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Toxicological information

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:
experimental study
Adequacy of study:
key study
Study period:
1994
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Meets scientific and regulatory standards, but acceptable restrictions (non-GLP test article certification, stability of test article not analysed and no certification of purity and stability

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1994
Report Date:
1994

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
yes
Principles of method if other than guideline:
Minor modifications
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
solid: crystalline
Details on test material:
- Name of test material (as cited in study report): diphenyl sulphone
- Substance type: monoconstituent
- Physical state: white crystalline solid
- Analytical purity: stated purity 99.99%
- Impurities (identity and concentrations): see confidential details
- Composition of test material, percentage of components: 99,99% pure
- Isomers composition: not applicable
- Purity test date: see confidential details
- Lot/batch No.: see confidential details
- Expiration date of the lot/batch: see confidential details
- Stability under test conditions: stable under normal storage and test conditions according the Sponsor
- Storage condition of test material: at ambient temperature in the dark until required
- Other: not applicable

Method

Target gene:
Thymidine kinase TK +/- gene
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
-Type and identity of media: RPMI 1640 with Hepes (GIBCO) supplemented with 4mM L-glutamine (GIBCO), 200 units/mL of penicillin and streptomycin (GIBCO) and 10% horse serum (GIBCO). The serum concentration was dropped to 5% during treatment and raises to 20% whenever the cells were dispensed into microwells.
-Properly maintained: yes
-Periodically checked for Mycoplasma contamination: yes (by ELISA)
-Periodically checked for karyotype stability: no data
-Periodically”cleansed” against high spontaneous background: no data
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
Dose range finding experiment: 125; 63; 31; 16 µg/mL
Experiment 1, +S9 and –S9: 125; 63; 31; 16 µg/mL
Experiment 2, +S9 and –S9: 125; 63; 31; 16 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: the test material was found to be soluble in DMSO at all concentrations.
Controls
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Remarks:
-S9:EMS (ethyl methanesulphonate 750 µg/mL); +S9: NDMA( N-nitrosodimethylamine 600 µg/mL)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium (microwell plates)
DURATION
- Preincubation period: 1 hour
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 72 hours
- Selection time (if incubation with a selection agent): 10-12 days

SELECTION AGENT (mutation assays): TFT ( trifluorothymidine)

NUMBER OF REPLICATIONS: duplicate

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth; other: Viability scored by cell growth; mutation scored by colony count
Evaluation criteria:
Test data from individual experiments were considered valid if:
(a) the spontaneous mutant frequencies in the presence and absence of S9-mix fell within an acceptable range
(b) the positive controls induced unequivocal positive responses.

A positive response in a valid individual experiment is recorded when statistically significant, dose related increases in mutant frequency are observed across a range of toxicity. Such increases in mutant frequency are considered not to be indicative of a positive response if the increases occur only at doses eliciting excessive toxicity, ie <10% survival. These increases must also be associated with increases in the numbers of mutants over those observed with the concurrent solvent control.

A negative result in a valid experiment is obtained when there is no significant dose related increase in mutant frequency compared to the solvent control.

A positive response in an individual experiment must be reproducible for the test sample to be considered positive (ie mutagenic) in this assay.
Statistics:
A statistical analysis is applied at the discretion of the Study Director in consultation with the Study Statistician. In this study, the data were examined by the Study Statistician and Study Director who considered that statistical analyses were not necessary.

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:
The highest concentration was based on solubility of the test sample in culture medium, with 125 µg/mL being just in excess of the limit of solubility in the dose range finding study.
Remarks on result:
other: strain/cell type: L5178Y cells TK +/-
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1. Summary of Data for Experiment 1

WITHOUT S9

WITH S9

Conc

(µg/mL)

Mean % Survival

Mean Mutant Frequency (x10-4)

Conc

(µg/mL)

Mean % Survival

Mean Mutant Frequency (x10-4)

DIPHENYL SULPHONE

DIPHENYL SULPHONE

125

116

2.8

125

125

1.4

63

120

1.9

63

149

1.4

31

109

2.5

31

124

1.4

16

119

2.2

16

118

1.4

SOLVENT CONTROL

SOLVENT CONTROL

DMSO (10µL/mL)

100

2.3

DMSO (10µL/mL)

100

1.9

POSITIVE CONTROL

POSITIVE CONTROL

EMS (750 µg/mL)

53

9.8

NDMA (600 µg/mL)

100

11.4

 

Table 2. Summary of Data for Experiment 2

WITHOUT S9

WITH S9

Conc

(µg/mL)

Mean % Survival

Mean Mutant Frequency (x10-4)

Conc

(µg/mL)

Mean % Survival

Mean Mutant Frequency (x10-4)

DIPHENYL SULPHONE

DIPHENYL SULPHONE

125

93

1.4

125

99

0.9

63

95

1.6

63

107

1.1

31

104

1.4

31

124

1.8

16

105

1.1

16

91

1.4

SOLVENT CONTROL

SOLVENT CONTROL

DMSO (10µL/mL)

100

1.4

DMSO (10µL/mL)

100

1.6

POSITIVE CONTROL

POSITIVE CONTROL

EMS  

(750 µg/mL)

30

21.6

NDMA  

(600 µg/mL)

73

19.9

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Under the conditions of this assay, diphenyl sulphone is non-mutagenic to L5178Y TK+/- cells as determined by selection in trifluorothymidine in both the presence and absence of an auxiliary metabolic activation system (S9-mix), when tested up to a concentration just in excess of the limit of solubility in the test medium.
Executive summary:

To assess the mutagenic potential of diphenyl sulphone to mammalian cells, L5178Y TK+/- mouse lymphoma cells were treated in vitro with various concentrations of test sample, both in presence and absence of a rat liver derived auxiliary metabolic activation system (S9-mix). Mutant frequencies were assessed by cell growth in the presence of trifluorothymidine (TFT) after a 72 hour expression time.

Two independent experiments were conducted using a range of concentrations of diphenyl sulphone in the presence and absence of S9-mix. The highest concentration of diphenyl sulphone tested in these two experiments was based on the solubility of the test sample in the culture medium, treatment with diphenyl sulphone at 125µg/mL being just in excess of the limit of solubility. No significant increases in the mutant frequency, compared to the solvent control values, were observed in either experiment in either the presence or absence of S9-mix.

It is therefore concluded that, under the conditions of this assay, diphenyl sulphone is non-mutagenic to L5178Y TK+/- cells in either the presence or absence of S9-mix when tested to a concentration just in excess of the limit of solubility.