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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
Diphenyl sulphone was non-mutagenic up to 5000µg/plate with/without S9 in the Ames reverse mutation test, with the highest concentration showing slight precipitation. Secondly, diphenyl sulphone was non-mutagenic to L5178Y TK+/- cells in either the presence or absence of S9-mix when tested to a concentration up to 125 µg/mL just in excess of the limit of solubility. Finally, it was not clastogenic to cultured human lymphocytes treated in vitro for 68 up to 92 hours with a range of concentrations between 1 and 500 µg/mL both with/without S9, with the highest concentration based on the limit of solubility of the test material.
Link to relevant study records
Reference
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
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
Target gene:
Thymidine kinase TK +/- gene
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.
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.
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'.

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

 

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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Diphenyl sulphone was evaluated in a key Salmonella mutagenicity assay according to OECD No. 471 (ICI, 1989). In both the presence and absence of S9 the compound did not induce any significant reproducible increases in the observed numbers of revertant colonies in any of the five tester strains used (TA1535, TA1537, TA1538, TA98 and TA100). Under the conditions of this assay, diphenyl sulphone therefore gave an unequivocal negative, ie non-mutagenic, response when tested to a maximum dose of 5000µg/plate, at which concentration significant precipitation of the compound was observed on the test plates. In addition, OECD QSAR toolbox (version 2012) was applied for the 2 strains that can detect cross-linking mutagens. The results were negative, and confirm absence of mutagenicity potential of diphenyl sulphone.

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 (ICI, 1994a), 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.

An evaluation of the clastogenic potential of diphenyl sulphone was made in human lymphocytes from two donors (one male and one female), treated in vitro for 68 up to 92 hours with a range of concentrations between 1 and 500 µg/mL of test material both in the presence and absence of S9 (ICI, 1994b). The highest concentration of diphenyl sulphone selected for chromosomal aberration analysis was based on the limit of solubility of the test material, compared to the solvent control cultures, and some cytotoxic effects on the chromosomal structure were observed in cultures from both donors treated with diphenyl sulphone at 500µg/mL.No statistically or biologically significant increases in the percentage of aberrant cells, compared to the solvent control values, were recorded at the 68 and 92 hour sampling time, in cultures from either donor treated in the presence or absence of S9-mix.


Justification for selection of genetic toxicity endpoint
Although 1 endpoints was selected above, all the 3 endpoints records are key studies for the different genetic toxicity endpoints: bacterial reverse mutation, chromosomal aberration and mammalian gene mutation.

Justification for classification or non-classification

Since diphenyl sulphone was negative for genotoxicity in various assays, classification for genotoxicity is not warranted according to the EC criteria for classification and labelling requirements for dangerous substances and preparations (Guidelines in Commission Directive 93/21/EEC) and CLP regulation (EC No. 1272/2008 of 16 December 2008).