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Genetic toxicity: in vitro

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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:
April 15, 1992 - June 25, 1992
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant. According to guidelines.
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1992
Report date:
1992

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Principles of method if other than guideline:
Not relevant
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay

Test material

Constituent 1
Reference substance name:
ESBO
IUPAC Name:
ESBO
Constituent 2
Reference substance name:
Epoxidised Soybean Oil
IUPAC Name:
Epoxidised Soybean Oil
Constituent 3
Reference substance name:
Soybean oil, epoxidized
EC Number:
232-391-0
EC Name:
Soybean oil, epoxidized
IUPAC Name:
232-391-0
Constituent 4
Reference substance name:
8013-07-8
Cas Number:
8013-07-8
IUPAC Name:
8013-07-8
Details on test material:
- Name of test material (as cited in study report): Epoxidised Soybean Oil
- Physical state: Yellow liquid
- Lot/batch No.: 08380306
- Storage condition of test material: Room temperature in the dark

Method

Target gene:
tk gene locus
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
L5178Y TK +/- mouse lymphoma cells were obtained from the American Type Culture Collection. They were stored as frozen stocks in liquid nitrogen. Each batch of cells was purged of TK mutants, checked for spontaneous mutant frequency and that they were mycoplasma free. For each experiment the vials were thawed rapidly, the cells were diluted in RPMI 10 and incubated in a humidified atmosphere of 5 % v/v CO2 in air. When the cells were growing well, subcultures were established in an appropriate number of flasks.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S-9 mammalian liver post-mitochondrial fraction
Test concentrations with justification for top dose:
The concentrations were selected using a cytotoxicity range-finder. See Table 1 below.
Vehicle / solvent:
Acetone was used as a solvent. The test item was added and the culture was then vortexed for approx. 10 seconds to obtain a good emulsion. A top dose of 5000 ug/ml was achievable using an emulsion.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Comprised of solvent, acetone, diluted 100-fold in the treatment medium.
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
Migrated to IUCLID6: Please see Below
Details on test system and experimental conditions:
METHOD OF APPLICATION:

Preparations of cofactor solutions with and without S-9

Quantity (ml)
With S-9 Without S-9
Glucose-6-phosphate (180 mg/ml) 1.0 -
NADP (25 mg/ml) 1.0 -
150 mM KCL 1.0 5.0
Rat Liver S-9 2.0 -

The above were used at the rate of 1.0 ml per 19 ml of cell culture containing the test chemical (to achieve the required final concentration in a total of 20 ml).


Three types of RPMI 1640 medium were prepared as follows:
Final Concentration in:
RPMI A RPMI 10 RPMI 20
Horse Serum (heat inactivated) 0 % v/v 10 % v/v 20 % v/v
Gentamycin 100 ug/ml 100 ug/ml 100 ug/ml
Fungizone 2.5 ug/ml 2.5 ug/ml 2.5 ug/ml
Pluronic 0.5 ug/ml 0.5 ug/ml -


DURATION
- Exposure duration: 9- 15 days
- Expression time (cells in growth medium): 2 days

NUMBER OF CELLS EVALUATED: 1 x E7 cells per flask

DETERMINATION OF CYTOTOXICITY
Following adjustment of the cultures to 2 x E5 cells/ml after treatment, samples from these were diluted to 8 cells/ml as seen in table 3. using a 8-channel pipette, 0.2 ml of concentration C of each culture was placed into each well of two 96-well microtitre plates (192 wells, at an average of 1.6 cells per wall). The plates were incubated at 37 C in a humidified incubator gassed with 5 % v/v CO2 in air until scorable (9-15 days). Wells containing viable clones were identified by eye using background illumination and counted.

Evaluation criteria:
At the end of the expression period the cell densities in the selected cultures were adjusted to 1 x E4/ml. TFT (300 ug/ml) was diluted 100-fold into these suspensions to give a final concentration of 3 ug/ml. Using a 8-channel pipette, 0.2 ml of each suspension was placed into each well of four 96-well microtitre plates (384 wells at 2 x E3 cells per well). Plates were incubated until scorable (13 days) and wells containing clones were identified as above and counted. In addition, the number of wells containing large colonies and the number containing small colonies were scored for the negative and positive controls and for doses of test chemical showing a significant increase in mutant frequency over the negative control.
Statistics:
All calculations were performed on a microcomputer.

Determination of survival or viability: Poison distribution, plating efficiency, % relative survival
Determination of mutant frequency: Mutant frequency, plating efficiency,
Statistical significance of mutant frequencies.

Results and discussion

Test results
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: Please see table 4 below
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 4: raw plate counts and % relative survival for Epoxidised Soybean Oil in the cytotoxicity range-finder.

Treatment ug/mL)

In the absence of S-9

In the presence of S-9

Survival (1) at Day 0*

% Relative survival

Survival (1) at day 0*

% relative survival

0

18

100.0

23

100.0

78.125

21

129.2

21

84.2

156.25

16

83.8

17

59.7

312.5

18

100.0

19

71.0

625

15

76.5

15

49.9

1250

15

76.5

15

49.9

2500

16

83.8

18

65.2

5000

19

109.0

23

100.0

(1) 1.6 cells/well plated

* 32 wells scored

Table 5: Summary of Results Experiment 1

Treatment (ug/ml)

Absence of S-9

Treatment (ug/mL)

Presence of S-9

% RS

Mutant Frequency

% RS

Mutant Frequency

0

100.0

312.57

0

100.0

401.83

312.5

95.8

389.82 NS

312.5

97.9

484.86 NS

625

86.6

326.20 NS

625

87.9

563.19 NS

1250

79.9

439.82 NS

1250

86.0

518.57 NS

2500

131.9

520.41 *

2500

152.9

426.40 NS

5000

143.9

467.96 *

5000

178.7

433.74 NS

Linear Trend

**

Linear Trend

NS

NQO

BP

 

 

0.05

83.5

1676.58

2

55.2

1794.55

0.1

45.3

2078.73

3

39.4

2122.84

 

Table 6: Summary of Results Experiment 2

Treatment (ug/ml)

Absence of S-9

Treatment (ug/mL)

Presence of S-9

% RS

Mutant Frequency

% RS

Mutant Frequency

0

100.0

410.98

0

100.0

304.98

312.5

112.9

334.82 NS

312.5

91.7

331.46 NS

625

87.7

466.23 NS

625

82.6

331.03 NS

1250

71.4

404.74 NS

1250

87.7

313.73 NS

2500

84.5

461.15 NS

2500

84.5

283.11 NS

5000 $$

100.7

 

5000

83.2

311.46 NS

Linear Trend

NS

Linear Trend

NS

NQO

BP

 

 

0.05

67.5

1136.27

2

59.5

883.78

0.1

37.7

1143.11

3

32.1

1443.04

 

NS Not significant

$$ Treatment excluded due to excessive heterogeneity

*, **, *** Significant at 5 %, 1% and 0.1 % level respectively

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

No evidence of mutagenicity was seen under the conditions of this assay.
Executive summary:

Epoxidised Soybean Oil (ESBO) was assayed for its ability to induce mutation at the tk locus (5-trifluorothymidine resistance) in mouse lymphoma cells using a fluctuation protocol. The study consisted of a cytotoxicity range-finder followed by 2 independent experiments, each conducted in the absence and presence of metabolic activation by an Aroclor 1254 induced rat liver post-mitochondrial fraction (S-9).

Following a wide range of treatments in the range-finder experiment, separated by 2-fold intervals and ranging from 78.125 to 5000µg/ml, cells survived all doses of ESBO yielding 109.0 % relative survival in the absence and 100.0 % relative survival in the presence of S-9 at the top dose.

Accordingly, 5 doses were chosen for the first experiment, separated by 2-fold intervals and ranging from 312.5 to 5000µg/ml. All doses were plated for viability and 5-trifluorothymidine resistance 2 days after treatment. The top doses plated yielded 143.9 % and 178.7 % relative survival in the absence and presence of S-9. In the second experiment the same dose range was selected. The top dose plated in this experiment was again 5000µg/ml in the absence and presence of S-9, which yielded 100.7 % and 83.2 % relative survival respectively.

Negative (solvent) and positive control treatments were included in each experiment in the absence and presence of S-9. Mutant frequencies in negative control cultures fell within normal ranges, and statistically significant increases in mutation were induced by the positive control chemicals 4-nitroquinoline 1-oxide (without S-9) and benzo(a)pyrene (with S-9). Therefore the study was accepted as valid.

In the absence of S-9, reproducible statistically significant and dose-related increases in mutant frequency were not observed in the 2 experiments over the dose range 312.5 to 2500µg/ml. At 5000µg/ml, a positive point was obtained in Experiment 1 and due to heterogeneity in the data this dose was excluded from analysis in Experiment 2. However, if each of the replicate cultures at 5000µg/ml in Experiment 2 are considered in turn, neither yields a statistically significant increase in mutant frequency. This, combined with the fact that there were no absolute increases in mutant numbers in Experiment 1 at 5000µg/ml and that carry over of the test compound was a problem at this dose, suggests that the increased mutant frequency seen in experiment 1 was not the result of chemically induced mutation.

In the presence of S-9, no statistically significant increases in mutant frequency were observed at any dose level tested in Experiment 1 or 2.

It is concluded that, under the conditions employed in this study, ESBO failed to demonstrate the ability to induce mutation at the tk locus of L5178Y mouse lymphoma cells in the absence and presence of S-9. Therefore, ESBO is not considered to be mutagenic.