Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 Dec 2018 - 08 Jan 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted in 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
adopted in 2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
adopted in 1998
Qualifier:
according to guideline
Guideline:
other: ICH S2(R1) guideline adopted June 2012 (ICH S2(R1) Federal Register
Version / remarks:
adopted in 2012
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
adopted in 2000
GLP compliance:
yes (incl. QA statement)
Remarks:
Medicines & Healthcare products Regulatory Agency, Department of Health of the Government of the United Kingdom
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
Reaction product of castor oil with glycerol
EC Number:
949-117-7
Molecular formula:
not applicable, substance is UVCB
IUPAC Name:
Reaction product of castor oil with glycerol
Test material form:
liquid

Method

Target gene:
his operon (for S. typhimurium strains) and trp operon (for E. coli strains)
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: Trinova Biochem GmbH, Germany; obtained: 27 Jun 2017
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: British Industrial Biological Research Association; obtained: 17 Aug 1987
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9 : Cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of male CD Sprague-Dawley rats (age: 6 - 8 weeks, weight: approx. 250 g) orally treated for 3 days with phenobarbitone/beta-naphthoflavone (80/100 mg/kg bw/day) in arachis oil as vehicle.
- date of preparation of S9 mix: 28 Oct 2018
- volume of S9 mix in the final culture medium: 0.5 mL
- quality controls of S9: enzymatic activity, sterility, metabolic capability: checked and certified
Test concentrations with justification for top dose:
Experiment 1 - Plate Incorporation Method: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
HIghest dose recommended in test guideline.

Experiment 2 - Pre-Incubation Method: 15, 50, 150, 500, 1500 and 5000 µg/plate
Concentrations selected based on the results of Experiment 1.
Vehicle / solvent:
- Vehicle: dimethyl sulfoxide (DMSO)
- Supplier: ThermoFisher Scientific
- Batch number: 184106
- Purity: 99.9%
- Expiry: May 2023

- Justification for choice of solvent/vehicle: Due to the fact that the test substance was only partially miscible with water, DMSO was selected as the vehicle.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
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:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration and number of independent experiments: triplicates each in two independent experiments

METHOD OF TREATMENT / EXPOSURE:
- Test substance added in medium; in agar (plate incorporation, Experiment 1) and preincubation (Experiment 2)

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 20 min at 37 ± 3 °C (only Experiment 1)
- Exposure duration/duration of treatment: 48 - 72 h at 37 ± 3 °C

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition

METHODS FOR MEASUREMENTS OF GENOTOXICIY
Genotoxicity in this reverse mutation assay was assessed by counting the number of revertant colonies compared to the negative controls.
Evaluation criteria:
Any, one, or all of the following can be used to determine the overall result of the study:

1. A dose-related increase in mutant frequency over the dose range tested.
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. A fold increase greater than two times the concurrent solvent control for TA100, TA98 and WP2uvrA or a three-fold increase for TA1535 and TA1537 (especially if accompanied by an out of historical range response).
5. Statistical analysis of data as determined by UKEMS.

A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Statistics:
Statistical significance was confirmed by using Dunnetts Regression Analysis (p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.

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:
valid
True 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
Remarks:
at and above 1500 µg/plate in the presence of S9 mix
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
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:
valid
True 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:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
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:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The test substance was only partially miscible with water.
- Precipitation and time of the determination: No precipitation was observed at any of the doses tested in either the presence or absence of metabolic activation.

STUDY RESULTS
- Concurrent vehicle negative and positive control data are reported in tabular form in the section 'Any other information on results incl. tables'.

Ames test:
- Mean number of revertant colonies per plate and standard deviation are reported in tabular form in the section 'Any other information on results incl. tables'.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive and negative (solvent/vehicle) historical control data are attached as pdf document in the section 'Attached background material'. Results obtained in the present study fell within the historical control data range.

Any other information on results incl. tables

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

Test Period

From: 18 December 2018

To: 21 December 2018

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)

118

101

100

(106)

10.1#

20

13

9

(14)

5.6

16

28

15

(20)

7.2

20

11

20

(17)

5.2

9

8

5

(7)

2.1

1.5 µg

120

135

106

(120)

14.5

9

12

21

(14)

6.2

20

20

18

(19)

1.2

16

12

16

(15)

2.3

11

6

10

(9)

2.6

5 µg

85

103

95

(94)

9.0

11

11

10

(11)

0.6

19

18

20

(19)

1.0

17

16

19

(17)

1.5

10

6

12

(9)

3.1

15 µg

109

98

84

(97)

12.5

20

11

14

(15)

4.6

21

21

18

(20)

1.7

16

11

16

(14)

2.9

10

11

12

(11)

1.0

50 µg

116

93

77

(95)

19.6

10

12

12

(11)

1.2

27

20

21

(23)

3.8

12

17

23

(17)

5.5

4

16

9

(10)

6.0

150 µg

106

77

111

(98)

18.4

19

26

7

(17)

9.6

28

28

18

(25)

5.8

24

24

15

(21)

5.2

6

16

9

(10)

5.1

500 µg

136

118

103

(119)

16.5

10

13

10

(11)

1.7

32

22

31

(28)

5.5

15

14

21

(17)

3.8

7

18

3

(9)

7.8

1500 µg

105

87

93

(95)

9.2

15

12

9

(12)

3.0

22

17

18

(19)

2.6

18

21

19

(19)

1.5

2

6

8

(5)

3.1

5000 µg

99

72

91

(87)

13.9

12

12

11

(12)

0.6

36

19

15

(23)

11.2

19

14

20

(18)

3.2

9

8

3

(7)

3.2

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

752

662

766

(727)

56.4

210

204

187

(200)

11.9

550

737

986

(758)

218.7

147

185

163

(165)

19.1

206

371

293

(290)

82.5

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

4NQO: 4-Nitroquinoline-1-oxide

9AA: 9-Aminoacridine

#: Standard deviation

Table 2: Test Results: Experiment 1 – With Metabolic Activation (Plate Incorporation)

Test Period

From: 18 December 2018

To: 21 December 2018

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)

110

132

124

(122)

11.1#

9

9

19

(12)

5.8

29

23

30

(27)

3.8

12

18

29

(20)

8.6

10

16

10

(12)

3.5

1.5 µg

114

120

104

(113)

8.1

7

12

11

(10)

2.6

27

26

36

(30)

5.5

19

16

22

(19)

3.0

11

12

9

(11)

1.5

5 µg

100

139

101

(113)

22.2

11

11

11

(11)

0.0

26

27

31

(28)

2.6

13

22

27

(21)

7.1

12

11

8

(10)

2.1

15 µg

117

95

123

(112)

14.7

9

13

13

(12)

2.3

10

12

35

(19)

13.9

19

21

24

(21)

2.5

8

11

7

(9)

2.1

50 µg

126

140

149

(138)

11.6

11

12

13

(12)

1.0

30

24

32

(29)

4.2

21

18

19

(19)

1.5

10

5

10

(8)

2.9

150 µg

132

142

107

(127)

18.0

15

10

13

(13)

2.5

26

29

34

(30)

4.0

31

19

20

(23)

6.7

18

13

9

(13)

4.5

500 µg

105

122

102

(110)

10.8

17

15

11

(14)

3.1

30

29

28

(29)

1.0

28

16

21

(22)

6.0

14

10

9

(11)

2.6

1500 µg

92

114

112

(106)

12.2

11

13

9

(11)

2.0

28

20

25

(24)

4.0

16

21

22

(20)

3.2

3

5

6

(5)

1.5

5000 µg

113

106

93

(104)

10.1

9

24

18

(17)

7.5

37

35

22

(31)

8.1

20

22

28

(23)

4.2

5

3

4

(4)

1.0

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

1693

1946

2005

(1881)

165.7

268

249

300

(272)

25.8

109

121

110

(113)

6.7

163

166

209

(179)

25.7

275

234

221

(243)

28.2

BP: Benzo(a)pyrene

2AA: 2-Aminoanthracene

#: Standard deviation

Table 3: Test Results: Experiment 2 – Without Metabolic Activation (Pre-Incubation)

Test Period

From: 04 January 2019

To: 07 January 2019

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)

99

121

112

(111)

11.1#

15

22

26

(21)

5.6

24

13

17

(18)

5.6

24

14

21

(20)

5.1

12

14

13

(13)

1.0

15 µg

120

111

128

(120)

8.5

26

19

26

(24)

4.0

12

12

25

(16)

7.5

20

20

19

(20)

0.6

8

9

9

(9)

0.6

50 µg

123

123

119

(122)

2.3

30

22

25

(26)

4.0

19

22

28

(23)

4.6

23

17

12

(17)

5.5

8

5

18

(10)

6.8

150 µg

135

123

99

(119)

18.3

22

29

20

(24)

4.7

11

15

16

(14)

2.6

22

22

22

(22)

0.0

10

8

11

(10)

1.5

500 µg

104

100

96

(100)

4.0

20

26

27

(24)

3.8

15

19

19

(18)

2.3

15

20

15

(17)

2.9

11

7

5

(8)

3.1

1500 µg

119

90

86

(98)

18.0

15

19

20

(18)

2.6

18

17

14

(16)

2.1

21

19

13

(18)

4.2

2

5

5

(4)

1.7

5000 µg

70

86

84

(80)

8.7

19

26

23

(23)

3.5

18

11

20

(16)

4.7

20

10

13

(14)

5.1

6 S

5 S

1 S

(4)

2.6

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

742

645

799

(729)

77.9

2127

2127

2101

(2118)

15.0

976

937

973

(962)

21.7

242

293

214

(250)

40.1

277

335

492

(368)

111.2

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

4NQO: 4-Nitroquinoline-1-oxide

9AA: 9-Aminoacridine

S: Sparse bacterial background lawn

#: Standard deviation

Table 4: Test Results: Experiment 2 – With Metabolic Activation (Pre-Incubation)

Test Period

From: 04 January 2019

To: 07 January 2019

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)

122

112

137

(124)

12.6#

17

21

25

(21)

4.0

31

23

32

(29)

4.9

29

26

24

(26)

2.5

14

11

12

(12)

1.5

15 µg

119

120

123

(121)

2.1

20

24

20

(21)

2.3

32

28

22

(27)

5.0

13

26

23

(21)

6.8

12

9

12

(11)

1.7

50 µg

120

107

138

(122)

15.6

13

16

22

(17)

4.6

23

21

23

(22)

1.2

33

25

13

(24)

10.1

9

10

9

(9)

0.6

150 µg

125

94

121

(113)

16.9

18

23

17

(19)

3.2

34

29

29

(31)

2.9

30

22

22

(25)

4.6

9

10

11

(10)

1.0

500 µg

125

137

125

(129)

6.9

17

29

11

(19)

9.2

20

24

19

(21)

2.6

21

21

24

(22)

1.7

17

15

16

(16)

1.0

1500 µg

108

109

93

(103)

9.0

9

18

18

(15)

5.2

24

28

29

(27)

2.6

24

18

19

(20)

3.2

12

11

11

(11)

0.6

5000 µg

71

84

80

(78)

6.7

24

16

20

(20)

4.0

25

22

18

(22)

3.5

19

24

32

(25)

6.6

3

3

8

(5)

2.9

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

949

1242

1082

(1091)

146.7

247

204

233

(228)

21.9

215

140

148

(168)

41.2

142

137

99

(126)

23.5

240

303

242

(262)

35.8

BP: Benzo(a)pyrene

2AA: 2-Aminoanthracene

#: Standard deviation

Applicant's summary and conclusion

Conclusions:
Interpretation of results: negative
Executive summary:

The available data on gene mutation in bacteria for reaction product of castor oil with glycerol do not meet the criteria for classification according to Regulation (EC) No. 1272/2008 (CLP). The data provide not sufficient evidence that would imply a classification & labelling with respect to mutagenicity/genotoxicity. However, as no information regarding gene mutation in mammalian cells and cytogenicity or chromosome aberration in mammalian cells are available, the overall conclusion for classification regarding mutagenicity/genotoxicity is ‘data lacking’.