 9.3 171.4
SD ±1.8 ±2.6 ±1.9 ±1.5 ±4.9
Min 18 98 18 7 159
Max 25 110 26 13 183

Pre incubation method

- Positive historical control data:
Tester strain TA 98 TA 100 TA 1535 TA 1537 E.COLI PKM 101 (With S9)
Mean 408.0 386.3 140.7 117.9 384.6
SD ±14.5 ±14.2 ±9.8 ±5.2 ±6.3
Min 290 298 103 102 363
Max 429 425 187 128 403
Tester strain TA 98 TA 100 TA 1535 TA 1537 E.COLI PKM 101 (Without S9)
Mean 404.8 383.1 141.9 119.1 385.3
SD ±20.8 ±15.8 ±11.9 ±5.5 ±6.0
Min 314 317 110 105 371
Max 428 450 200 134 418

- Negative (solvent/vehicle) historical control data:
Tester strain TA 98 TA 100 TA 1535 TA 1537 E.COLI PKM 101 (With S9)
Mean 20.6 102.2 22.0 9.5 171.5
SD ±1.5 ±2.5 ±1.9 ±1.4 ±4.5
Min 18 97 18 7 161
Max 24 107 25 12 182
Tester strain TA 98 TA 100 TA 1535 TA 1537 E.COLI PKM 101 (Without S9)
Mean 20.9 102.4 21.5 9.3 170.6
SD ±1.7 ±2.4 ±2.2 ±1.4 ±4.6
Min 18 97 17 7 158
Max 24 110 25 12 181

Any other information on results incl. tables

 

 Table 1: SUMMARY OF INITIAL CYTOTOXICITY TEST-SALMONELLA TYPHIMURIUM TA100

Test Item Concentration (mg/plate)

No. of Revertants/plate

With S9

Without S9

R1

R2

R3

Average

±SD

Bacterial Lawn Intensity

R1

R2

R3

Average

±SD

Bacterial Lawn Intensity

Vehicle Control

99

87

106

97

9.6

4+

105

112

96

104

8.0

4+

2

102

107

103

104

2.6

4+

110

106

104

107

3.1

4+

1

106

96

102

101

5.0

4+

101

95

89

95

6.0

4+

0.9

88

105

101

98

8.9

4+

104

91

97

97

6.5

4+

0.8

93

97

84

91

6.7

4+

96

89

104

96

7.5

4+

0.7

103

96

91

97

6.0

4+

94

107

97

99

6.8

4+

Values of Revertants are in Mean±SD

Lawn intensity: 4+= Thick lawns: Distinguished by a healthy (Normal) background lawn comparable tovehiclecontrol plates.

Table 2: SUMMARY OF COLONY COUNTS OF REVERTANTS OF TRIAL-I, PLATE INCORPORATION METHOD

Treatment

Test Concentration  (mg/plate)

No. of Revertants (Mean of 3 Plates)

With S9

 

Without S9

Salmonella typhimurium

E.coliWP2 uvrA (pKM 101)

Salmonella typhimurium

E.coliWP2 uvrA (pKM 101)

TA

98

TA 100

TA 1535

TA 1537

TA

98

TA 100

TA 1535

TA 1537

Vehicle Control

100 µL of Distilled Water

Mean

25.7

98.0

20.3

9.3

165.3

27.3

94.0

21.3

9.7

175.0

±SD

5.7

9.5

3.5

4.2

7.8

1.5

9.8

2.1

1.5

10.1

Lawn Intensity

4+

4+

4+

4+

4+

4+

4+

4+

4+

4+

Hostacerin DGMS

2

Mean

24.7

95.0

20.7

8.7

171.7

21.7

96.0

18.3

6.3

169.3

±SD

4.7

7.9

2.5

1.5

7.4

3.5

8.9

3.5

2.5

11.0

Fold Increase

1.0

1.0

1.0

0.9

1.0

0.8

1.0

0.9

0.7

1.0

Lawn Intensity

4+

4+

4+

4+

4+

4+

4+

4+

4+

4+

0.63

Mean

23.3

94.3

17.3

7.3

173.0

21.0

97.3

20.0

9.7

175.3

±SD

5.7

11.6

2.1

2.1

13.1

4.4

4.0

3.0

2.5

7.8

Fold Increase

0.9

1.0

0.9

0.8

1.0

0.8

1.0

0.9

1.0

1.0

Lawn Intensity

4+

4+

4+

4+

4+

4+

4+

4+

4+

4+

0.2

Mean

20.3

87.3

22.0

9.0

170.0

21.3

96.0

19.3

7.0

172.7

±SD

4.2

8.0

1.0

2.6

4.6

4.9

15.1

3.2

2.6

9.3

Fold Increase

0.8

0.9

1.1

1.0

1.0

0.8

1.0

0.9

0.7

1.0

Lawn Intensity

4+

4+

4+

4+

4+

4+

4+

4+

4+

4+

0.06

Mean

21.3

92.7

19.7

8.0

170.7

21.3

95.3

20.7

7.7

179.0

±SD

2.5

7.4

4.9

3.6

5.7

6.0

9.6

3.1

1.5

1.7

Fold Increase

0.8

0.9

1.0

0.9

1.0

0.8

1.0

1.0

0.8

1.0

Lawn Intensity

4+

4+

4+

4+

4+

4+

4+

4+

4+

4+

0.02

Mean

26.3

91.3

20.3

8.3

177.7

19.0

88.7

19.3

8.7

172.7

±SD

4.7

9.1

1.5

4.9

6.8

3.0

5.5

4.7

3.1

6.0

Fold Increase

1.0

0.9

1.0

0.9

1.1

0.7

0.9

0.9

0.9

1.0

Lawn Intensity

4+

4+

4+

4+

4+

4+

4+

4+

4+

4+

Positive Control

100 µL of respective Positive Control

Mean

353.0

335.3

133.7

130.0

360.7

357.3

351.3

132.0

120.0

361.0

±SD

31.1

20.5

5.0

8.0

35.3

38.5

20.4

15.1

28.4

20.7

Fold Increase

13.8

3.4

6.6

13.9

2.2

13.1

3.7

6.2

12.4

2.1

Lawn Intensity

4+

4+

4+

4+

4+

4+

4+

4+

4+

4+

Values of Revertants are in Mean±SD

Positive controls:

For with S9:

ForSalmonella typhimuriumTA98, TA100, TA1535 and TA1537 = 4 µg/plate of 2-Aminoanthracene

ForEscherichia coliWP2 uvrA (pKM101)strain +S9 = 30µg/plate of 2-Aminoanthracene

For without S9:

For TA98: 2 µg/plate of 2-Nitrofluorene

For TA100 and TA1535: 1 µg/plate of Sodium azide.

For TA1537: 50 µg/plate of 9-Aminoacridine

ForEscherichia coliWP2 uvrA (pKM101): 5 µg/plate of4-Nitroquinoline 1-oxide

Table 3: SUMMARY OF COLONY COUNTS OF REVERTANTS OF TRIAL-I, PREINCUBATION METHOD

Treatment

Test Concentration  (mg/plate)

No. of Revertants (Mean of 3 Plates)

With S9

 

Without S9

Salmonella typhimurium

E.coliWP2 uvrA (pKM 101)

Salmonella typhimurium

E.coliWP2 uvrA (pKM 101)

TA

98

TA 100

TA 1535

TA 1537

TA 98

TA 100

TA 1535

TA 1537

Vehicle Control

100 µL of Distilled Water

Mean

19.7

106.0

20.0

8.0

174.3

22.7

98.3

18.7

9.3

172.3

±SD

6.7

8.0

2.6

2.6

5.5

6.0

3.1

2.5

3.2

5.0

Lawn Intensity

4+

4+

4+

4+

4+

4+

4+

4+

4+

4+

Hostacerin DGMS

2

Mean

15.3

85.3

19.3

7.3

173.3

21.3

98.0

16.7

8.3

170.3

±SD

1.2

11.1

0.6

4.0

12.5

3.2

9.8

1.5

5.1

1.2

Fold Increase

0.6

0.9

1.0

0.8

1.0

0.8

1.0

0.8

0.9

1.0

Lawn Intensity

4+

4+

4+

4+

4+

4+

4+

4+

4+

4+

0.63

Mean

25.7

93.3

20.0

9.3

164.0

21.7

98.0

19.0

8.0

170.3

±SD

2.5

4.5

4.4

1.5

6.0

5.5

3.5

4.4

3.6

11.2

Fold Increase

1.0

1.0

1.0

1.0

1.0

0.8

1.0

0.9

0.8

1.0

Lawn Intensity

4+

4+

4+

4+

4+

4+

4+

4+

4+

4+

0.2

Mean

17.7

93.7

19.7

8.7

167.3

22.7

97.0

17.0

11.0

169.3

±SD

3.1

8.1

5.1

4.2

6.1

4.7

9.8

5.3

2.0

6.5

Fold Increase

0.7

1.0

1.0

0.9

1.0

0.8

1.0

0.8

1.1

1.0

Lawn Intensity

4+

4+

4+

4+

4+

4+

4+

4+

4+

4+

0.06

Mean

18.7

101.7

19.7

10.3

171.0

20.0

86.7

20.7

9.3

168.0

±SD

4.2

8.7

2.5

4.7

15.7

4.4

7.1

2.5

2.1

5.3

Fold Increase

0.7

1.0

1.0

1.1

1.0

0.7

0.9

1.0

1.0

1.0

Lawn Intensity

4+

4+

4+

4+

4+

4+

4+

4+

4+

4+

0.02

Mean

15.3

94.7

16.7

7.3

169.3

19.0

89.0

16.7

8.0

166.0

±SD

1.2

13.3

5.5

3.1

7.4

3.6

7.2

3.1

1.7

6.2

Fold Increase

0.6

1.0

0.8

0.8

1.0

0.7

0.9

0.8

0.8

0.9

Lawn Intensity

4+

4+

4+

4+

4+

4+

4+

4+

4+

4+

Positive Control

100 µL of respective Positive Control

Mean

330.3

367.7

134.3

111.0

379.0

397.3

351.7

133.7

115.7

380.0

±SD

14.3

8.1

11.0

13.7

15.5

35.0

33.7

13.3

13.2

17.7

Fold Increase

12.9

3.8

6.6

11.9

2.3

14.5

3.7

6.3

12.0

2.2

Lawn Intensity

4+

4+

4+

4+

4+

4+

4+

4+

4+

4+

Values of Revertants are in Mean±SD

Positive controls:

For with S9:

ForSalmonella typhimuriumTA98, TA100, TA1535 and TA1537 = 4 µg/plate of 2-Aminoanthracene

ForEscherichia coliWP2 uvrA (pKM101)strain +S9 = 30µg/plate of 2-Aminoanthracene

For without S9:

For TA98: 2 µg/plate of 2-Nitrofluorene

For TA100 and TA1535: 1 µg/plate of Sodium azide.

For TA1537: 50 µg/plate of 9-Aminoacridine

ForEscherichia coliWP2 uvrA (pKM101): 5 µg/plate of4-Nitroquinoline 1-oxide

Applicant's summary and conclusion

Conclusions:
Based on the results of the study, it can be concluded that the registration substance is “non-mutagenic” in the Bacterial Reverse Mutation Test up to the highest tested concentration of 2 mg/plate under the test conditions.
Executive summary:

The genotoxicity of the registration substance was investigated according to the Guideline OECD 471. No mutagenic activity was observed with and without metabolic activation of the concentration of 2 mg/plate in two independent experiments.