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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation in bacteria (OECD 471, Ames test): S. typhimurium TA 1535, TA 1537, TA 98, and TA 100: negative with and without metabolic activation

Cytogenicity in mammalian cells (OECD 473, Chromosome aberration test): primary rat hepatocytes: negative

Gene mutation in mammalian cells (OECD 476, HGPRT Test): Chinese hamster lung fibroblasts (V79): 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:
17 Jan - 4 Feb 1990
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
only four strains tested
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
his operon
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254
Test concentrations with justification for top dose:
Dose range finding test: 5, 50, 500 and 5000 µg/plate with and without metabolic activation
Main test: 50, 150, 500, 1500, 5000 µg/plate with and without metabolic activation
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 9-Aminoacridine (80 µg/pltae ) for TA1537; N-Ethyl-N'-nitrosoguanidine (3 µg/plate) for TA100, (5 µg/plate) for TA1535; 2-Nitrofluorene (1 µg/plate) for TA98.
Remarks:
Without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: With S9: 2-Aminoanthracene (0.5 µg/plate) for TA98, (1 µg/plate) for TA100, (2 µg/plate) for TA1535 and TA1537
Remarks:
With metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation

- Preincubation period: 1 h, 37 °C
- Exposure duration: 24 h (range finding test), 72 h (main test), 37 °C

NUMBER OF REPLICATIONS: triplicates, each in two experiments

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

OTHER:
- S9: added at 3% (main test) and 10% (range finding test and main test). In a rerun S9 of the main test S9 was added at 10% and 30%, respectively
Evaluation criteria:
The test substance is considered to show evidence of mutagenic activity, if treatment with a test material produces an increase in revertant colony numbers of at least twice the concurrent solvent controls, with some evidence of a positive dose-relationship, in two separate experiments, with any bacterial strain either in the presence or absence of S9 mix.
The test substance is considered to show no evidence of mutagenic activity, if treatment with a test material does not produce reproducible increases of at least 1.5 times the concurrent solvent controls, at any dose level with any bacterial strain.
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: at 5000µg/plate for all strains in the presence and absence of S9
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Results main experiment:

Experiment 1:

Dose Level (µg/plate) Liver S-9 mix TA 1535 TA1537 TA98 TA100
mean  SD mean  SD mean  SD mean  SD
5000 - 10 1.0 10 2.1 19 0.0 64 5.6
1500 - 11 1.7 9 0.6 23 3.0 90 1.0
500 - 14 2.5 10 2.1 20 1.5 96 2.5
150 - 12 2.0 9 1.5 22 3.1 102 2.6
50 - 11 1.0 9 0.6 21 2.0 101 3.1
0 - 17 1.0 10 0.6 24 5.0 104 4.5
Solvent - 15 1.0 10 1.0 26 4.0 112 2.5
ENNG (5 µg/plate) - 458 67.1            
9-AC (80 µg/plate) -     1108 51.9        
NF (1 µg/plate) -         85 9.0    
ENNG (3 µg/plate) -             349 44.3
                   
5000 3% 12 1.5 10 0.6 24 2.1 72 1.5
1500 3% 11 4.4 10 1.2 21 2.1 92 2.0
500 3% 14 1.0 9 1.0 19 1.0 99 6.1
150 3% 11 1.0 8 1.0 22 2.6 101 5.0
50 3% 13 1.0 10 1.2 20 0.6 100 2.0
0 3% 18 1.7 10 2.5 17 2.1 99 6.4
Solvent 3% 11 2.5 9 1.0 20 1.2 102 4.5
AA (0.5 µg/plate) 3%         161 20.0    
AA (1 µg/plate) 3%             295 21.6
AA (2 µg/plate) 3% 56 3.8 80 11.5        
                   
5000 10% 12 1.5 10 2.1 22 1.5 73 1.0
1500 10% 11 1.2 9 1.0 20 1.0 93 4.2
500 10% 14 2.5 12 2.1 20 3.8 97 7.6
150 10% 15 2.8 10 1.5 22 3.2 101 2.3
50 10% 11 2.5 10 1.5 21 1.4 108 2.0
0 10% 14 1.0 8 1.5 23 3.6 104 10.2
Solvent 10% 14 1.5 11 1.5 24 3.2 101 2.3
AA (0.5 µg/plate) 10%         143 11.0    
AA (1 µg/plate) 10%             353 10.1
AA (2 µg/plate) 10% 57 7.6 70 8.0        

Experiment 2:

Dose Level (µg/plate) Liver S-9 mix TA 1535 TA1537 TA98 TA100
mean  SD mean  SD mean  SD mean  SD
5000 - 10 3.2 10 1.0 19 3.1 82 6.1
1500 - 10 1.0 14 0.6 21 0.0 104 7.5
500 - 9 0.6 10 2.5 21 2.5 100 11.2
150 - 12 1.0 11 2.6 19 1.0 95 5.6
50 - 10 1.0 14 1.5 22 0.6 100 2.5
0 - 9 0.6 11 1.2 23 3.0 89 7.6
Solvent - 11 0.6 12 3.2 22 1.2 100 14.5
ENNG (5 µg/plate) - 439 48.8            
9-AC (80 µg/plate) -     x x        
NF (1 µg/plate) -         80 8.0    
ENNG (3 µg/plate) -             424 56.0
                   
5000 10% 11 2.0 17 3.0 19 2.1 92 4.0
1500 10% 14 2.0 16 0.6 22 2.5 100 12.7
500 10% 11 2.1 13 1.2 20 0.0 107 3.1
150 10% 10 0.6 15 1.5 22 1.0 108 4.0
50 10% 12 2.5 12 2.1 23 1.5 107 3.1
0 10% 10 2.0 16 4.2 20 2.0 100 10.4
Solvent 10% 10 1.7 15 2.5 22 2.5 105 10.5
AA (0.5 µg/plate) 10%         87 1.2    
AA (1 µg/plate) 10%             358 48.0
AA (2 µg/plate) 10% 71 18.8 68 12.8        
                   
5000 30% 9 0.6 17 1.2 19 3.5 101 7.6
1500 30% 9 0.6 16 2.1 23 2.0 106 5.3
500 30% 10 1.5 14 2.0 21 3.1 107 4.5
150 30% 11 0.6 14 2.3 24 0.0 110 3.0
50 30% 12 1.7 14 2.9 27 1.0 110 9.0
0 30% 12 1.0 16 0.6 21 2.1 118 8.6
Solvent 30% 12 2.1 17 2.5 21 1.2 110 10.7
AA (0.5 µg/plate) 30%         83 6.1    
AA (1 µg/plate) 30%             299 30.2
AA (2 µg/plate) 30% 74 9.3 61 8.5        

SD: standard deviation

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

9 -AC: 9 -aminoacridine

AA: 2 -aminoanthracene

NF: 2 -nitrofluorene

x: too many colonies to count accurately

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:
26 Feb - 2 May 1990
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
GLP compliance:
yes
Type of assay:
other: in vitro mammalian chromosome aberration test
Target gene:
Not applicable
Species / strain / cell type:
lymphocytes: cultured human lymphocytes
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640 with 20% Foetal Calf Serum (Gibco) and 2% phytohaemagglutinin
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254
Test concentrations with justification for top dose:
7.8, 15.6, 31.3, 62.5, 125, 250, 500, 1000, 2000 and 4000 µg/mL with and without metabolic activation
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Without S9: Ethylmethane sulphonate (Sigma London Chemical Company Ltd.) at 750 µg/mL in DMSO; with S9: Cyclophosphamide (Sigma London Chemical Company Ltd.) at 20 µg/mL in distilled water
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 24 h
- Fixation time (start of exposure up to fixation or harvest of cells): 22

SPINDLE INHIBITOR (cytogenetic assays): cholchicine, 0.25 µg/mL
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: in duplicates

NUMBER OF CELLS EVALUATED: 100 from each culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER
S9: two hours after dosing sulture medium with S9 was replaced with fresh medium
Statistics:
Fisher's test
Key result
Species / strain:
lymphocytes: cultured human lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The test compound was miscible with ethanol at a concentration of 470 mg/mL, the highest tested. However, the final concentrations above 4000 µg/mL were immiscible with aqueous tissue culture medium, when dosed at 1% v/v. 4000 µg/mL was regarded and referred to as the maximum achievable concentration in the test.

COMPARISON WITH HISTORICAL CONTROL DATA:
The aberration frequency of 1.5% at a concentration of 1000 µg/mL in the presence of metabolic activation fell well within the historical control ranges observed in the laboratory
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Results:

Metabolic activation

Test item

Concentration (µg/mL)

No. Of analyzed cells

Aberrant Cells (mean %)

Relative mitotic index (%)

with gaps

without gaps

-

Ethanol

10 µl/mL

100

0

0

100

-

Glycerol trioctanoate

7.8

-

-

-

121

-

15.6

-

-

-

100

-

31.3

-

-

-

125

-

62.5

-

-

-

104

-

125

-

-

-

121

-

250

-

-

-

129

-

500

100

0.5

0.5

118

-

1000

100

0.5

0.5

104

-

2000

100

1.0

1.0

150

-

4000

100

0.0

0.0

125

-

EMS

750

100

15.5***

14.5***

-

+

Ethanol

10 µl/mL

100

0.0

0.0

100

+

 

7.8

-

-

-

82

+

 

15.6

-

-

-

94

+

 

31.3

-

-

-

97

+

 

62.5

-

-

-

0

+

 

125

-

-

-

90

+

 

250

-

-

-

85

+

Glycerol trioctanoate

500

100

1.0

1.0

91

+

1000

100

1.5*

1.5*

94

+

2000

100

0.0

0.0

89

+

4000

100

0.0

0.0

61

+

CP

750

100

16.0***

16.0***

-

* p<0.05; *** p<0.001

EMS: Ethylmethane sulphonate

CP: Cyclophosphamide

mean%: total abberant cells *100 / total cells scored

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:
12 Jul - 26 Sep 2018
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)
Version / remarks:
adopted 29 Jul 2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Medicines and Healthcare Products Regulatory Agency, Department of Health, London, United Kingdom
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature, dark
Target gene:
HPRT locus
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Harlan CCR
- Suitability of cells: Cell type selected is listed as one of the recommended cell types in OECD guideline 476
- Methods for maintenance in cell culture: Freshly thawed cells from stock cultures were maintained in 225 cm2 culture flasks in minimal essential medium (MEM) and cultured at a humidified atmosphere of 5% CO2 and at 37 °C incubation temperature.
- Cell cycle length, doubling time or proliferation index: 12 - 16 h
- Modal number of chromosomes: 22

MEDIA USED
- Type and identity of media: Eagles Minimal Essential (MEM) 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffered, including pen/strep and 10% fetal bovine serum (FBS), 5% CO2
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically 'cleansed' against high spontaneous background: yes
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital/b-naphtho flavone
Test concentrations with justification for top dose:
Without metabolic activation: 0.25, 0.5, 1, 2, 4, 8, 16, 32 µg/mL (4h)
With metabolic activation: 0.25, 0.5, 1, 2, 4, 8, 16, 32 µg/mL (4h)
The selection of the concentrations used in the main experiments was based on data from the range-finding experiment. Precipitation was observed at and above 15.63 µg/mL during the 4 h treatment with and without metabolic activation. The maximum concentrations selected for the main mutagenicity experiment were therefore limited by the onset of the test item precipitate, in both, the asbsence and presence of metabolic activation.
Vehicle / solvent:
Acetone
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding: 1 x 10E7 cells per 225 cm2 flask

DURATION :
- Exposure duration: 4 h exposure with and without S9 mix
- Expression time (cells in growth medium): At the end of the treatment period the cells were plated for determination of the cloning efficiency and the mutation frequency and incubated for 6 or 7 days.
- Selection time: 6 - 7 days
- Fixation time (start of exposure up to fixation or harvest of cells): 12 - 14 days

SELECTION AGENT: 11 µg/mL 6-Thioguanine (6-TG)

STAIN: Giemsa

NUMBER OF REPLICATIONS: triplicates each in two independent experiments

DETERMINATION OF CYTOTOXICITY :
- Method: relative survival and cloning efficiecy
Evaluation criteria:
A test item is considered to be clearly positive if, in any of the experimental conditions examined:
- at least one of the test concentration exhibits a statistically significant increae compared with the concurrent vehicle control
- the increase is considered to be concentration-related
- the results are outside the range of the historical negative control data for the test item concentrations

A test item is considered to be clearly negative if, in all of the experimental conditions examined:
- none of the test concentrations exhibits a statistically significant increase compared with the concurrent vehicle control
- there is no concentration related increase
- the results for the test item concentration are within the range of the historical negative control data

In case the response is neither clearly negative nor clearly positive or in order to assist in establishing the biological relevance of a result, the data should be evaluated by expert judgement and/or further investigations.
Statistics:
The Student's t-test was applied to compare the mutation data of each individual concentration with the vehicle control.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
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 osmolality: The osmolality did not increase by more than 50 mOsm at the concentration levels investigated
- Precipitation: Preicipitation of the test item was observed at and above 15.63 µg/mL in both the presence and absence of metabolic activation

RANGE-FINDING/SCREENING STUDIES:
A concentration range of 0.49 to 125 µg/mL was used in a preliminary cytotoxicity test. At the end of the exposure period, precipitation of the test item was observed at and above 15.63 µg/mL in both the absence and presence of metabolic activation. There was no evidence of any marked concentration related-reductions in relative survival in either the presence or absence of metabolic activation.

HISTORICAL CONTROL DATA
- Positive historical control data: The mean number of mutants with metabolic activation is 334.40 ± 114.17 (range: 209 - 774, n=30) for 1 µg/mL DMBA and 484.97 ± 174.83 (range: 103 - 925, n=30) for 2 µg/mL DMBA. The mean number of mutants without metabolic activation is 261.30 ± 36.82 (range: 171 - 346, n=30) for 500 µg/mL EMS and 417.54 ± 63.80 (range: 291 - 574, n=28) for 750 µg/mL (see Table 3 under "Any other information results incl tables"). The results of the test fall within the historical control data range.
- Vehicle historical control data: The mean number of mutants with metabolic activation is 12.07 ± 4.44 (range: 5 - 23, n=30). The mean number of mutants without metabolic acativation is 12.47 ± 3.95 (range: 5 - 24, n=30) (see Table 4 under "Any other information results incl tables"). The results of the test fall within the historical control data range.


Table 1: Main mutation experiment, 4 hour exposure without metabolic activation (S9)

 

 

Day 0 Viability

Day 7 Viability

Day 7 Mutant

Dose (µg/mL)

 

Colonies/flask (200 cells plated/flask)

 

Adjusted CE

 

 

Colonies/flask (200 cells plated/flask)

 

 

Mean % Control

 

 

 

 

Group MFS 10-6

% CE

RS

Mean RS

% CE

% Control

Colonies/flask (2x105 cells plated/flask)

MF

MFS 10-6

SD

0

A

139

122

166

71.2

57.5

100

100

137

156

170

77.2

100

100

0

6

3

0

2

5

2

2

0

5

12.5

16.2

1.93

15

B

158

153

176

81.2

63.3

100

149

168

154

78.5

100

3

4

0

4

4

0

2

2

3

0

11

14

0.25

A

Not plated, surplus to requirements

 

Not plated, surplus to requirements

 

Not plated, surplus to requirements

 

 

B

Not plated, surplus to requirements

Not plated, surplus to requirements

Not plated, surplus to requirements

0.5

A

142

162

166

78.3

63.3

110

103

133

163

159

75.8

98.3

97

1

2

3

3

5

0

0

3

3

0

10

13.2

1.5

10

B

162

158

150

78.3

61.1

96.5

160

137

151

74.7

95.1

0

0

1

1

1

0

3

3

0

1

5

6.7

1

A

160

171

173

84

67.9

118

105

154

148

156

76.3

98.9

99

0

0

4

5

3

3

1

4

2

0

11

14.4

1.56

11

B

142

154

149

74.2

57.9

91.4

153

149

162

77.3

98.5

0

0

2

1

2

3

1

2

0

1

6

7.8

2

A

130

145

155

71.7

57.9

101

110

140

123

153

69.3

89.8

94

2

2

3

5

0

4

3

2

2

3

13

18.8

2.02

19

B

190

172

216

96.3

75.1

119

150

160

155

77.5

98.7

6

7

0

1

3

1

3

3

6

0

15

19.4

4

A

197

150

171

86.3

69.8

121

113

148

145

148

73.5

95.2

96

4

2

0

3

5

0

4

1

1

4

12

16.3

2.08

18

B

166

173

168

84.5

65.9

104

161

141

152

75.7

96.4

3

4

4

0

4

7

2

0

5

0

14.5

19.2

8

A

165

153

148

77.7

62.8

109

103

149

187

168

84

108.9

106

4

4

1

0

2

2

4

1

2

1

10.5

12.5

1.79

13

B

168

140

159

77.8

60.7

95.9

170

150

162

80.3

102.3

3

4

1

0

5

1

1

1

6

0

11

13.7

16 P

A

155

161

167

80.5

65.1

113

112

136

161

192

81.5

105.6

102

4

4

3

1

2

4

5

0

2

3

14

17.2

1.24

16

B

166

196

175

89.5

69.8

110

137

160

170

77.8

99.2

3

1

2

1

3

3

2

2

2

3

11

14.1

32 P

A

Not plated due to precipitate

 

Not plated due to precipitate

 

Not plated due to precipitate

 

 

B

Not plated due to precipitate

Not plated due to precipitate

Not plated due to precipitate

EMS 500

A

110

114

122

57.7

46.6

81

84

179

148

132

76.5

99.1

80

55

35

28

40

53

32

36

42

54

30

203

264.7

8.51

352

B

151

134

143

71.3

55.6

87.9

100

74

111

47.5

60.5

45

36

46

32

38

56

36

42

40

46

209

438.9

EMS 750

A

84

73

107

44

35.6

61.8

64

132

142

130

67.3

87.3

74

72

54

44

57

61

40

50

56

41

50

263

389.9

12.1

562

B

86

131

109

54.3

42.4

66.9

74

108

101

47.2

60.1

73

63

84

63

58

70

66

70

77

69

347

734.6

DMBA = Dimethyl benzanthracene
CE = Cloning efficiency
RS = Relative survival
MF = Mutant frequency
MFS = Mutant frequency per survivor
SD = Standard deviation


Table 2: Main mutation experiment, 4 hour exposure with metabolic activation (S9)

 

 

Day 0 Viability

Day 7 Viability

Day 7 Mutant

 

 

Colonies/flask (200 cells plated/flask)

 

 

 

 

Colonies/flask (200 cells plated/flask)

 

 

 

 

 

 

 

Group MFS 10-6

Dose (µg/mL)

% CE

Adjusted CE

RS

Mean RS

% CE

% Control

Mean % Control

Colonies/flask (2x105 cells plated/flask)

MF

MFS 10-6

SD

0

A

181

145

100

71

48.3

100

100

186

179

174

89.8

100

100

3

1

2

1

0

0

4

3

3

3

10

11.1

1.64

15

B

181

137

150

78

58.8

100

169

150

164

80.5

100

3

3

0

5

0

3

4

5

2

4

14.5

18

0.25

A

Not plated, surplus to requirements

 

Not plated, surplus to requirements

 

Not plated, surplus to requirements

 

 

B

Not plated, surplus to requirements

Not plated, surplus to requirements

Not plated, surplus to requirements

0.5

A

142

126

126

65.7

44.7

92.5

93

203

166

178

91.2

101.5

98

1

3

0

4

4

2

3

2

4

3

13

14.3

1.18

14

B

147

146

140

72.2

54.4

92.5

148

162

149

76.5

95

0

2

3

2

2

3

3

3

2

1

10.5

13.7

1

A

182

129

152

77.2

52.5

108.7

105

163

188

174

87.5

97.4

98

1

0

4

2

2

5

2

2

1

4

11.5

13.1

1.5

15

B

172

160

141

78.8

59.4

101.1

168

171

142

80.2

99.6

1

3

2

4

3

0

3

4

2

5

13.5

16.8

2

A

122

120

130

62

42.2

87.3

100

202

189

207

99.7

110.9

105

2

3

4

0

5

0

4

3

3

4

14

14

1.66

15

B

184

171

170

87.5

65.9

112.2

170

149

159

79.7

99

2

0

3

4

2

4

0

4

5

2

13

16.3

4

A

126

146

160

72

49

101.4

109

176

172

174

87

96.8

98

4

1

4

0

4

1

2

3

2

3

12

13.8

1.4

15

B

184

178

183

90.8

68.5

116.5

168

149

160

79.5

98.8

2

4

2

3

2

0

5

4

2

2

13

16.4

8

A

149

126

115

65

44.3

91.5

95

202

184

186

95.3

106.1

104

2

4

3

0

3

2

3

4

4

1

13

13.6

1.28

14

B

159

172

132

77.2

58.2

98.9

170

168

150

81.3

101

0

3

2

3

4

2

4

3

2

1

12

14.8

16

A

140

130

139

68.2

46.4

96

100

182

168

154

84

93.5

97

4

2

5

0

2

2

4

3

2

2

13

15.5

1.45

16

B

170

155

160

80.8

60.9

103.6

172

152

163

81.2

100.8

5

2

2

3

4

1

0

4

3

4

14

17.2

32

A

Not plated due to precipitate

 

Not plated due to precipitate

 

Not plated due to precipitate

 

 

B

Not plated due to precipitate

Not plated due to precipitate

Not plated due to precipitate

DMBA 1

A

52

60

69

30.2

20.5

42.5

74

189

180

186

92.5

103

100

66

76

61

63

63

61

48

58

53

72

311

335.7

13.43

441

B

192

169

137

83

62.6

106.4

147

157

160

77.3

96.1

90

76

83

91

86

84

77

91

89

78

423

546.3

DMBA 2

A

40

35

30

17.5

11.9

24.6

38

160

178

157

82.5

91.8

86

83

66

59

62

46

56

80

68

73

62

328

397

17.8

532

B

64

99

79

40.3

30.4

51.7

120

140

132

65.3

81.2

74

81

99

110

101

101

75

93

54

84

436

667.3

DMBA = Dimethyl benzanthracene
CE = Cloning efficiency
RS = Relative survival
MF = Mutant frequency
MFS = Mutant frequency per survivor
SD = Standard deviation

Table 3: Historical control ranges for vehicle control cultures

Vehicle Control Mutant Frequencies per survivor x 10-6
  4-Hour Without S9 4-Hour With S9
Minimum 5 5
Maximum 23 24
Mean 12.07 12.47
Standard Deviation 4.44 3.95
Number of Experiments 30 30

Table 4: Historical control ranges for positive control cultures

Positive Control Mutant Frequencies per survivor x 10-6

 

4-Hour Without S9 (EMS)

4-Hour Without S9 (EMS)

4-Hour With S9
 (DMBA)

4-Hour With S9
(DMBA)

500 µg/mL

750 µg/mL

1 µg/mL

2 µg/mL

Minimum

171

291

209

103

Maximum

346

574

774

925

Mean

261.3

417.54

334.4

484.97

Standard Deviation

36.82

63.8

114.17

174.83

Number of Experiments

30

28

30

30

DMBA = Dimethyl benzanthracene
EMS = Ethyl methane sulphonate

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

In vitro Gene mutation in bacteria

Gene mutation in bacteria by propane-1,2,3-triyl- 2-ethylhexanoate was analysed in an Ames test performed according to GLP and OECD guideline 471, with the deviation that only four bacteria strains were used (Huntingdon, 1990a). Bacteria strains S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 were treated with the test substance (diluted in DMSO) at concentrations of 5, 50, 500 and 5000 µg/plate in the dose-range finding test and 50, 150, 500, 1500, 5000 µg/plate in the main test, with and without metabolic activation. As no cytotoxicity and no increase in revertant colonies was noted in any strain and concentration tested, propane-1,2,3-triyl- 2-ethylhexanoate was considered to be not mutagenic in bacteria under the condition of this test.

 

In vitro cytogenicity in mammalian cells

In vitro cytogenicity was evaluated in an in vitro mammalian chromosome aberration test performed according to GLP and OECD guideline 473 (Huntingdon, 1990b). Cultured human lymphocytes were treated with diluted test substance (vehicle: ethanol) at concentration of 7.8, 15.6, 31.3, 62.5, 125, 250, 500, 1000, 2000 and 4000 µg/mL, with and without metabolic activation (S9 mix). Since no increase in the number of aberrant cells was noted at any concentration tested, propane-1,2,3-triyl- 2-ethylhexanoate was considered to be not clastogenic under the conditions of this study.

 

In vitro gene mutation in mammalian cells

The test item was analysed for its mutagenic potential in mammalian cells according to OECD guideline 476 and in compliance with GLP (Envigo, 2017). Gene mutation in the HPRT locus were investigated in Chinese Hamster Lung Fibroblasts (V79 cells) in the presence and absence of metabolic activation (S9-mix from rats treated with phenobarbital/b-naphtoflavone). A preliminary range-finding study revealed no concentration-related effects up to a concentration of 125 µg/mL with and without metabolic activation. However, precipitation of the test item was observed at and above 15.63 µg/mL. In two independent experiments, the cells were exposed to the test item at eight different concentrations (range 0.25 to 32 µg/mL) for four hours with and without S9-mix. Concurrent vehicle controls and positive control substances were included. Relative survival and mutation frequencies in the absence and presence of metabolic activation were investigated.

The vehicle control values were within the range of historical control data. The positive control substances (ethyl metahane sulphonate - S9-mix and dimethyl benzanthracene + S9-mix) markedly increased mutant frequencies, demonstrating the sensitivity of the test and the activity of the metabolic activation system. Treatment with the test item did not significantly induce the number of forward mutations at the HPRT locus of V79 cells, neither in the presence, nor in the absence of metabolic activation. Under the conditions of the study, propane-1,2,3-triyl 2-ethylhexanoate revealed no mutagenic potential in Chinese Hamster V79 cells in vitro.

 

Conclusion

In conclusion, assessment of the available experimental data on gene mutation in bacteria, gene mutation in mammalian cells and chromosome aberration in mammalian cells suggests that propane-1,2,3-triyl- 2-ethylhexanoate is neither mutagenic nor clastogenic in vitro.

Justification for classification or non-classification

The available data on gene mutation do not meet the criteria for classification according to Regulation (EC) No. 1272/2008, and are therefore conclusive but not sufficient for classification.