Docusate sodium

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to internationally accepted test guidelines and is considered relevant, adequate and reliable.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1993

Report date:

1993

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

minor deviation in treatment volume

Principles of method if other than guideline:

The use of acetone as a solvent for this study necessitated a reduction in the treatment volume from 0.1 mL to 0.05 mL ( to avoid toxic effects of the solvent). A corresponding reduction was therefore made for positive control treatments. In order to achieve the same final concentration of positive control per plate, the stock positive control solution concentrations specified in the protocol were doubled.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Docusate sodium

EC Number:

209-406-4

EC Name:

Docusate sodium

Cas Number:

577-11-7

Molecular formula:

C20H38O7S.Na

IUPAC Name:

sodium 1,4-bis[(2-ethylhexyl)oxy]-1,4-dioxobutane-2-sulfonate

Details on test material:

- Name of test material (as cited in study report): Sodium dioctyl sulphosuccinate , Aerosol OT-100
- Physical state: White, waxy solid
- Analytical purity: >97%
- Lot/batch No.: HJ2601
- Storage condition of test material: in the dark at room temperature

Method

Target gene:

histidine

Species / strain

Species / strain / cell type:

S. typhimurium, other: TA98,TA100, TA1535, TA1537, TA102

Additional strain / cell type characteristics:

other: histidine -requiring

Metabolic activation:

with and without

Metabolic activation system:

S-9 (mammalian liver post-mitochondrial fraction) used for metabollic activation was prepared from male Sprague-Dawley rats induced with Aroclor 1254 and obtained from Molecular Toxicology Inc., Annapolis, Maryland, USA.

Test concentrations with justification for top dose:

Toxicity Range-finder Experiment: 8, 40, 200, 1000, 5000 µg/plate
Mutation Experiment 1 (-S9): 1.6, 8.0, 40, 200, 1000 µg/plate
Mutation Experiment 1 (+S9): 4, 20, 100, 500, 2500 µg/plate
Mutation Experiment 2 (-S9): 62.5, 125, 250, 500, 1000 µg/plate
Mutation Experiment 2 (+S9): 156.25, 312.50, 625.00, 1250.0, 2500.0 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: The use of acetone as a solvent for this study
necessitated a reduction in the treatment volume from 0.1 ml to 0.05 ml (to avoid toxic effects of
the solvent). A corresponding reduction was therefore made for positive control treatments. In
order to achieve the same final concentration of positive control per plate, the stock positive control
solution concentrations specified in the protocol were doubled.

Controls

Untreated negative controls:

yes

Remarks:

treatments with the solvent: acetone

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-nitrofluorene (2NF), Sodium azide (NaN3), 9-aminoacridine (AAC), Glutaraldehyde (GLU), 2-aminoanthracene (AAN)

Remarks:

With the exception of NAN3 and GLU, which were prepared in water, all stock solutions were prepared in sterile anhydrous analytical grade dimethyl sulphoxide (DMSO), and stored in aliquots at 0-5°C in the dark.

Details on test system and experimental conditions:

Experiment 1: plate incorporation
Experiment 2 (+S-9): pre-incubation than plate incorporation
Experiment 2 (-S-9): plate incorparation

DURATION
- Pre-incubation period: 1 hour at 37°C (in Experiment 2 and +S-9)
- Exposure duration:
Incubation time in Toxicity range-finder Experiment= 3 days
Incubation time in Mutagenicity Experiment 1: 3days
Experiment 2+S-9 has a pre-incubation step of 1 hour
Incubation time in Mutagenicity Experiment 2= 3 days
- Expression time (cells in growth medium):
- Fixation time (start of exposure up to fixation or harvest of cells):

NUMBER OF REPLICATIONS:
Mutation Experiment 1, 2(-S-9): 5+3+3+3+3+3+3 (TA98, TA100, TA 1535, TA1537, TA102)
Mutation Experiment 1, 2 (+S-9): 5+3+3+3+3+3+3 (TA98, TA100)
Mutation Experiment 1, 2 (+S-9): 5+3+3+3+3+3 (TA 1535, TA1537, TA102) – no positive controls

NUMBER OF CELLS EVALUATED:

DETERMINATION OF CYTOTOXICITY
- Method: colony counting

OTHER EXAMINATIONS:
- Other: m-statistic, Dunnett’s test, linear regression analysis

OTHER:

Evaluation criteria:

A test compound was considered to be mutagenic if:
i) the assay was valid ( see 2.4.2.)
ii) Dunnett’s test gave significant response (p≤ 0.01) , and the data set showed a significant dose-correlation
iii) The positive responses described in (ii) were reproducible.

Statistics:

For evaluation of test chemical and positive control data there are many statistical methods in use, and several are acceptable (7,8). The m-statistic was first calculated to check that the data were Poisson-distributed (8), and then Dunnett’s test was used to compare the counts of each dose with the control. The presence or otherwise of a dose-response was then examined using linear regression analysis (8).

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 98

Remarks:

Experiment I

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 98

Remarks:

Experiment I

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 100

Remarks:

Exp I

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 1535

Remarks:

Experiment I

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 1537

Remarks:

experiment I

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 6

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 7

Species / strain:

S. typhimurium TA 102

Remarks:

Experiment I

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Test results 8

Species / strain:

S. typhimurium TA 98

Remarks:

Experiment II

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 9

Species / strain:

S. typhimurium TA 100

Remarks:

Experiment II

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 10

Species / strain:

S. typhimurium TA 1535

Remarks:

Experiment II

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 11

Species / strain:

S. typhimurium TA 1537

Remarks:

Experiment II

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 12

Species / strain:

S. typhimurium TA 102

Remarks:

Experiment II

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: some thinning of the background lawn at 1000 µg/plate
- Other confounding effects: no

RANGE-FINDING/SCREENING STUDIES:
An initial toxicity range-finder experiment was carried out in
TA100 only, using final concentrations of sodium dioctyl sulphosuccinate at 8, 40, 200, 1000 and
5000 µg/plate plus a solvent and positive control. In the absence S-9, complete killing of the test
bacteria was observed at the highest concentration of 5000µg/plate. In addition, a thinning of the
background lawn at the second highest concentration (1000µg/plate) also indicated toxicity. In the
presence of S-9, toxicity was only observed at the highest dose. In view of these results , maximum
test concentrations of 1000 and 2500µg/plate were chosen for Experiment 1 treatments, in absence
and presence of S-9 respectively.

COMPARISON WITH HISTORICAL CONTROL DATA:
Individual plate counts from both experiments were recorded separately and the mean and
standard deviation of the plate counts for each treatment were determined.

Remarks on result:

other: all strains/cell types tested

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative
It is concluded that docusate sodium failed to induce mutation in 5 strains of Salmonella thyphimurium, when tested up to concentrations close to or within the toxic range, in the absence and presence of a rat liver metabolic activation system.

Executive summary:

An initial toxicity range-finder experiment was carried out in TA100 only, with docusate sodium concentrations of 8, 40, 200, 1000 and 5000 µg/plate plus a solvent and positive control. In the absence S-9, cytotoxicity was observed at the highest concentration of 5000µg/plate. In addition, a thinning of the background lawn at the second highest concentration (1000µg/plate) also indicated toxicity. In the presence of S9, toxicity was only observed at the highest dose. In view of these results, maximum test concentrations of 1000 and 2500 µg/plate were chosen for the main experiment 1, in absence  and presence of S9 respectively. In experiment 1, concentrations were close to the limit of toxicity, therefore for experiment 2, concentrations for all strains were maximally 2000 µg/plate without S9 and 2500 µg/plate with S9. In both experiments, docusate sodium did not result in statistically significant increases in revertant number of colonies, both with and without S9.