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Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro gene mutation in bacteria (OECD 471): negative with and without metabolic activation

In vitro cytogenicity / chromosome aberration (OECD 473): negative with and without metabolic activation

In vitro gene mutation in mamallian cells (OECD 476): negative with and without metabolic activation

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 Apr - 18 Jun 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
The Department of Health of the Government of the United Kingdom
Type of assay:
bacterial reverse mutation assay
Target gene:
his operon for S. typhimurium strains and trp operon for E. coli strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbitone/β-naphthoflavone
Test concentrations with justification for top dose:
Preliminary toxicity test: 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate with and without metabolic activation
Experiment 1 (plate incorporation): 50, 150, 500, 1500, 5000 µg/plate with and without metabolic activation
Experiment 2 (pre-incubation): 50, 150, 500, 1500, 5000 µg/plate with and without metabolic activation
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: The test item was immiscible in sterile distilled water and dimethyl sulphoxide at 50 mg/mL but was fully miscible in acetone at 100 mg/ml in solubility checks performed in-house. Acetone was therefore selected as the vehicle.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
acetone
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:in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 20 min (Experiment 2 only)
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: triplicates each in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: inspection of the bacterial background lawn
Evaluation criteria:
There are several criteria for determining a positive result. 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. Statistical analysis of data.
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgement about test item activity. Results of this type will be reported as equivocal.
Statistics:
Mean values and standard deviation were calculated.
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
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, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
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, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
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, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
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, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The test item was immiscible in sterile distilled water at 50 mg/ml.
- Precipitation: A test item precipitate (oily in appearance) was observed at and above 1500 µg/plate, this observation did not prevent the scoring of revertant colonies.
- Stability in vehicle: There was no visible change in test item formulations throughout each experiment, therefore, it was assumed to be stable under the test conditions.

RANGE-FINDING/SCREENING STUDIES:
The preliminary toxicity test and Experiment 1 can be considered as range-finding studies. No cytotoxicity and no effects on the number of revertant colonies were observed at any test concentration in both tests. Therefore, in Experiment 2 the test item concentration range was the same as the range-finding tests, i.e. up to the limit concentration of 5000 µg/plate.
COMPARISON WITH HISTORICAL CONTROL DATA:
The number of revertant colonies in the vehicle, untreated and positive controls was within the ranges of the reported history profile of vehicle (combined historical negative and solvent control ranges) and positive control values of the testing facility for the two last calendar years.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the preliminary toxicity test, the test item was non-toxic to the strains of bacteria used (TA100 and WP2uvrA). The test item formulation and S9-mix used in this experiment were both shown to be sterile.
No cytotoxic effects (reduction in the growth of the bacterial background lawn) were observed at any concentration in Experiments 1 and 2.

Table 1. Preliminary toxicity test (numbers of revertant colonies).

With (+) or

without (-)

S9-mix

Strain

Dose (µg/plate)

 

 

0

0.15

0.5

1.5

5

15

50

150

500

1500

5000

-

TA100

65

63

69

68

65

66

62

63

63

66P

69P

+

TA100

73

71

62

63

73

64

79

67

69

65P

69P

-

WP2uvrA

29

27

27

25

24

29

31

35

36

33P

36P

+

WP2uvrA

43

44

44

41

48

43

27

34

38

46P

49P

P: Precipitate

 

Table 2. Spontaneous mutation rates (concurrent negative controls)

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

EXPERIMENT 1 (plate incorporation)

TA 100

TA 1535

WP2uvrA

TA 98

TA 1537

106

99

136

(114)

23

31

24

(26)

44

41

28

(38)

35

28

31

(31)

11

12

13

(12)

EXPERIMENT 2 (pre-incubation)

134

128

110

(124)

27

20

21

(23)

57

31

44

(44)

40

33

25

(33)

11

15

7

(11)

 

Table 3. Test results Experiment 1

EXPERIMENT 1 (plate incorporation)

Number of revertants (mean ± standard deviation)

S9-Mix

Without

Test item (µg/plate)

TA 100

TA 1535

WP2uvrA

TA 98

TA1537

Solvent control

(acetone)

112

120

138

(123 ± 13.3)

19

24

17

(20 ± 3.6)

51

52

40

(48 ± 6.7)

35

24

36

(32 ± 6.7)

16

15

16

(16 ± 0.6)

50

114

124

112

(117 ± 6.4)

17

19

21

(19 ± 2.0)

37

49

43

(43 ± 6.0)

32

31

32

(32 ± 0.6)

16

15

15

(15 ± 0.6)

150

111

114

115

(113 ± 2.1)

17

17

19

(18 ± 1.2)

47

43

35

(42 ± 6.1)

29

27

36

(31 ± 4.7)

15

16

17

(16 ± 1.0)

500

120

122

130

(124 ± 5.3)

17

16

23

(19 ± 3.8)

43

41

44

(43 ± 1.5)

29

27

21

(26 ± 4.2)

19

19

17

(18 ± 1.2)

1500

134 P

120 P

110 P

(121 ± 12.1)

25 P

17 P

24 P

(22 ± 4.4)

40 P

52 P

41 P

(44 ± 6.7)

31 P

31 P

20 P

(27 ± 6.4)

11 P

8 P

12 P

(10 ± 2.1)

5000

123 P

114 P

111 P

(116 ± 6.2)

24 P

13 P

13 P

(17 ± 6.4)

39 P

33 P

33 P

(35 ± 3.5)

29 P

24 P

29 P

(27 ± 2.9)

16 P

9 P

8 P

(11 ± 4.4)

ENNG

649

549

575

(591 ± 51.9)

640

678

1024

(781 ± 211.6)

942

1016

956

(971 ± 39.3)

-

-

4NQO

-

-

-

167

174

158

(166 ± 8.0)

-

9AA

-

-

-

-

474

289

516

(426 ± 120.8)

 

S9-Mix

With

Test item (µg/plate)

TA 100

TA 1535

WP2uvrA

TA 98

TA1537

Solvent control

(acetone)

95

119

86

(100 ± 17.1)

11

5

13

(10 ± 4.2)

55

49

55

(53 ± 3.5)

27

27

33

(29 ± 3.5)

15

13

17

(15 ± 2.0)

50

102

69

116

(96 ± 24.1)

19

8

13

(13 ± 5.5)

55

41

49

(48 ± 7.0)

27

24

29

(27 ± 2.5)

12

19

16

(16 ± 3.5)

150

90

131

103

(108 ± 21.0)

13

17

8

(13 ± 4.5)

48

43

48

(46 ± 2.9)

27

35

37

(33 ± 5.3)

12

8

11

(10 ± 2.1)

500

123

79

91

(98 ± 22.7)

15

16

13

(15 ± 1.5)

48

52

53

(51 ± 2.6)

27

25

27

(26 ± 1.2)

15

16

17

(16 ± 1.0)

1500

90 P

112 P

99 P

(100 ± 11.1)

15 P

9 P

15 P

(13 ± 3.5)

44 P

47 P

48 P

(46 ± 2.1)

39 P

25 P

25 P

(30 ± 8.1)

15 P

12 P

19 P

(15 ± 3.5)

5000

106 P

103 P

103 P

(104 ± 1.7)

9 P

15 P

9 P

(11 ± 3.5)

48 P

43 P

43 P

(45 ± 2.9)

31 P

31 P

29 P

(30 ± 1.2)

13 P

15 P

15 P

(14 ± 1.2)

2AA

1407

1922

1370

(1566 ± 308.6)

238

253

243

(245 ± 7.6)

513

537

581

(544 ± 34.5)

-

249

385

295

(310 ± 69.2)

BP

-

-

-

200

262

210

(224 ± 33.3)

-

ENNG: N-ethyl-N'-nitro-N-nitrosoguanidine: 2 µg/plate for WP2uvrA, 3 µg/plate for TA100, 5 µg/plate for TA1535

9AA: 9-Aminoacridine: 80 µg/plate for TA1537

4NQO: 4-Nitroquinoline-1-oxide: 0.2 µg/plate for TA98

2AA: 2-Aminoanthracene: 1 µg/plate for TA100, 2 µg/plate for TA1535 and TA1537, 10 µg/plate for WP2uvrA

BP: Benzo(a)pyrene: 5 µg/plate for TA98

P: Precipitate

 

Table 4. Test results Experiment 2

EXPERIMENT 2 (pre-incubation)

Number of revertants (mean ± standard deviation)

S9-Mix

Without

 

Test item (µg/plate)

TA 100

TA 1535

WP2uvrA

TA 98

TA1537

Solvent control

(acetone)

118

132

114

(121 ± 9.5)

25

23

25

(24 ± 1.2)

57

27

21

(35 ± 19.3)

35

41

33

(36 ± 4.2)

17

8

13

(13 ± 4.5)

50

112

119

120

(117 ± 4.4)

19

27

13

(20 ± 7.0)

40

35

49

(41 ± 7.1)

25

41

27

(31 ± 8.7)

13

12

7

(11 ± 3.2)

150

114

111

122

(116 ± 5.7)

21

24

17

(21 ± 3.5)

41

31

41

(38 ± 5.8)

23

32

15

(23 ± 8.5)

11

8

12

(10 ± 2.1)

500

124

127

136

(129 ± 6.2)

28

29

29

(29 ± 0.6)

41

37

43

(40 ± 3.1)

31

28

31

(30 ± 1.7)

9

13

4

(9 ± 4.5)

1500

115 P

126 P

126 P

(122 ± 6.4)

23 P

25 P

20 P

(23 ± 2.5)

39 P

39 P

32 P

(37 ± 4.0)

31 P

29 P

33 P

(31 ± 2.0)

9 P

16 P

12 P

(12 ± 3.5)

5000

107 P

104 P

116 P

(109 ± 6.2)

16 P

31 P

19 P

(22 ± 7.9)

31 P

49 P

49 P

(43 ± 10.4)

33 P

35 P

23 P

(30 ± 6.4)

17 P

8 P

13 P

(13 ± 4.5)

ENNG

448

453

502

(468 ± 29.8)

233

192

231

(219 ± 23.1)

541

492

501

(511 ± 26.1)

 -

-

4NQO

-

-

-

158

140

106

(135 ± 26.4)

-

9AA

-

-

-

-

604

442

535

(527 ± 81.3)

 

S9-Mix

With

Test item (µg/plate)

TA 100

TA 1535

WP2uvrA

TA 98

TA1537

Solvent control

(acetone)

130

132

128

(130 ± 2.0)

9

9

23

(14 ± 8.1)

53

59

41

(51 ± 9.2)

32

29

33

(31 ± 2.1)

12

8

12

(11 ± 2.3)

50

120

114

115

(116 ± 3.2)

13

13

21

(16 ± 4.6)

39

49

41

(43 ± 5.3)

27

41

31

(33 ± 7.2)

8

19

19

(15 ± 6.4)

150

130

134

116

(127 ± 9.5)

17

17

15

(16 ± 1.2)

44

48

56

(49 ± 6.1)

45

33

40

(39 ± 6.0)

16

20

9

(15 ± 5.6)

500

114

124

135

(124 ± 10.5)

16

9

8

(11 ± 4.4)

53

48

53

(51 ± 2.9)

25

32

39

(32 ± 7.0)

17

19

16

(17 ± 1.5)

1500

123 P

118 P

108 P

(116 ± 7.6)

9 P

9 P

8 P

(9 ± 0.6)

45 P

43 P

43 P

(44 ± 1.2)

37 P

39 P

33 P

(36 ± 3.1)

15 P

23 P

13 P

(17 ± 5.3)

5000

122 P

126 P

115 P

(121 ± 5.6)

12 P

11 P

8 P

(10 ± 2.1)

51 P

49 P

49 P

(50 ± 1.2)

35 P

32 P

31 P

(33 ± 2.1)

11 P

15 P

19 P

(15 ± 4.0)

2AA

1270

1262

1407

(1313 ± 81.5)

184

212

234

(210 ± 25.1)

275

255

350

(293 ± 50.1)

-

279

277

289

(282 ± 6.4)

BP

-

-

-

176

204

203

(194 ± 15.9)

-

ENNG: N-ethyl-N'-nitro-N-nitrosoguanidine: 2 µg/plate for WP2uvrA, 3 µg/plate for TA100, 5 µg/plate for TA1535

9AA: 9-Aminoacridine: 80 µg/plate for TA1537

4NQO: 4-Nitroquinoline-1-oxide: 0.2 µg/plate for TA98

2AA: 2-Aminoanthracene: 1 µg/plate for TA100, 2 µg/plate for TA1535 and TA1537, 10 µg/plate for WP2uvrA

BP: Benzo(a)pyrene: 5 µg/plate for TA98

P: Precipitate

Conclusions:
Interpretation of results: negative
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 Aug 2012 - 31 Jan 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
The Department of Health of the Government of the United Kingdom
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
Not applicable
Species / strain / cell type:
lymphocytes: cultured peripheral human lymphocytes
Details on mammalian cell type (if applicable):
- Type and identity of media: Eagle's minimal essential medium with HEPES buffer (MEM), supplemented with L-glutamine, penicillin/streptomycin, amphotericin B and 10% foetal bovine serum (FBS).
Duplicate lymphocyte cultures were established for each dose level by mixing the following components:
9.05 – 9.15 mL MEM, 10% (FBS)
0.1 mL Li-heparin
0.1 mL phytohaemagglutinin
0.65 - 0.75 mL heparinised whole blood
- Properly maintained: yes
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbitone and β-naphthoflavone
Test concentrations with justification for top dose:
10, 20, 40, 80, 160 and 320 µg/mL with and without metabolic activation
Vehicle / solvent:
- Vehicle(s)/solvent(s) acetone
- Justification for choice of solvent/vehicle: The test item was insoluble in sterile distilled water at 50 mg/ml and dimethyl sulphoxide at 500 mg/mL but was fully soluble in acetone at 500 mg/mL in solubility checks performed at the testing laboratory. Acetone was therefore selected as the vehicle.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
Experiment 1: 4 h with and without metabolic activation
Experiment 2: 4 h with metabolic activation, 24 h without metabolic activation
- Expression time (cells in growth medium):
Experiment 1: 20 h after exposure with and without metabolic activation
Experiment 2: 20 h after exposure with metabolic activation, 24 h (continuous) exposure without metabolic activation
- Fixation time (start of exposure up to fixation or harvest of cells): 24 h (Experiments 1 and 2)

SPINDLE INHIBITOR (cytogenetic assays): Colcemid (0.1 µg/mL)
STAIN (for cytogenetic assays): 5% Giemsa

NUMBER OF REPLICATIONS: 2 each in Experiment 1 and 2, respectively.

NUMBER OF CELLS EVALUATED: 100 per culture in vehicle control and treatment groups; 50 per culture in positive control groups

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index of 2000 cells

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
Evaluation criteria:
A positive response was recorded for a particular treatment if the percentage of cells with aberrations, excluding gaps, markedly exceeded that seen in the concurrent control, either with or without a clear dose-relationship. For modest increases in aberration frequency a dose response relationship was generally required and appropriate statistical tests were applied in order to record a positive response.
Statistics:
The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test.
Key result
Species / strain:
lymphocytes: cultured peripheral human lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
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: There was no significant change in pH when the test item was dosed into culture media. The pH value increased with test item concentration from 7.41 at 19.5 µg/mL to 7.47 at 5000 µg/mL.
- Effects of osmolality: The osmolality did not increase by more than 50 mOsm.
- Water solubility: The test item was insoluble in sterile distilled water at 50 mg/mL
- Precipitation: In Experiment 1, greasy/oily precipitate was observed in the whole blood cultures at the end of the exposure period at and above 40 µg/mL and 80 µg/mL in the presence and absence of S9, respectively. In Experiment 2, greasy/oily precipitate was observed in the whole blood cultures at the end of the exposure period at and above 160 µg/mL in the absence of S9 only. No precipitate was observed in the 4(20)-hour exposure group with S9.
- Other confounding effects: The lack of precipitate being recorded in the presence of S9 was considered to be due differences between technical staff. However, the modest toxicity observed was similar to that seen in the preliminary toxicity test and Experiment 1 and, therefore, the test item was considered to have been adequately tested.

RANGE-FINDING/SCREENING STUDIES:
A preliminary toxicity test was performed on cell cultures using a 4-hour exposure time with and without metabolic activation followed by a 20-hour recovery period, and a continuous exposure of 24 hours without metabolic activation. The dose range of test item used was 19.5, 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500 and 5000 µg/mL. Parallel flasks, containing culture medium without whole blood, were established for the three exposure conditions so that test item precipitate observations could be made. Precipitate observations were recorded at the beginning and end of the exposure periods.
Using a qualitative microscopic evaluation of the microscope slide preparations from each treatment culture, appropriate dose levels were selected for mitotic index evaluation. Mitotic index data was used to estimate test item toxicity and for selection of the dose levels for the main test.
Precipitate observations were made from the blood-free cultures at the end of the exposure period. In the exposure group 4(+20)-hour in the absence of S9, precipitate was noted at the end of exposure at and above 78.13 µg/mL, which became greasy/oily at and above 156.25 µg/mL. However, in the 4(+20)-hour exposure group in the presence of S9, precipitate was observed at and above 78.13 µg/mL, becoming greasy/oily at and above 312.5 µg/mL. In the 24-hour continuous exposure group, precipitate was observed at and above 156.25 µg/mL, becoming greasy/oily at and above 312.5 µg/mL. However, precipitate observations made in the whole blood cultures indicated a greasy/oily precipitate was observed at and above 78.13 µg/mL in both of the 4(+20)-hour exposure groups, and at and above 312.5 µg/mL in the 24-hour continuous exposure group. It was thought that the presence of the blood improved the visibility of the precipitate.
Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 5000 µg/mL in all exposure groups. The determined mitotic index showed that there was only modest evidence of toxicity observed, and in general maximum exposure was considered to be achieved at and around 312.5 µg/mL where the formation of the greasy/oily precipitate was observed in two of the exposure conditions.
The selection of the maximum dose level was based on the onset of the precipitate, particularly the formation of a greasy/oily precipitate, in all exposure groups tested.

COMPARISON WITH HISTORICAL CONTROL DATA:
Vehicle and positive controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes according the historical control data of the testing laboratory.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Experiment 1: No dose-related inhibition of mitotic index was observed in either exposure group (with and without S9). Therefore, the maximum dose level selected for metaphase analysis was 320 µg/mL for both exposure groups.
Experiment 2: No inhibition of mitotic index was observed in either exposure group (with and without S9). The maximum dose level selected for metaphase analysis was therefore 320 µg/mL for both exposure groups. The ‘B’ replicate of the 320 µg/mL dose level of the 24-hour exposure group was lost during the harvesting process and therefore extra metaphases were scored from the ‘A’ replicate to compensate. This is acceptable under the test guidelines.

Table 1. Mitotic index – Preliminary toxicity test

Concentration (µg/ml)

4(+20)h without s9

4(+20)h with s9

24h without s9

Mitotic index

% of control

Mitotic index

% of control

Mitotic index

% of control

0

5.70

100

6.35

100

8.35

100

19.5

-

-

-

-

-

-

39.06

6.10

107

5.45

86

-

-

78.13

4.90 P

86

5.40 P

85

6.60

79

156.25

5.65 P GP

99

5.30 P

83

4.90 P

59

312.5

4.30 P GP

75

5.60 P GP

88

5.50 P GP

66

625

- P GP

-

- P GP

-

4.95 P GP

59

1250

- P GP

-

- P GP

-

- P GP

-

2500

4.25 GP

75

3.25 GP

51

7.85 P GP

94

5000

5.15 GP

90

6.80 GP

107

5.80 GP

69

- = Not assessed for mitotic index.

P = Precipitate observed at end of exposure period in the blood-free cultures

GP = Greasy/oily precipitate observed at end of exposure period in the blood-free cultures

 

Table 2. Mitotic index - Experiment 1

Concentration µg/ml

4 hours treatment without s9

4 hours treatment with s9

 

A

B

Mean

% of control

A

B

Mean

% of control

0

2.40

2.15

2.28

100

2.65

4.60

3.63

100

10

-

-

-

-

-

-

-

-

20

3.60

2.55

3.08

135

3.85

2.80

3.33

92

40

3.25

3.40

3.33

146

2.90 GP

2.55 GP

2.73

75

80

2.60 GP

3.45 GP

3.03

133

- GP

- GP

-

-

160

- GP

- GP

-

-

2.85 GP

3.00 GP

2.93

81

320

3.25 GP

2.35 GP

2.80

123

4.25 GP

4.50 GP

4.38

121

MMC 0.4

0.20

0.65

0.43

19

NA

NA

NA

NA

CP 5

NA

NA

NA

NA

1.15

1.10

1.13

31

MMC = Mitomycin C

CP = Cyclophosphamide

NA = Not applicable

- = Not assessed for mitotic index

GP = Greasy/oily precipitate observed at end of exposure period

 

Table 3. Mitotic index - Experiment 2

Concentration µg/ml

4 hours treatment without s9

4 hours treatment with s9

 

A

B

Mean

% of control

A

B

Mean

% of control

0

5.30

6.00

5.65

100

8.70

9.15

8.93

100

10

-

-

-

-

-

-

-

-

20

-

-

-

-

-

-

-

-

40

-

-

-

-

-

-

-

-

80

4.45

4.20

4.33

77

9.80

10.50

10.15

114

160

5.70 P GP

5.40 P GP

5.55

98

7.60

7.55

7.58

85

320

4.55 P GP

- P GP

4.55

81

8.35

7.45

7.90

89

MMC 0.2

1.90

2.40

2.15

38

NA

NA

NA

NA

CP 5

NA

NA

NA

NA

5.10

5.15

5.13

57

CP = Cyclophosphamide

MMC = Mitomycin C

NA = Not applicable

- = Not assessed for mitotic index

P = Precipitate observed at end of exposure period

GP = Greasy/oily precipitate observed at end of exposure period

 

Table 4. Results of Chromosome Aberration Test - Experiment 1: 4 h treatment without metabolic activation (S9)

Concentration µg/mL

No. of cells observed

Percentage of cells with structural chromosome aberrations (%)

Gaps (% cells)

Mitotic Index (%)

Percentage of cells with numerical aberrations (%)

ctb

cte

csb

cse

Others

Total

Solvent

200

0.0

0.0

0.0

0.0

0.0

0.0

0.0

100

0.0

20

200

0.5

0.0

0.0

0.0

0.0

0.5

0.0

135

0.0

40

200

0.5

0.0

0.0

0.0

0.5

1.0

0.0

146

0.0

80

200

1.5

0.0

0.5

0.0

0.0

1.5

0.5

133

0.0

320

200

0.0

0.0

0.0

0.0

0.0

0.0

0.0

123

0.0

MMC 0.4

100

41.0

32.0

1.0

1.0

1.0

57.0*

5.0

19

0.0

MMC = Mitomycin C

* = P < 0.001

ctb = chromatide breaks; csb = chromosome breaks; cte = chromatide exchanges; cse = chromosome exchanges

 

 

Table 5. Results of Chromosome Aberration Test - Experiment 1: 4 h treatment with metabolic activation (S9)

Concentration µg/mL

No. of cells observed

Percentage of cells with structural chromosome aberrations (%)

Gaps (% cells)

Mitotic Index (%)

Percentage of cells with numerical aberrations (%)

ctb

cte

csb

cse

Others

Total

Solvent

200

0.0

0.0

0.0

0.0

0.0

0.0

0.0

100

0.0

20

200

0.0

0.0

0.0

0.0

0.0

0.0

0.0

92

0.0

40

200

0.5

0.0

0.0

0.0

0.0

0.5

0.0

75

0.0

160

200

0.5

0.0

0.0

0.0

0.0

0.5

0.0

81

0.0

320

200

0.0

0.0

0.0

0.0

0.0

0.0

0.0

121

0.0

CP 5

100

24.0

6.0

3.0

0.0

0.0

29.0*

2.0

31

0.0

CP = Cyclophosphamide

* = P < 0.001

ctb = chromatide breaks; csb = chromosome breaks; cte = chromatide exchanges; cse = chromosome exchanges

 

 

Table 6. Results of Chromosome Aberration Test - Experiment 2: 24 h treatment without metabolic activation (S9)

Concentration µg/mL

No. of cells observed

Percentage of cells with structural chromosome aberrations (%)

Gaps (% cells)

Mitotic Index (%)

Percentage of cells with numerical aberrations (%)

ctb

cte

csb

cse

Others

Total

Solvent

200

0.0

0.0

0.0

0.0

0.0

0.0

1.5

100

0.0

80

200

1.0

0.0

0.0

0.0

0.0

1.0

0.0

77

0.0

160

200

0.0

0.0

0.0

0.0

0.0

0.0

0.0

98

0.0

320

200(a)

0.0

0.0

0.5

0.0

0.0

0.5

1.5

81

0.0

MMC 0.2

100

21.0

19.0

1.0

0.0

0.0

35.0*

2.0

38

0.0

MMC = Mitomycin C

(a) = Additional 100 cells from ‘A’ culture scored due to loss of the ‘B’ culture.

* = P < 0.001

ctb = chromatide breaks; csb = chromosome breaks; cte = chromatide exchanges; cse = chromosome exchanges

 

Table 7. Results of Chromosome Aberration Test - Experiment 2: 4 h treatment with metabolic activation (S9)

Concentration µg/mL

No. of cells observed

Percentage of cells with structural chromosome aberrations (%)

Gaps (% cells)

Mitotic Index (%)

Percentage of cells with numerical aberrations (%)

ctb

cte

csb

cse

Others

Total

Solvent

200

0.5

0.0

0.0

0.0

0.0

0.5

0.0

100

0.0

80

200

0.0

0.0

0.0

0.0

0.0

0.0

0.0

114

0.0

160

200

0.5

0.0

0.0

0.0

0.0

0.5

0.5

85

0.0

320

200

1.0

0.0

0.0

0.0

0.0

1.0

0.0

89

0.0

CP 5

100

20.0

7.0

3.0

1.0

0.0

28.0*

5.0

57

0.0

CP = Cyclophosphamide

* = P < 0.001

ctb = chromatide breaks; csb = chromosome breaks; cte = chromatide exchanges; cse = chromosome exchanges

Conclusions:
Interpretation of results: negative
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 Jul - 22 Oct 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Deviations:
no
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
The Department of Health of the Government of the United Kingdom, UK
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene
Target gene:
TK locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media:
R10 medium: RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/ml), Streptomycin (100 µg/ml), Sodium pyruvate (1 mM), Amphotericin B (2.5 µg/ml) and 10% donor horse serum.
R0 medium: RPMI 1640
R20 medium: RPMI 1640 with 20% donor horse serum
Cells were routinely cultured in R10 medium at 37 °C with 5% CO2in air. The cells have a generation time of approximately 12 hours and were subcultured accordingly.

- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital/β-naphthoflavone
Test concentrations with justification for top dose:
5, 10, 20, 40, 60 and 80 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: following solubility checks performed at the testing laboratory
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 h (Experiment 1), 4 and 24 h (Experiment 2, with and without metabolic activation, respectively)
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 10 - 14 days
- Fixation time (start of exposure up to fixation or harvest of cells): 12 - 17 days

SELECTION AGENT (mutation assays): 4 µg/mL trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: duplicates each in two independent experiments in 96-well microtitre plates

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency and relative total growth

OTHER:
Small and large colonies were differentiated, as small colonies are capable to indicate chromosomal mutations.
Evaluation criteria:
For a test item to demonstrate a mutagenic response, it must produce a statistically significant increase in the induced mutant frequency (IMF) over the concurrent vehicle mutant frequency value. The IMF must exceed the Global Evaluation Factor (GEF) value, which is based on the global background MF for the method used. The GEF value was set at 126E-06 for the microwell method. Therefore, any test item dose level that has a mutation frequency value that is greater than the corresponding vehicle control by the GEF of 126E-6 and demonstrates a positive linear trend will be considered positive. However, if a test item produces a modest increase in mutant frequency, which only marginally exceeds the GEF value and is not reproducible or part of a dose-related response, then it may be considered to have no toxicological significance. Conversely, when a test item induces modest reproducible increases in the mutation frequencies that do not exceed the GEF value then scientific judgement will be applied. If the reproducible responses are significantly dose-related and include increases in the absolute numbers of mutant colonies then they may be considered to be toxicologically significant.
Small significant increases designated by the UKEMS statistical package are reviewed using the above criteria, and may be disregarded at the Study Director's discretion.
Statistics:
The experimental data was analysed using a dedicated computer program, Mutant 240C by York Electronic Research, which follows the statistical guidelines recommended by the UKEMS (Robinson W D et al (1989) Statistical evaluation of bacterial/mammalian fluctuation tests. In: Statistical Evaluation of Mutagenicity Test Data, UKEMS sub-committee on guidelines for mutagenicity testing (Kirkland D J Ed.), Cambridge University Press Report part III, pp102-140).
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
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: There was no marked change in pH when the test item was dosed into media.
- Effects of osmolality: The osmolality did not increase by more than 50 mOsm.
- Precipitation:
Preliminary toxicity test: A cloudy precipitate of the test item was observed at and above 39.06 µg/mL, which became greasy/oily in appearance at and above 156.25 µg/mL in all exposure groups. Due to the presence of excessive precipitate where no test item-induced toxicity was observed using %RSG values, the maximum concentration in the subsequent mutagenicity test was limited to 80 µg/mL.
Experiment 1: A precipitate of the test item was observed at and above 40 µg/mL both in the presence and absence of metabolic activation.
Experiment 2: A precipitate of the test item was observed at and above 60 µg/mL both in the presence and absence of metabolic activation.
- Other confounding effects: In Experiment 1, the test item induced a small but statistically significant dose-related (linear trend) increase in the mutant frequency x10E-06 per viable cell in the absence of metabolic activation. However, these values were within the acceptable range for a vehicle control and the GEF was not exceeded. The result was considered to be spurious and of no toxicological relevance. In Experiment 2, there were no statistically significant or dose-related (linear trend) increases in the mutant frequency x10E-06 per viable cell either in the presence or absence of metabolic activation.

RANGE-FINDING/SCREENING STUDIES:
The concentration range of the test item used in the preliminary toxicity test was 9.77 to 2500 µg/mL. In all three exposure groups (4 h ± S9-mix and 24 h -S9 mix), there was no evidence of concentration-related reductions in the Relative Suspension Growth (%RSG) of cells treated with the test item when compared to the concurrent vehicle controls.

COMPARISON WITH HISTORICAL CONTROL DATA:
The normal range for mutant frequency per survivor is 50-170E-06 for the TK+/- locus in L5178Y cells at the testing facility. Vehicle controls results should ideally be within this range, although minor errors in cell counting and dilution or exposure to the metabolic activation system may cause this to be slightly elevated. Experiments where the vehicle control values are markedly greater than 200E-06 mutant frequency per survivor are not normally acceptable and would be repeated.
Positive control chemicals should induce at least three to five fold increases in mutant frequency greater than the corresponding vehicle control.
None of the vehicle control mutant frequency values were outside the acceptable range of 50 to 200E-06 viable cells. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional.

Table 1. Summary of results

Experiment 1

Treatment (µg/mL)

4 h (-S9)

Treatment (µg/mL)

4 h (+S9)

 

%RSG

RTG

MF§

 

%RSG

RTG

MF§

0

100

1.00

159.43

0

100

1.00

163.58

5

98

1.37

98.00

5

94

0.94

141.72

10

101

1.60

96.32

10

90

1.05

153.43

20

101

1.17

148.24

20

104

1.18

153.59

40

98

1.07

154.46

40

98

1.14

162.50

60

102

1.03

172.31

60

97

1.10

161.40

80

101

0.92

176.88

80

98

1.05

132.84

Linear trend

**

Linear trend

NS

EMS 400

73

0.54

1387.80

CP 2

76

0.45

1706.15

Experiment 2

Treatment (µg/mL)

24 h (-S9)

Treatment (µg/mL)

4 h (+S9)

 

%RSG

RTG

MF§

 

%RSG

RTG

MF§

0

100

1.00

177.70

0

100

1.00

156.35

5

94

1.06

160.29

5

98

1.08

143.50

10

98

1.25

130.32

10

103

1.10

155.25

20

66

0.82

171.30

20

108

1.08

154.66

40

75

0.90

181.49

40

99

1.14

151.66

60

89

0.99

172.55

60

100

1.21

139.04

80

81

1.00

167.51

80

93

1.06

144.53

Linear trend

NS

Linear trend

NS

EMS 150

64

0.51

1561.68

CP 2

81

0.50

1329.95

%RSG= Relative Suspension Growth

RTG = Relative Total Growth

MF§ = 5-TFT resistant mutants/1E06 viable cells 2 days after treatment

** = P < 0.01

NS = Not significant

EMS = Ethylmethanesulphonate

CP = Cyclophosphamide

Conclusions:
Interpretation of results: negative
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Gene mutation in bacteria

Propane-1,2,3-triyl 3,5,5-trimethylhexanoate was tested for potential induction of gene mutation in bacteria in an Ames test conducted according to OECD guideline 471 and under GLP conditions (Harlan, 2012d). S. typhimurium and E. coli tester strains (TA 1535, TA 1537, TA 98, TA 100 and WPS2 uvrA) were treated with the test material dissolved in acetone at five concentrations up to 5000 µg/plate, using both the plate incorporation and pre-incubation methods and in the presence and absence of metabolic activation (S9 mix). Negative, vehicle and appropriate positive controls were included.

No cytotoxicity (reduction of the bacterial background lawn) was observed up to the highest test concentration. An oily precipitate was noted at 1500 µg/plate and above, without preventing the scoring of revertant colonies. No significant increases in the frequency of revertant colonies were recorded for any of the tester strains, at any concentration either in the presence or absence of metabolic activation. The negative, vehicle and positive controls yielded the expected results and the respective frequency of revertant colonies was within the range of the reported historical control values.

Based on the study results, the test substance was considered to be non-mutagenic under the conditions of the test.

Cytogenicity in vitro

A study was conducted with propane-1,2,3-triyl 3,5,5-trimethylhexanoate to detect the potential induction of structural chromosomal aberrations in cultured mammalian cells according to OECD guideline 473 and in compliance with GLP (Harlan, 2013a). Duplicate cultures of human lymphocytes, treated with the test material, were evaluated for chromosome aberrations at six concentrations (10, 20, 40, 80, 160 and 320 µg/mL), along with vehicle (acetone) and positive controls, both in the presence and absence of metabolic activation (S9 mix). The concentrations used in the main experiments were selected using data from the preliminary toxicity test and the selection of the maximum concentration was based on the onset of precipitate, particularly the formation of a greasy/oily precipitate at 156.25 and above, in all exposure groups tested. Two independent experiments were performed. In the first experiment, cells were treated with the test material for 4 h (with and without S9 mix) and harvested after a 20-hour expression period. In the second experiment, the 4 h exposure with metabolic activation and 20-hour expression period was repeated, while in the absence of metabolic activation exposure was increased to 24 h. No cytotoxicity was observed at up to 320 µg/mL (precipitation observed at 40 µg/mL and above). The test item did not induce any statistically significant increases in the frequency of cells with aberrations in the exposure groups either in the presence or absence of S9, which generally included at least one precipitating concentration. All vehicle controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All positive control materials induced statistically significant increases in the frequency of cells with aberrations. The test material was thus considered to be non-clastogenic to human lymphocytes in vitro.

Gene mutation in mammalian cells

The potential mutagenicity of propane-1,2,3-triyl 3,5,5-trimethylhexanoate on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line was assessed in a GLP-compliant study conducted according to OECD guideline 476 (Harlan, 2013b). L5178Y TK +/- 3.7.2c mouse lymphoma cells were treated with the test material at six concentrations (5, 10, 20, 40, 60 and 80 µg/mL) with and without metabolic activation system (S9 mix). Vehicle (acetone) and positive controls were included. Two independent experiments were performed. In the first experiment, cells were exposed to the test material for 4 h in the presence and absence of S9 mix. In the second experiment, cells were treated for 4 h with metabolic activation and for 24 h without metabolic activation. No cytotoxic effects were noted. The test material did not induce any toxicologically significant concentration-related increases in the mutant frequency at any concentration, either with or without metabolic activation. With no evidence of any marked toxicity in the preliminary toxicity test, the maximum concentration used in the mutagenicity test was limited by the onset of greasy/oily precipitate effectively reducing exposure of the test item to the cells. Overall, precipitate of test item was observed at and above 40 µg/mL. The vehicle controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control items induced marked increases in the mutant frequency. The test material was thus considered to be non-mutagenic to L5178Y cells under the conditions of the test.

Conclusions for genetic toxicity in vitro

Propane-1,2,3-triyl 3,5,5-trimethylhexanoate has been tested for the induction of gene mutations in bacteria and mammalian cells as well as for the induction of chromosome aberrations. All three tests yielded negative results. Therefore, the substance is considered to be not mutagenic and not clastogenic in vitro.

Justification for classification or non-classification

The available data on the genetic toxicity in vitro of propane-1,2,3-triyl 3,5,5-trimethylhexanoate do not meet the classification criteria according to Regulation (EC) No. 1272/2008 and are therefore conclusive but not sufficient for classification.