Diphenyl sulphone

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Diphenyl sulphone was non-mutagenic with/without S9 in the Ames reverse mutation test in any of the  five strains used  TA1535,TA1537,TA 1538, TA 98, and TA 100, with the highest concentration showing slight precipitation( OECD 471 ICI 1989). In addition Diphenyl sulphone was also non-mutagenic with/without S9 in the Escherichia Coli strain, in the Reverse Mutation assay (OECD 471, Proviron 2021) Secondly, diphenyl sulphone was non-mutagenic to L5178Y TK+/- cells (MLA) in either the presence or absence of S9-mix when tested to a concentration up to 150 µg/mL (OECD 490, Proviron 2021). 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(OECD 473, ICI 1994b).

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:

supporting 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

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

not applicable

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. Further the mutagenic  activity  of  Diphenyl sulphone was also  evaluated in a Escherichia Coli Reverse Mutation  Assay ( Proviron ,2021), in both the presence and absence of S9 .Diphenyl sulphone gave a negative response. 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 (Proviron , 2021), both in presence and absence of a rat liver derived auxiliary metabolic activation system (S9-mix). In a first  experiment the test item was tested  up to concentrations of  200 µg/ml in the absence  and presence of S-9 mix. The  incubation time  3 hours. No toxicity was  observed at this dose level in the  absence and  presence of  S-9 mix. The test  item precipitated in the culture medium at this  dose level. In the  second experiment, the test item was tested up to concentrations of  150 µg/ml in the  absence of S9 mix. The incubation time was 24 hours.  No toxicity was  observed at this dose level in the  absence of  S-9 mix. The test  item  precipitated in the culture medium at this dose level.                                                                                                                           In the absence of S9-mix, the test item did not induce a biologically relevant increase in the mutant frequency in the first experiment. This result was confirmed in a repeat experiment with modification in the duration of treatment.                                                                                                                       In the presence of S9-mix, the test item did not induce a biologically relevant increase in the mutant frequency. In conclusion, Diphenyl  sulphone  is not mutagenic in the  mouse lymphoma  L5178Y test system.(OECD 490, Proviron 2021)

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 (OECD 473,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).