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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 October 11 - 2018 January 30
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Guideline study under GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes (incl. QA statement)
Remarks:
Envigo Research Limited, Shardlow Business Park, Shardlow, Derbyshire DE72 2GD UK
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
3-hydroxy-2,2-bis(hydroxymethyl)propyl (R)-12-hydroxyoleate
EC Number:
201-095-3
EC Name:
3-hydroxy-2,2-bis(hydroxymethyl)propyl (R)-12-hydroxyoleate
Cas Number:
78-22-8
Molecular formula:
C23H44O6
IUPAC Name:
3-hydroxy-2,2-bis(hydroxymethyl)propyl 12-hydroxyoctadec-9-enoate
Test material form:
liquid: viscous
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 0000150418
- Expiration date of the lot/batch: 14 August 2018
- Purity test date: Not reported

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: N/A
- Specific activity: N/A
- Locations of the label: N/A
- Expiration date of radiochemical substance: N/A

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark
- Stability under test conditions: Expected to be stable
- Solubility and stability of the test substance in the solvent/vehicle: Not reported
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: N/A

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: N/A
- Preliminary purification step (if any): N/A
- Final dilution of a dissolved solid, stock liquid or gel: N/A
- Final preparation of a solid: N/A

FORM AS APPLIED IN THE TEST (if different from that of starting material) : liquid

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable): N/A

OTHER SPECIFICS:

Method

Target gene:
histidine for the S. typhimurium strains, and tryptophan for 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:
S-9 liver fraction (10% v/v), from male rats induced with phenobarbital and 5,6-benzoflavone
Test concentrations with justification for top dose:
The test item was tested using the following method. The maximum concentration was 5000 ug/plate (the maximum recommended dose level). Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 ug/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.
Vehicle / solvent:
DMSO, FIsher Scientific, batch 1714907, >99% pure
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
concurrent
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
other: 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: plate incorporation (first test) and preincubation (second test)
- Cell density at seeding (if applicable): 0.1 ml of a 10-h bacterial culture (having a density of at least 10E9/mL)

DURATION
- Preincubation period: 0.5 h
- Exposure duration: 48-72 h for plate incorporation; preincubation for 30 m before plating in top agar
- Expression time (cells in growth medium):
- Fixation time (start of exposure up to fixation or harvest of cells): cells not fixed

SELECTION AGENT (mutation assays): The Ames assay employs, as an indicator of mutation, observable (and countable) growth (colonies) in agar deficient in histidine. Colonies of bacteria represent mutants which have back-reverted to a histidine auxotroph. An automated colony counter is used.


NUMBER OF REPLICATIONS: 3 (triplicate)

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: not applicable


DETERMINATION OF CYTOTOXICITY
- Method: reduction in bacterial lawn

Rationale for test conditions:
The first plate incorporation test included 7 concentrations, in triplicate. Plates were also prepared without the addition of bacteria in order to assess the sterility of the test item, S9 mix and phosphate buffer. All plates were incubated at 34 to 39°C for 48-72 hours. After this period, the appearance of the background bacterial lawn were examined and revertant colonies counted using an automated colony counter. Colonies were counted manually if automated counting is not possible (e.g. if dense precipitate is present).
Any toxic effects of the test item were detected as thinning or absence of the background lawn of non-revertant colonies, and/or reduction in revertant colony numbers to ≤ 50% of the concurrent vehicle control count. In the absence of any toxic effects the maximum concentration used in the second test were the same as that used in the first. If toxic effects were observed at more than one concentration, a lower concentration was chosen. A minimum of four non-toxic concentrations were obtained. If this was not achieved then the first test was repeated using a more appropriate concentration range. If a negative or equivocal response was obtained a variation on the above procedure will be used, with a minimum of five concentrations of test item used.
If the required number of non-toxic concentrations was not obtained, the second test was repeated using a more appropriate concentration range. It may also have been repeated to confirm a positive response or to confirm a dose-response.
Evaluation criteria:
For a test material to be classified as toxic, the test item must cause a reduction in the number of spontaneous revertants (below a factor of 0.5 fold under the concurrent solvent control) and/or the bacterial lawn should exhibit evidence of thinning when viewed microscopically.

For a test to be considered valid, the mean of the vehicle control revertant colony numbers for each strain should lie within or close to the current historical control range of the laboratory unless otherwise justified by the Study Director.

The positive control compounds must produce an increase in mean revertant colony numbers of at least twice that of the concurrent vehicle controls.

Criteria for a positive result (any, one, or all of the following may be used to determine the overall results of the study, weighing the results in terms of their biological significance. The criteria are listed in order of priority:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase in mean revertant colony numbers at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Fold increase greater than two times thef the concurrent solvent control for any tester strain (especially if accompanied by an out-of historical range response (Cariello and Piegorsch, 1996).
5. Statistical analysis of data as determined by the UKEMS (Mahon et al., 1989)

A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
If the results obtained fail to satisfy the criteria for a clear “positive” or “negative” response, the test data should be evalueated by expert judgment and or further possible investigations.

Fold-increase: (three times in the case of strains TA1535 and TA1537) those of the concurrent vehicle controls.
Statistics:
The automated scoring system (Delta Building Monitoring System, Ames Study Manager and Sorcerer Imaging System) automatically conducts statistical analysis during the scoring process using Dunnetts Regression Analysis. Statistical significance will be confirmed for those values that indicate statistically significant increases in the frequency of revertant colonies compared with the concurrent solvent control (* = p < 0.05). Values that the program concludes are statististically significant but are within the in-house historical vehicle/untreated control range will not be reported.
See (Mahon et al, 1989, Analysis of data from microbial colony assays in: Kirkland, D.J. (Ed.). UKEMS Sub-committee on Guidelines for Mutagenicity Testing. Report. Part III. Statistical Evaluation of Mutagenicity Test Data, Cambridge University Press, Cambridge, pp.26-65.).

Results and discussion

Test resultsopen allclose all
Key result
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 applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
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 applicable
Positive controls validity:
valid
Key result
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 applicable
Positive controls validity:
valid
Key result
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 applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A pKM 101
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 applicable
Positive controls validity:
valid

Any other information on results incl. tables

Representative results:

Table 1            Spontaneous Mutation Rates (Concurrent Negative Controls)

Experiment 1

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA

TA98

TA1537

72

 

29

 

24

 

18

 

10

 

98

(93)

25

(28)

18

(19)

18

(17)

9

(9)

108

 

29

 

15

 

14

 

9

 

Table 2            Test Results: Experiment 1 – Without Metabolic Activation(Plate Incorporation)

Test Period

From: 14 November 2017

To: 17 November 2017

S9-Mix

(-)

Dose Level

Per Plate

Number of revertants (mean) +/- SD

Base-pair substitution strains

Frameshift strains

TA100

TA1535

WP2uvrA

TA98

TA1537

Solvent Control

(DMSO)

96

98

76

(90)

12.2#

23

38

21

(27)

9.3

32

24

16

(24)

8.0

21

21

15

(19)

3.5

9

9

12

(10)

1.7

1.5 µg

108

97

95

(100)

7.0

40

24

26

(30)

8.7

29

20

22

(24)

4.7

9

9

13

(10)

2.3

14

8

13

(12)

3.2

5 µg

94

83

90

(89)

5.6

33

24

23

(27)

5.5

19

26

26

(24)

4.0

21

20

19

(20)

1.0

14

13

13

(13)

0.6

15 µg

87

88

82

(86)

3.2

42

33

25

(33)

8.5

29

25

23

(26)

3.1

19

11

12

(14)

4.4

8

13

12

(11)

2.6

50 µg

93

89

91

(91)

2.0

42

33

38

(38)

4.5

23

24

23

(23)

0.6

19

19

18

(19)

0.6

15

12

15

(14)

1.7

150 µg

89

92

98

(93)

4.6

25

33

30

(29)

4.0

33

21

22

(25)

6.7

20

19

16

(18)

2.1

15

11

14

(13)

2.1

500 µg

77

89

89

(85)

6.9

28

31

30

(30)

1.5

25

25

23

(24)

1.2

21

25

24

(23)

2.1

12

13

11

(12)

1.0

1500 µg

80

92

87

(86)

6.0

28

40

25

(31)

7.9

27

24

16

(22)

5.7

19

16

12

(16)

3.5

9

10

13

(11)

2.1

5000 µg

81 F

74 F

79 F

(78)

3.6

32 F

18 F

13 F

(21)

9.8

24 F

22 F

16 F

(21)

4.2

19 F

27 F

10 F

(19)

8.5

15 F

2 F

6 F

(8)

6.7

Positive controls

S9-Mix

(-)

Name

Dose Level

No. of Revertants

ENNG

ENNG

ENNG

4NQO

9AA

3 µg

5 µg

2 µg

0.2 µg

80 µg

665

538

606

(603)

63.6

465

450

489

(468)

19.7

848

735

822

(802)

59.2

160

139

163

(154)

13.1

540

324

395

(420)

110.1

Table 3            Test Results: Experiment 1 – With Metabolic Activation(Plate Incorporation)

Test Period

From: 14 November 2017

To: 17 November 2017

S9-Mix

(+)

Dose Level

Per Plate

Number of revertants (mean) +/- SD

Base-pair substitution strains

Frameshift strains

TA100

TA1535

WP2uvrA

TA98

TA1537

Solvent Control

(DMSO)

84

114

116

(105)

17.9#

22

36

28

(29)

7.0

27

26

25

(26)

1.0

34

19

35

(29)

9.0

12

18

14

(15)

3.1

1.5 µg

106

93

93

(97)

7.5

30

25

32

(29)

3.6

25

25

31

(27)

3.5

15

19

21

(18)

3.1

19

5

12

(12)

7.0

5 µg

105

98

105

(103)

4.0

25

32

35

(31)

5.1

30

32

26

(29)

3.1

26

30

27

(28)

2.1

13

19

8

(13)

5.5

15 µg

103

91

96

(97)

6.0

27

21

21

(23)

3.5

25

26

28

(26)

1.5

17

21

19

(19)

2.0

14

19

13

(15)

3.2

50 µg

97

98

102

(99)

2.6

30

20

30

(27)

5.8

26

25

28

(26)

1.5

18

15

16

(16)

1.5

16

19

12

(16)

3.5

150 µg

92

106

112

(103)

10.3

40

23

28

(30)

8.7

28

36

25

(30)

5.7

12

23

25

(20)

7.0

12

15

9

(12)

3.0

500 µg

116

94

96

(102)

12.2

25

23

26

(25)

1.5

30

28

30

(29)

1.2

22

22

14

(19)

4.6

10

10

11

(10)

0.6

1500 µg

94

113

90

(99)

12.3

30

22

20

(24)

5.3

28

30

26

(28)

2.0

26

28

23

(26)

2.5

14

8

10

(11)

3.1

5000 µg

103 F

97 F

113 F

(104)

8.1

15 F

17 F

17 F

(16)

1.2

18 F

26 F

18 F

(21)

4.6

19 F

11 F

24 F

(18)

6.6

4 F

12 F

7 F

(8)

4.0

Positive controls

S9-Mix

(+)

Name

Dose Level

No. of Revertants

2AA

2AA

2AA

BP

2AA

1 µg

2 µg

10 µg

5 µg

2 µg

2395

2473

2494

(2454)

52.2

276

298

268

(281)

15.5

181

175

212

(189)

19.9

194

184

202

(193)

9.0

338

346

365

(350)

13.9

ENNG     N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO     4-Nitroquinoline-1-oxide

9AA        9-Aminoacridine

2AA   2 -Aminoanthracene

BP           Benzo(a)pyrene

Applicant's summary and conclusion

Conclusions:
The substance was tested in a guideline Ames Assay (OECD 471) and found to be non-mutagenic. The substance does not meet the criteria for classification as a mutagen according to Regulation EC No. 1272/2008.