Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
13 Jul - 19 Dec 2012
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP - Guideline study. According to the ECHA guidance document "Practical guide 6: How to report read-across and categories (Dec 2012)", the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
adopted in 2008
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
The department of health of the government of the United Kingdom
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): Heptanoic acid ester of neopentlyglycol
- Physical state: clear colourless liquid
- Analytical purity: > 99%
- Lot/batch No.: 595859
- Expiration date of the lot/batch: 08 Oct 2013
- Storage condition of test material: room temperature in the dark

Method

Target gene:
TK locus
Species / strain
Species / strain:
mouse lymphoma L5178Y cells
Details on mammalian cell lines (if applicable):
- Type and identity of media: RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/mL) and 10% donor horse serum (giving R10 media. RPMI 1640 with 20% donor horse serum (R20) and without serum (R0) were used during the course of the study
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbonel/beta-naphthoflavone
Test concentrations with justification for top dose:
Experiment I
Without S9 mix: 1.6, 3.2, 6.41, 12.81, 25.63, 51.25, 76.88, and 102.5 µg/mL (4 h)
With S9 mix: 1.6, 3.2, 6.41, 12.81, 25.63, 51.25, 76.88, and 102.5 µg/mL (4 h)
Experiment II
Without S9 mix: 1.6, 3.2, 6.41, 12.81, 25.63, 51.25, 76.88, and 102.5 µg/mL (24 h)
With S9 mix: 3.2, 6.41, 12.81, 25.63, 51.25, 76.88, 102.5, and 205.0 µg/mL (4h)
Vehicle:
- Vehicle(s)/solvent(s) used: acetone
Controls
Negative controls:
no
Solvent controls:
yes
Remarks:
Acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Remarks:
cyclophosphamide, 2 µg/mL, +S9; ethylmethanesulphonate 400 µg/mL in Exp1 and 150 µg/mL in Exp 2, –S9.
Details on test system and conditions:
METHOD OF APPLICATION: in medium

DURATION
1st experiment: 4 h exposure with and without S9 mix.
2nd experiment: 4 h exposure with S9 mix and 24 h with and without S9 mix.
- Expression time (cells in growth medium): 2 days after the end of the reatment, cells were plated for determination of the mutation frequency in 96-well microtitre plates containing TFT selective medium. Cells were also diluted to 10 cells/mL and plated (2 cells/well) for viability (%V) in non-selective medium. The microtitre plates were incubated for 10 to 14 days.
- Selection time (if incubation with a selection agent): 10-14 days
- Fixation time (start of exposure up to fixation or harvest of cells): 12-14 days
SELECTION AGENT (mutation assays): 4µg/mL trifluorothymidine (TFT)
NUMBER OF REPLICATIONS: duplicates each in two independent experiments
DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Evaluation criteria:
See any other information on materials and methods incl. tables
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).

Results and discussion

Test results
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
see additional information on cytotoxicity
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'. Remarks: mouse lymphoma L5178Y cells
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 in the solubility test.
RANGE-FINDING/SCREENING STUDIES:
A preliminary toxicity test was performed on cell cultures at 5 x 10E5 cell/mL, using a 4h exposure-period with both and without S9, and at 1.5 x 10E5 cells/mL using a 24h exposure-period without S9. The dose range used in the preliminary toxicity test was 6.41 to 1640 µg/mL for all three of the exposure groups.
Results from the preliminary toxicity test were used to set the test item dose levels for the mutagenicity experiments. Maximum dose levels were selected using the following criteria:
1) Maximum recommended dose level, 5000 µg/mL or 10 mM.
2) The presence of excessive precipitate where no test item-induced toxicity was observed.
3) Test item-induced toxicity, where the maximum dose level used should produce 10 to 20% survival (the maximum level of toxicity required).
COMPARISON WITH HISTORICAL CONTROL DATA:
The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for L5178Y cell line at the TK +/- locus.
ADDITIONAL INFORMATION ON CYTOTOXICITY:
The daily cell counts were used to obtain a Relative Suspension Growth (%RSG) values that gives an indication of post treatment toxicity during the expression period as a comparison to the vehicle control, and when combined with the Viability (%V) data a Relative Total Growth (RTG) value.
In the Experiment 1 there was evidence of marked dose-related toxicity following exposure to the test item in the absence of metabolic activation, as indicated by the RTG and % RSG values. In the presence of metabolic activation there was no evidence of the test item induced toxicity which varied from the toxicity observed in the preliminary toxicity test.
There was no evidence of a marked reduction in viability (%V) in the absence or presence of metabolic activation therefore indicating that residual toxicity had not occurred. Near to optimum levels of toxicity were achieved in the absence of metabolic activation only. The test item proved to be non-toxic in the presence of metabolic activation, therefore optimum toxicity could not be achieved. Acceptable levels of toxicity were seen with both positive control substances.
In Experiment 2 there was evidence of marked toxicity following exposure to the test item in the absence of metabolic activation, as indicated by RTG and %RSG values. In the presence of metabolic activation there was evidence of slight test item induced toxicity at the top dose level (205 µg/mL). Based on the toxicity results from the preliminary toxicity testing and Experiment 1 it was considered that the test item had been tested up to the limit of its exposure to cells and therefore, adequately tested. There was evidence of slight reductions in viability (%V) in the absence of metabolic activation, therefore indicating that residual toxicity had occurred. Based on the %RSG and RTG values observed, it was considered that optimum levels of toxicity had been achieved in the absence of metabolic activation only. The excessive toxicity observed at and above 76.9 µg/mL in the absence of metabolic activation resulted in these dose levels not being plated for viability of 5-TFT resistance. Acceptable levels of toxicity were seen with both positive control substances.
OTHER:
Small and large colonies were differentiated, as small colonies are capable to indicate chromosomal mutations.

Any other information on results incl. tables

Table 1: Experiment I - 4 h exposure - Without Metabolic Activation

Concentration
[µg/mL]

Relative Total Growth [%]

Relative Total Growth

Mutation factor

0 (acetone)

100

1.00

181.34

1.6

96

--

--

3.2

97

--

--

6.41

91

1.03

207.35

12.81

88

0.98

175.16

25.63

74

0.83

189.77

51.25

43

0.42

220-94

76.88

35

0.40

194.97

102.5

28

0.47

139.22

EMS, 400

69

0.87

705.33

EMS     Ethyl methane sulphonate

Table 2: Experiment I - 4 h exposure - With Metabolic Activation

Concentration
[µg/mL]

Relative Total Growth [%]

Relative Total Growth

Mutation factor

0 (acetone)

100

1.00

177.48

1.6

105

--

--

3.2

107

--

--

6.41

101

0.86

204.41

12.81

106

1.02

164.09

25.63

99

0.91

190.08

51.25

96

0.93

183.37

76.88

103

1.05

166.33

102.5

96

0.94

191.22

CP, 2

63

0.36

1320.55

CP         cyclophosphamide

Table 3: Experiment II - 24 h Exposure - Without Metabolic Activation

Concentration
[µg/mL]

Relative Total Growth [%]

Relative Total Growth

Mutation factor

0 (acetone)

100

1.00

173.24

1.6

91

0.89

159.79

3.2

87

0.83

165.80

6.41

80

0.89

142.67

12.81

76

0.89

127.24

25.63

49

0.63

158.76

51.25

15

0.27

162.70

76.88

3

 

 

102.5

2

 

 

EMS, 150

65

0.54

1563.99

EMS     Ethyl methane sulphonate

Table 4: Experiment II - 4 h Exposure – With Metabolic Activation

Concentration
[µg/mL]

Relative Total Growth [%]

Relative Total Growth

Mutation factor

0 (acetone)

100

1.00

144.47

3.2

86

 

 

6.4

83

 

 

12.8

86

0.85

143.05

25.6

86

0.81

172.98

51.3

82

0.93

127.62

76.9

87

0.95

144.64

102.5

83

0.85

149.46

205

78

0.89

132.37

CP, 2

62

0.45

1059.59

CP         cyclophosphamide

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative