Docusate sodium

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Docusate sodium did not induce mutations in the Ames test up to concentrations close to toxic range, both with/without liver metabolic activation (S9) system. A marginal increase in chromosome aberrations was observed in CHO cells in the presence of S9 only and at the highest dose levels near cytotoxicity, which was likely related to an indirect mechanism. Read across with a category group substances did not reveal chromosome aberrations up to cytotoxic concentrations, nor were there positive effects in the mouse lymphoma assay. Finally, an in vivo Micronucleus assay with Docusate sodium tested up to the maximum tolerated dose level of 2000 or 1000 mg/kg bw/day in males and female rats, respectively, that produced clinical signs of toxicity and cytotoxicity in bone marrow, following repeated oral administration showed no genotoxic properties in the rat bone marrow micronucleus test at a sampling time of 24 hours after the last dosing.

Link to relevant study records

Reference

Reference 1

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.

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

Target gene:

histidine

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.

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).

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

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

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

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

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

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

Species / strain:

S. typhimurium TA 102

Remarks:

Experiment I

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

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

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

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

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

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

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.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1994

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.

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Not applicable

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media: supplied by Dr. S. Gallowy, West Point, PA, USA
- Properly maintained: yes: in tissue culture flasks containing McCoy's 5A medium incl. 10% foetal calf serum, 100 µg/mL gentamycin.
- Periodically "cleansed" against high spontaneous background: yes: regularly subcultured at low density.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

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

Test concentrations with justification for top dose:

Experiment 1- trial 1: 27.09, 38.71, 55.29 µg/mL (20h+ 0h recovery, -S9); 0.125 µg/mL NQO(4-Nitroquinoline 1-oxide)
Experiment 1- trial 2: 100, 110, 120 µg/mL (2h + 18h recovery, +S9); 25 µg/mL CPA(Cyclophosphamide)
Experiment 2- trial 1a: 60, 70, 80 µg/mL (20h + 0h recovery, -S9);
Experiment 2- trial 1b: 60µg/mL (44h +0h recovery, -S9);
Experiment 2 -trial 2: 110, 120, 130µg/mL ( 2h +18h recovery, +S9);25 µg/mL CPA
Experiment 2- trial 3 (repeat): 120, 125, 130µg/mL (2h + 18h recovery, +S9); 25µg/mL CPA

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: Preliminary solubility data indicated that sodium dioctyl sulphosuccinate was soluble in acetone at a
concentration of at least 500mg/ml. When this solution was diluted 100 fold into culture medium, precipitation was observed. Precipitation was only slight when an acetone solution at approximately 47 mg/ml was diluted similarly (to give a final concentration of approximately 470µg/ml) and 470µg/ml was therefore chosen as an appropriate maximum dose level for the chromosome aberration assay.

Untreated negative controls:

yes

Remarks:

untreated medium

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

Positive control +S-9: 25µg/mL

Untreated negative controls:

yes

Remarks:

untreated medium

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Positive control -S9: 0.125 µg

Details on test system and experimental conditions:

METHOD OF APPLICATION: in tissue flasks containing McCoy’s 5A medium, including 10% v/v foetal
calf serum (FCS) and 100µg/ml gentamycin

DURATION
- Preincubation period: 2 days (experiment 2, trial 3), 1 day (all other trials)
- Exposure duration (See also Table 1):
exp. 1, trial 1 (-S9): 20 hours
exp. 1; trial 2 (+S9): 2 hours (18 hours recovery)
exp. 2; trial 1a (-S9): 20 hours
exp. 2; trial 1b (-S9): 44h
exp.2, trial 2 (+S9): 2 hours (18 hours recovery)
exp.2, trial 3 (+S9): 2 hours (18 hours recovery)
- Fixation time (start of exposure up to fixation or harvest of cells): 20, 44, 20 hours

SPINDLE INHIBITOR (cytogenetic assays):colchicine
STAIN (for cytogenetic assays): 5 minutes in 4% (v/v) Giemsa in pH 6.8 buffer

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 200 per dose

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index
OTHER EXAMINATIONS:
- Determination of polyploidy:yes
- Determination of endoreplication: yes
- Determination of hyperdiploid cells: yes
-Determination of cells with structural aberrations including gaps: yes
-Determination of cells with structural aberrations excluding gaps: yes

Evaluation criteria:

The test chemical was considered as clearly positive in this assay if:
1) Statistically significant increases in the proportion of cells with structural aberrations (excluding gaps) occurred at one or more
concentrations
2) The proportion of aberrant cells at such data points exceeded the normal range
3) The results were confirmed in the second experiment

Statistics:

After completion of scoring slides were decoded. The aberrant cells in each culture were categorized as follows:
1 cells with structural aberrations including gaps
2 cells with structural aberrations excluding gaps
3 polyploid, endoreduplicated or hyperdiploid cells.
The totals for category 2 in negative control cultures was used to determine whether the assay was acceptable or not.
The proportions of aberrant cells in each replicate were used to establish acceptable heterogeneity between replicates by means of a binomial dispersion test.
The proportion of cells in category 2 for each test treatment condition, were compared with the proportion in negative controls using Fischer’s exact test. Probability values of p ≤ 0.05 were accepted as significant.
The proportion of cells in categories 1 and 3 were examined in relation to historical control (normal) ranges.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

ambiguous

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
- Water solubility: 470 µg/ml
- Precipitation: no
- Other confounding effects: no

RANGE-FINDING/SCREENING STUDIES: no

COMPARISON WITH HISTORICAL CONTROL DATA: yes

ADDITIONAL INFORMATION ON CYTOTOXICITY: no

In Experiment 1, treatment in the absence of S-9 was continuous for 20 hours (20+0h). Treatment in the presence of S-9 was for 2 hours only followed by a 18 hour recovery period prior to harvest (2+18h). The test compound dose levels for chromosome analysis were selected by evaluating the effect of sodium dioctyl sulphosuccinate on mitotic index. Chromosome aberrations were analyzed at 3 consecutive dose levels. The highest concentration chosen for analysis, 55.29 and 120 µg/mL, induced approximately 52% and 62% mitotic inhibition in the absence and presence of S-9respectively.

 

Similar to higher treatment regimens were used in Experiment 2, with in addition a delayed sampling time following treatment in the absence of S-9 included (44+0h). Chromosome aberrations were analyzed in cells receiving 20+0 h treatments in the absence of S-9 and 2+18 hour treatments in its presence at 3 consecutive dose levels. The highest concentrations chosen for analysis were, 80 and 130 µg/mL, which included approximately 79% and 34% mitotic inhibition respectively. A concentration inducing 50-75% mitotic inhibition was, therefore not identified following treatment in the presence of S-9, cytotoxicity in terms of a decrease in the yield of cells on the slides was, however, apparent at this dose and it seemed prudent to accept this as the top concentration for analysis.

 

The effects of a single concentration only, 60 µg/ml (without S-9) was investigated at the delayed(44+0h) sampling time. (A delayed harvest following treatment in the presence of S-9 (2+42h) was not included in Experiment 2 because a positive effect was apparent at the 20 hour sample). Appropriate negative (solvent and untreated) control cultures were included in the test system under each treatment condition. Acceptable frequencies of cells with structural aberrations were observed in solvent control cultures. Untreated controls were not scored. 4-Nitroquinoline 1-oxide (NQO) and cyclophosphamide (CPA) were employed as positive control chemicals following 20+0h hour treatments in the absence and 2+18 hour treatments in the presence of liver S-9 respectively, in both experiments. Both compounds induced statistically significant increases in the proportion of cells with structural aberrations.

 

Treatment of cultures with sodium dioctyl sulphosuccinate in the absence of S-9 resulted in frequencies of cells with aberration which were similar to and not significantly different from those in concurrent negative controls. Numbers of aberrant cells in all treated cultures fell within (or very close to) the historical negative control range.

 

Cultures treated with sodium dioctyl sulphosuccinate in the presence of S-9 in Experiment 1 had significantly increased frequencies of cells with aberrations at the highest dose level chosen for analysis (120µg/ml). Numbers of aberrant cells in both replicates receiving this concentration fell outside the historical negative control range. In contrast, cultures treated and sampled under these conditions in Experiment 2 had normal frequencies of aberrant cells at all dose levels analyzed. It will be recalled that the highest scorable in Experiment 2 failed to induce 50% mitotic inhibition. This part of the assay was therefore repeated int trial 3 using an interval between doses of max. 5 µg/ml. On neither occasion, however, was it possible to achieve a concentration at which 50-75% mitotic inhibition was apparent. The highest scorable dose was 130µg/ml and slides from these cultures were analyzed. Although small, but statistically significant increase in cells with aberrations were seen at both 125 and 130 µg/ml, the effect was considered to be of marginal biological significance because numbers of aberrant cells fell outside the normal range in only a single replicate at the highest dose.

 

Sodium dioctyl sulphosuccinate demonstrated a clear threshold for the induction of toxicity following treatment in the presence of S-9. Marginal reductions in mitotic activity occurred with increasing concentration until a threshold was reached at which point cell division ceased. The fact that chromosome aberrations were only clearly seen at a dose very close to this toxic threshold implies that the mechanism of induction was probably via an indirect rather than direct mechanism, that is, involved a target other than DNA.

Conclusions:

Interpretation of results: ambiguous with metabolic activation
It is concluded that docusate sodium was able to induce chromosome aberrations in CHO cells. The effect, however, was only seen following treatment in the presence of S-9 and only at doses very close to the threshold of toxicity. It is likely that aberration induction involved an indirect mechanism.

Executive summary:

Docusate sodium was tested in an in vitro cytogenetics assay using duplicate cultures of CHO cells in 2 independent experiments . Treatments covering a broad range of doses, separated by narrow intervals, were performed both in the absence and presence of metabolic activation by a rat liver post-mitochondrial fraction (S-9) from Aroclor 1254 induced animals. The highest dose level used, 470µg/mL was close to the solubility limit of sodium dioctyl sulphosuccinate in culture medium.

Treatment of cultures with docusate sodium in the absence of S-9 resulted in frequencies of cells with aberration which were similar to and not significantly different from those in concurrent negative controls. Numbers of aberrant cells in all treated cultures fell within (or very close to) the historical negative control range. Cultures treated with sodium dioctyl sulphosuccinate in the presence of S9 in Experiment 1 had significantly increased frequencies of cells with aberrations at the highest dose level chosen for analysis (120 µg/mL). Numbers of aberrant cells in both replicates receiving this concentration fell outside the historical negative control range. In contrast, cultures treated and sampled under these conditions in Experiment 2 had normal frequencies of aberrant cells at all dose levels analysed, however the highest concentration failed to induce 50% mitotic inhibition. This part of the assay was therefore repeated on 2 further occasions using a smaller interval, however, it was not possible to achieve 50-75% mitotic inhibition. The highest scorable dose in the second trial was 130 µg/mL and slides from these cultures were analyzed. Due to applicant and auther, small but statistically significant increase in cells with aberrations were seen at both 125 and 130 µg/mL, however the effect was considered to be of marginal biological significance because numbers of aberrant cells fell outside the normal range in only a single replicate at the highest dose.It is concluded that docusate sodium was able to induce chromosome aberrations in CHO cells. The effect was, however, only seen following treatment in the presence of S-9 and only at doses very close to the threshold of toxicity. It is likely that aberration induction involved an indirect mechanism.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

See read accross justification attached in Section 13.

Reason / purpose for cross-reference:

read-across source

Species / strain:

Chinese hamster lung fibroblasts (V79)

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
- Effects of pH: no effect
- Effects of osmolality:no effect

RANGE-FINDING/SCREENING STUDIES: yes

COMPARISON WITH HISTORICAL CONTROL DATA: yes

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Using reduced cell numbers as an indiator for toxicity in the pre-test, clear toxic effects were observed afer 4h treatment with 625 µg/mL and above in the absence and the presence of S-9 mix. Considering the toxicity data of the pre-test, 800 µg/mL (without S9) and 1000 µg/mL (with S-9) were chosen as top concentrations in the main experiment I.
Dose selection of experiment II was also influenced by test item toxicity. In the range finding experiment clearly reduced cell numbers were observed after 24h exposure withe 312,5 µg/mL and above. Therefore 600 µg/mL was chosen as top treatment concentration for continuous exposure in the absence of S-9 and 800 µg/mL in the presence of S-9.

Remarks on result:

other: all strains/cell types tested

In both experiments, no biologically relevant increase in the number of cell carrying structural chromosome aberration was observed after treatment with the test item. The observed satistical significance's and dose-dependency are regarded as being biologically irrelevant. No increase in frequencies of polyplid metaphases was found after treatment with the test item as compared to the frequencies of the controls. Appropriate mutagens were used as positive control; the induced statistically signifcant increases in cells with structural chromosome aberrations.

Conclusions:

Interpretation of results: negative
In conclusion, it can be stated that under the experimental conditions reported, the read-across test item did not induce structural chromosome aberrations as determined by the chromosome aberration test in V79 cells ( Chinese hamster cell line) in vitro.
Therefore, C-SAT 030041 is considered to be non clastogenic in this chromosome aberration test with and without S9 mix when tested up to cytotoxic concentrations.

Executive summary:

Read-across test item CAS No. 2373-38-8 (C-SAT 030041) was tested in an in vitro cytogenetics assay using duplicate cultures of CHO cells in 2 independent experiments. Treatments covering a broad range of doses, separated by narrow intervals, were performed both in the absence and presence of metabolic activation by a rat liver post-mitochondrial fraction (S-9) from Aroclor 1254 induced animals. A pre-test for cytotoxicity testing was performed up to 5000µg/mL, resulting in reduced cell numbers afer 4h treatment with 625 µg/mL and above in the absence and the presence of S-9 mix. Considering the toxicity data of the pre-test, 800 µg/mL (without S9) and 1000 µg/mL (with S-9) were chosen as top concentrations in the main experiment I. Dose selection of experiment II was also influencef by test item toxicity. In the range finding experiment clearly reduced cell numbers were observed after 24h exposure withe 312,5 µg/mL and above. Therefore 600 µg/mL was chosen as top treatment concentration for continuous exposure in the absence of S-9 and 800 µg/mL in the presence of S-9.

In both experiments, no biologically relevant increase in the number of cell carrying structural chromosome aberration was observed after treatment with the test item. No increase in frequencies of polyplid metaphases was found after treatment with the test item as compared to the frequencies of the controls. Appropriate mutagens were used as positive control; the induced statistically signifcant increases in cells with structural chromosome aberrations.

In conclusion, it can be stated that under the experimental conditions in CHO cells in vitro, C-SAT 030041 is considered to be non clastogenic in this chromosome aberration test with and without S9 mix when tested up to cytotoxic concentrations.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

See read accross justification attached in Section 13.

Reason / purpose for cross-reference:

read-across source

Species / strain:

mouse lymphoma L5178Y cells

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

Remarks on result:

other: all strains/cell types tested

The highest concentration used in the pre-test was chosen with regard to the purity (80 % active substance) and the molecular weight of the test item (400 g/mol). Test item concentrations between 39.1 and 5000 μg/mL (≈10 mM) were used to evaluate toxicity in the presence (4 h treatment) and absence (4 h and 24 h treatment) of metabolic activation. Following 4 hour treatment distinct toxic effects leading to RSG (relative suspension growth) values below 50 % were observed at 156.3 μg/mL and above in the absence and at 312.5 μg/mL and above in the presence of metabolic activation. Following continuous treatment (24 hours) a reduced relative suspension growth was determined at 312.5 μg/mL and above. The test medium was checked for precipitation at the end of each treatment period (4 or 24 hours) before the test item was removed. Precipitation was noted at 2500 μg/mL and above at the end of the 4 h treatment. Following 24 hours treatment, precipitation was observed at the maximum concentration of 5000 μg/mL.

The dose range of the first main experiment was selected according to the data generated in the pre-experiment. However, the onset of toxicity shifted and the analysable toxic range was not covered in the first experiment. Therefore, this experiment was repeated at higher concentrations without metabolic activation and at more narrowly spaced concentrations in the presence of metabolic activation. To overcome problems with possible deviations in toxicity both main experiments were started with more than four concentrations.

Relevant toxic effects indicated by a relative cloning efficiency 1 (survival) and/or a relative total growth (RTG) of less than 50 % in both parallel cultures were observed at 150 μg/mL and above in experiment IA without metabolic activation. The recommended toxic range of approximately 10 – 20 % of survival or RTG was covered. In the presence of metabolic activation toxic effects as described above were noted at 300 μg/mL. Although the recommended 10 – 20 % range of toxicity was not covered severe toxic effects occurred. The cell growth compared to the corresponding solvent control was severely reduced 24 h after treatment (20.8 - 21.3%). The surviving cells recovered and grew at a quite normal rate resulting in higher values of survival and RTG. Still, a reduction of the cell population down to about 20 % of the initial value is indicating severe toxicity, any further reduction may lead to undesired selection processes resulting in irreproducible artefacts.

No substantial and reproducible dose dependent increase of the mutation frequency was observed up to the maximum concentration with and without metabolic activation. The threshold of 126 plus each solvent control count was not reached or exceeded at any test point even at toxicity levels below 10 % of survival or RTG.

Conclusions:

Interpretation of results: negative
In conclusion it can be stated that under experimental conditions reported the read-across test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the abscence and presence of metabolic activation.

Executive summary:

Read-across substance C-SAT 06003 was studied in the mouse lymphoma thymidine kinase locus using the cell line L5178Y. The assay was performed in two independent experiments, using two parallel cultures each. Both main experiments were performed with and without liver microsomal activation and a treatment period of 4 h. The second experiment (experiment IA) was required to verify the results obtained in experiment I and to cover highly toxic concentrations using an adjusted concentration range. The highest applied concentration in the pre-test on toxicity (5000 μg/mL) was chosen with regard to the molecular weight of the test item. The dose range of the main experiments was limited by toxicity of the test item.

No substantial and reproducible dose dependent increase in mutant colony numbers was observed in both main experiments. No relevant shift of the ratio of small versus large colonies was observed up to the maximum concentration of the test item. Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced mutant colonies, indicating that the tests were sensitive and valid. In conclusion it can be stated that during the mutagenicity test described and under the experimental conditions reported the test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation. Therefore, C-SAT 060033 is considered to be non-mutagenic in this mouse lymphoma assay.

Reference 5

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

See read accross justification attached in Section 13.

Reason / purpose for cross-reference:

read-across source

Remarks:

Weight of evidence

Species / strain:

lymphocytes: human peripheral blood lymphocytes

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
- Effects of pH and osmolality:
The pH and osmolality of the negative control and all test item formulations in the medium were determined for each experiment employing the methods given below:
pH values: using a digital pH meter type WTW pH 525 (series no. 51039051),
Osmolality: with a semi-micro osmometer.
No relevant changes in pH or osmolality of the formulations were noted.
- Water solubility: The test item was completely dissolved in aqua ad iniectabilia.

RANGE-FINDING/SCREENING STUDIES:
In this preliminary experiment without and with metabolic activation test item concentrations of 10, 25, 100, 250, 1000, 2500 and 5000 µg test item/mL medium were employed. Cytotoxicity was noted starting at the concentration of 250 µg test item/mL. Hence, 250 µg/mL were employed as the top concentration for the mutagenicity tests without and with metabolic activation.

COMPARISON WITH HISTORICAL CONTROL DATA:
Data from vehicle and positive controls are used to establish historical control ranges. These values are used in deciding the adequacy of the concurrent vehicle controls or positive controls for an experiment.
The micronucleus frequencies of the vehicle controls without and with metabolic activation for the last 8 or 7 studies (most recent background data, not audited by the QAU-department) are given as follows:
Micronucleus frequency per 1000 cells
Without metabolic activation (4-h or 20-h exposure)
Untreated control (n = 8)
Mean: 4.9
SD: 2.0
range: 1 - 9
Vehicle control (n = 8)
Mean: 7.2
SD: 4.6
range: 1 - 18
Mitomycin C Positive control (n = 7)
Mean: 95.8
SD: 66.1
range: 24 - 286
Colchicine Positive control (n = 7)
Mean: 25.4
SD: 10.2
range: 7 - 43
With metabolic activation (4-h exposure)
Vehicle control (n = 8)
Mean: 10.8
SD: 6.2
range: 2 - 25
Cyclophosphamide Positive control (n = 7)
Mean: 60.3
SD: 37.8
range: 20 – 147
ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the main study cytotoxicity was noted at the top concentration of 250 µg/mL in the experiments without and with metabolic activation.

Remarks on result:

other: all strains/cell types tested

Conclusions:

Interpretation of results:
negative with metabolic activation
negative without metabolic activation
Under the present test conditions, the test item tested up to cytotoxic concentrations of 250 µg read-across test item/mL medium, in the absence and in the presence of metabolic activation employing two exposure times (without S9) and one exposure time (with S9) revealed no indications of chromosomal damage in the in vitro micronucleus test.
In the same test, Mitomycin C and cyclophosphamide induced significant chromosomal damage and colchicine induced significant damage to the cell division apparatus, respectively.

Executive summary:

Test sample of read-across substance Sodium bis(C11-14-isoalkyl, C13-rich) sulfosuccinate was assayed in an in vitro micronucleus test using human peripheral lymphocytes both in the presence and absence of metabolic activation by a rat liver post-mitochondrial fraction (S9 mix) from Aroclor 1254 induced animals.

The test was carried out employing 2 exposure times without S9 mix: 4 and 20 hours, and 1 exposure time with S9 mix: 4 hours. The experiment with S9 mix was carried out twice. The harvesting time was 20 hours after the end of exposure. The study was conducted in duplicate.

The test item was completely dissolved in aqua ad iniectabilia. Aqua ad iniectabilia served as the vehicle control.

The concentrations employed were chosen based on the results of a cytotoxicity study. In this preliminary experiment without and with metabolic activation test item concentrations of 10, 25, 100, 250, 1000, 2500 and 5000 µg/mL medium were employed. Cytotoxicity was noted starting at the concentration of 250 µg test item/mL. Hence, 250 µg/mL were employed as the top concentration for the mutagenicity tests without and with metabolic activation.

In the main study cytotoxicity was noted at the top concentration of 250 µg/mL in the experiments without and with metabolic activation.

Mitomycin C and colchicine were employed as positive controls in the absence and cyclophosphamide in the presence of metabolic activation.

Tests without metabolic activation (4- and 20-hour exposure)

The micronucleus frequencies of cultures treated with the test item at concentrations of 31.3, 62.5 or 125 µg test item/mL medium (4 h or 20-h exposure) in the absence of metabolic activation ranged from 5.0 to 8.5 micronuclei per 1000 binucleated cells. There was no dose related increase in micronuclei up to the cytotoxic concentrations. The dose level of 250 µg test item/mL medium led to cytotoxicity, no cells of sufficient quality were available for evaluation.

Vehicle controls should give reproducibly low and consistent micronuclei frequencies, typically 5 - 25 micronuclei per 1000 cells according to OECD 487. In this test the following frequencies were observed: vehicle control: 8.0 or 5.0 micronuclei per 1000 binucleated cells and untreated controls: 9.5 or 8.0 micronuclei per 1000 binucleated cells (4-hour and 20-hour exposure, respectively). Vehicle and untreated control values fell within acceptation ranges.

Test with metabolic activation (4-hour exposure)

The micronucleus frequencies of cultures treated with the test item at concentrations of 31.3, 62.5 or 125 µg test item/mL medium (4-h exposure) in the presence of metabolic activation ranged from 2.5 to 10.0 micronuclei per 1000 binucleated cells. There was no dose related increase in micronuclei up to the cytotoxic concentration. The dose level of 250 µg test item/mL medium led to cytotoxicity, not enough cells of sufficient quality were available for evaluation.

Vehicle controls should give reproducibly low and consistent micronuclei frequencies, typically 5 - 25 micronuclei per 1000 cells according to OECD 487. In this test the following frequencies were obsereved: vehicle control: 8.5 or 8.0 micronuclei per 1000 binucleated cells and untreated controls: 10.0 or 8.5 micronuclei per 1000 binucleated cells. Vehicle and untreated control values fell within acceptation ranges.

Under the present test conditions, the test item tested up to cytotoxic concentrations of 250 µg test item/mL medium, in the absence and in the presence of metabolic activation employing two exposure times (without S9) and one exposure time (with S9) revealed no indications of chromosomal damage in the in vitro micronucleus test.

In the same test, Mitomycin C and cyclophosphamide induced significant chromosomal damage and colchicine induced significant damage to the cell division apparatus, respectively.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

An in vivo cytogenetics assay was requested based on ECHA decision number CCH-D-2114330559-45-01/F.

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

adopted July 29, 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

dated May 30, 2008.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: mammalian erythrocyte micronucleus test

Species:

rat

Strain:

other: Crl: CD(SD)

Details on species / strain selection:

The rat is a rodent commonly used for this type of study.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age at start of administration:
Main Study: males: 53 days; females: 55 - 60 days
Satellite animals (males): 53 days
- Weight at study initiation:
Main Study: males: 257 - 296 g; females: 189 - 233 g
Satellite animals (males): 262 - 295 g
- Assigned to test groups randomly: yes, under following basis: body weight randomisation
- Housing: The animals were kept in groups of 2 - 3 in MAKROLON cages (type III plus). Granulated textured wood (Granulat A2, J. Brandenburg, 49424 Goldenstedt, Germany) was used as bedding material for the cages. The cages were changed and cleaned twice a week.
- Diet (e.g. ad libitum): Commercial ssniff® R/M-H V1534 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany) served as food. Feeding was discontinued approx. 16 hours before administration; only tap water was then available ad libitum.
- Water (e.g. ad libitum): Drinking water in drinking bottles was offered ad libitum.
- Acclimation period: At least 5 adaptation days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C  3°C (maximum range)
- Humidity (%): 55%  15% (maximum range). Deviations from the maximum range caused for example during cleaning procedures were dealt with in SOPs.
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: To:
Start of the experimental phase: July 17, 2017
Termination of the experimental phase: October 16, 2017
Period of treatment: September - October 2017

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: tap water
- Justification for choice of solvent/vehicle: water soluble
- Concentration of test material in vehicle: 25, 50, 100 mg/mL
- Amount of vehicle (if gavage or dermal): 20 mL/kg b.w

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Docusate Sodium was suspended in tap water. The administration volume was 20 mL/kg bw.

Duration of treatment / exposure:

Repeated oral administration of the test item on two days at a 24-hour interval by oral gavage.

Frequency of treatment:

Two times at 0 and 24 hours

Post exposure period:

24 hours after the last dose for the vehicle control and test item-treated animals
48 hours after single administration for the positive reference item

Dose / conc.:

500 mg/kg bw/day (actual dose received)

Remarks:

Docusate sodium

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

Docusate sodium

Dose / conc.:

2 000 mg/kg bw/day (actual dose received)

Remarks:

Docusate sodium

No. of animals per sex per dose:

Main study: 25 males and 15 females
Satellite animals: 12 males

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Route of administration: intraperitoneal (single administration)
- Doses / concentrations: 27 mg/kg bw; in 0.9% NaCl solution; 20 mL/kg bw administration volume

Tissues and cell types examined:

bone marrow polychromatic erythrocytes and normochromatic erythrocytes
The satellite animals were used for bone marrow and blood plasma sampling for possible toxicokinetics only). Following sacrifice and sampling, no further examinations were carried out.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
-Preliminary toxicity test: The dose levels for the main study had been selected in agreement with the Sponsor based on the results of a preliminary dose-range-finding study in rats to determine the maximum tolerated dose (MTD) employing one animal per sex and dose. The oral dose levels of 500, 1000 and 2000 mg/kg b.w./day were tested. The administration was carried out on two days at a 24-hour interval. The administration volume was 20 mL/kg bw The animals were observed for 3 days. No signs of systemic toxicity were noted up to the highest reasonable dose level of 2000 mg per kg b.w./day. No animal died prematurely.
-Main study: Hence, three ascending oral dose levels of 500, 1000 and 2000 mg Docusate Sodium/kg bw/day and the vehicle (tap water) were administered two times at 0 and 24 hours. The positive reference item cyclophosphamide (27 mg CPA/kg bw, i.p. in 0.9% NaCl solution) was employed by single intraperitoneal administration. 25 male and 15 female animals were employed. Females were compared to males for the vehicle control and high dose group due to differences in toxicity observed between male and female rats in the 2-week dietary toxicity study (LPT Study No. 34271), where males showed increased liver (+30%) and kidney weights (+12%), whereas this was not marked in females.
Hence, in this study each group consisted of 5 male rats and, in addition, 5 females each were employed for the vehicle control and high dose group. The administration volume was 20 mL/kg bw.
The dose levels cover a range from the maximum to little or no toxicity. The maximum tolerated dose level (MTD) is defined the highest dose that is tolerated without evidence of study-limiting toxicity, relative to the duration of the study period (for example, by inducing body weight), depression or hematopoietic system cytotoxicity, but not death or evidence of pain, suffering or distress. The highest dose may also be defined as a dose that produces some indication of toxicity of the bone marrow (e.g. reduction in the proportion of immature erythrocytes among total erythrocytes in the bone marrow) or for solutions the maximum feasible application volume.
In the high dosed females (2000 mg Docusate Sodium per kg bw/day) 4/5 females died prematurely within 3 days and were not suitable for the micronucleus assay. Hence, 1000 mg Docusate Sodium per kg bw/day were administered to 5 female rats as the maximum tolerated dose level for females.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): 24 hours after the last dosing.

DETAILS OF SLIDE PREPARATION:
The main study animals were sacrificed. After removing some of the muscles the femurs were excised below the knee and at the iliac joint. The bone marrow was flushed out with calf serum and centrifuged at 850 x g for 3 to 5 minutes. The supernatant was removed and the sediment re-suspended in a drop of calf serum by using a Pasteur Pipette. Then two smears of 30 to 60 mm length were prepared and allowed to air dry.
Once dry, the preparations were stained for 1.5 minutes in May-Grünwald solution and then for 1 minute in 50% May-Grünwald solution in Weise buffer. Afterwards the cells were rinsed with Weise buffer and then further stained in 10% Giemsa solution in Weise buffer for 20 minutes. Subsequently, the slides were rinsed with Weise buffer and aqua demineralisata. The lower side of the slides were cleaned with Methanol. The slides were left to air-dry before being covered with Eukitt® (Fluka) and a cover slip.

METHOD OF ANALYSIS:
The slides were coded and randomised before microscopic analysis.
Four thousand (4000) polychromatic erythrocytes per animal were scored for the incidence of micronuclei, and the ratio of polychromatic (PCE) to normochromatic erythrocytes (NCE) was determined for each animal by counting a total of 500 erythrocytes.

Evaluation criteria:

Providing that all acceptability criteria are fulfilled, a test chemical is considered clearly positive if:
- At least one of the treatment groups exhibits a statistically significant increase in the frequency of micronucleated immature erythrocytes compared with the concurrent negative control,
- this increase is dose-related when evaluated with an appropriate trend test, and
- any of these results are outside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits).
Providing that all acceptability criteria are fulfilled, a test chemical is considered clearly negative if, in all experimental conditions examined:
a) None of the treatment groups exhibits a statistically significant increase in the frequency of micronucleated immature erythrocytes compared with the concurrent negative control;
b) there is no dose-related increase at any sampling time when evaluated by an appropriate trend test;
c) all results are inside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits), and
d) bone marrow exposure to the test item occurred.

Statistics:

The assessment is carried out by a comparison of the samples combined with the positive and the vehicle reference items, using a chi-squared test corrected for continuity according to YATES (COLQUHOUN, 1971) as recommended by the UKEMS guidelines (The United Kingdom Branch of the European Environmental Mutagen Society: Report of the UKEMS subcommittee on guidelines for mutagenicity testing, part III, 1989: Statistical evaluation of mutagenicity test data).
. For each group, inter-individual variation in the numbers of micronucleated PCE was evaluated by means of a heterogeneity chi-square test. The numbers of micronucleated PCE in each treated group were then compared with the numbers in the vehicle control groups by using a 2 x 2 contingency table to determine chi-square. Probability values of p ≤ 0.05 were accepted as significant. The Spearman's rank correlation coefficient was employed for investigation of a possible dose-relationship.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Sex:

female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Table 1. Preliminary test of the mutagenicity study

Animal No./Sex	Body weight (g)		Number of polychromatic erythrocytes scored	Micronucleated polychromatic erythrocytes		PCE	NCE	Ratio PCE/NCE*
	TD 1	TD 2		Number/4000 PCE	Number/1000 PCE
500 mg Docusate sodium/kg bw/day, p.o.
1 m	211	215	4000	4	1.0	172	328	0.52
2 f	204	211	4000	3	0.8	120	380	0.32
mean				3.5	0.9			0.42
SD				0.7	0.1			0.14
1000 mg Docusate sodium/kg bw/day, p.o.
3 m	213	214	4000	4	1.0	156	344	0.45
4 f	205	206	4000	1	0.3	112	388	0.29
mean				2.5	0.7			0.37
SD				2.1	0.5			0.11
2000 mg Docusate sodium/kg bw/day, p.o.
5 m	223	213	4000	4	1.0	122	378	0.32
6 f	205	195	4000	5	1.3	92	408	0.23
mean			-	4.5	1.2			0.28
SD				0.7	0.2			0.06

PCE polychromatic erythrocytes

NCE normochromatic erythrocytes

*      per 500 counted cells

SD    standard deviation

m     male

f       female

p.o. per oral

TD   test day

 

Table 2. Summarised data of the genotoxicity study

Docusate sodium (mg/kg bw/day, p.o.)	Number of polychromatic erythrocytes scored per group (n=5)	Ratio PCE/NCE^#1		Micronucleated polychromatic erythrocytes mean frequency
				per 4000 PCE		per 1000 PCE
		m	f	m	f	m	f
		mean (n=5)		mean (n=5)		mean (n=5)
0	2000	0.93	0.54	3.6	4.0	0.9	1.0
500	2000	0.64		4.8		1.2
1000	2000	0.68*	0.31**	3.6	4.0	0.9	1.0
2000	2000	0.36**		3.4		0.9
Cyclophosphamide
27 mg/kg bw, i.p.	2000	0.11**		119.0**		29.8**

*      significant at p ≤ 0.05 compared to control

**    significant at p ≤ 0.01 compared to control

PCE polychromatic erythrocytes

NCE normochromatic erythrocytes

^#1      per 500 counted cells

M    male

f       female

p.o. per oral

i.p.   intraperitoneal

Conclusions:

Docusate Sodium did not increase the incidence of micronucleated polychromatic erythrocytes (PCE) at any of the three tested dose levels of 500, 1000 or 2000 mg test item/kg bw/day.

Executive summary:

Docusate Sodium was assayed in an in vivo bone marrow micronucleus test in the rat for the detection of damage to the chromosomes or the mitotic apparatus following repeated oral administration of the test item on two days at a 24-hour interval. In addition, blood samples and bone marrow samples were taken for possible bioanalysis.

Preliminary dose-range-finding study

Docusate Sodium was suspended in tap water. The dose levels for the main study had been selected in agreement with the Sponsor based on the results of a preliminary dose-range-finding study in rats to determine the maximum tolerated dose (MTD) employing one animal per sex and dose. The highest dose was also defined as a dose that produces cytotoxicity in the bone marrow, a reduction in the proportion of immature erythrocytes among total erythrocytes. In case of no dose-relateddecrease of PCE/NCE ratiosfurther bioanalysis would have been necessary to verify exposure of bone marrow to the test substance. The oral dose levels of 500, 1000 and 2000 mg/kg bw/day were tested. The administration was carried out on two days at a 24-hour interval. The administration volume was 20 mL/kg bw. The animals were observed for 3 days. No signs of systemic toxicity were noted up to the highest reasonable dose level of 2000 mg per kg bw/day. No animal died prematurely.

Main study

Hence, three ascending oral dose levels of 500, 1000 and 2000 mg Docusate Sodium/kg bw/day and the vehicle (tap water) were administered two times at 0 and 24 hours. The positive reference item cyclophosphamide (27 mg CPA/kg bw, i.p. in 0.9% NaCl solution) was employed by single intraperitoneal administration. 25 male and 15 female animals were employed. Females were compared to males for the vehicle control and high dose group due to differences in toxicity observed between male and female rats in the 2-week dietary toxicity study (LPT Study No. 34271), where males showed increased liver (+30%) and kidney weights (+12%), whereas this was not marked in females.

Hence, in this study each group consisted of 5 male rats and, in addition, 5 females each were employed for the vehicle control and high dose groups (1000 and 2000 mg/kg bw/day). The administration volume was 20 mL/kg bw.

The male animals treated with 500 or 1000 mg Docusate Sodium per kg bw/day revealed soft faeces on Test days 2 and 3, respectively. 1000 mg/kg bw/day caused slightly reduced motility, slight ataxia and slight dyspnoea in the males 6 hours after the first administration until Test day 3. The high dosed male and female animals with 2000 mg Docusate Sodium per kg bw/day revealed soft, mucous and evil-smelling faeces, slightly to moderately reduced motility, slight to moderate ataxia and slight to moderate dyspnoea 6 hours after administration until Test day 3 in all 5 of 5 male and 5 of 5 female animals. Four of 5 female animals died prematurely within three days and were not suitable for the micronucleus assay. Hence, 1000 mg Docusate Sodium per kg bw/day were administered to 5 female rats as the maximum tolerated dose level for females. 1000 mg/kg bw/day caused pilo-erection and soft faeces on Test days 2 and 3 in all 5 female rats.

All high dosed animals (males: 2000 mg per kg bw/day and females: 1000 mg/kg) and, in addition, 3 of 5 male animals dosed with 1000 mg per kg bw/day lost body weight within 3 days.

Bone marrow was sampled on Test day 3 (24 hours after the last dose for the vehicle control and test item-treated animals and 48 hours after single administration for the positive reference item).

Four thousand (4000) polychromatic erythrocytes per animal were scored for the incidence of micronuclei, and the ratio of PCE to NCE was determined for each animal. The PCE/NCE ratios of the high dosed male (2000 mg/kg b.w./day) and female animals (1000 mg/kg b.w./day) were statistically significantly decreased (at p < 0.01) and of the males treated with 1000 mg/kg b.w./day significantly decreased at p £ 0.05 compared to the control animals which demonstrate cytotoxicity and, hence, bone marrow exposure to the test item.Therefore no further bioanalysis was necessary.

Docusate Sodium did not increase the incidence of micronucleated polychromatic erythrocytes (PCE) at any of the three tested dose levels of 500, 1000 or 2000 mg test item/kg bw/day. The incidence of micronucleated PCE was 0.9 or 1.0 per 1000 PCEs for the high dosed samples for males (2000 mg/kg bw/day) and females (1000 mg/kg bw/day). The corresponding micronucleus frequency of the vehicle control (negative reference) was 0.9 or 1.0 per 1000 PCEs (males and females, respectively). Administration of cyclophosphamide (positive reference item) significantly increased the number of micronucleated PCE to 29.8 per 1000 PCEs.

In conclusion, under the present test conditions, Docusate Sodium tested up to the maximum tolerated dose level of 2000 or 1000 mg/kg bw/day in males and females, respectively, following repeated oral administration showed no genotoxic properties in the rat bone marrow micronucleus test at a sampling time of 24 hours after the last dosing.

In the same system, cyclophosphamide (positive reference item) induced significant damage.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

A key study for bacterial mutagenicity was performed in an Ames reverse mutation test with 5 Salmonella typhimurium strains (Cytec, Clare 1993); the study was conducted according to OECD 471 and GLP guidelines, and was considered to be reliable, adequate and relevant. After a range-finder experiment showing cytotoxicity at the highest concentration of 5000 µg/plate, maximum test concentrations of 1000 and 2500 µg/plate were chosen for the main experiment 1. 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. In addition, a literature study was available with Docusate sodium tested at concentrations of 1, 10 and 100 µg/plate, which did also not result in statistically significant increases in revertant number of colonies, both with and without S9 (Literature, Bonin & Baker 1980). It is not further taken into account due to the low concentrations.

 

A key study was available for the mutation assay at the thymidine kinase locus (TK+/-) in mouse lymphoma L5178Y cells with sodium dihexylsulfosuccinate (CAS 2373-38-8); the study was conducted according to OECD 476 and GLP guidelines, and was considered to be reliable, adequate and relevant (Cognis, Wollny 2006). The assay was performed in two independent experiments, using two parallel cultures each. Both main experiments were performed with and without liver microsomal activation and a treatment period of 4 h. The second experiment (experiment IA) was required to verify the results obtained in experiment I and to cover highly toxic concentrations using an adjusted concentration range. The highest applied concentration in the pre-test on toxicity (5000 µg/mL) was chosen with regard to the molecular weight of the test item. The dose range of the main experiments was limited by toxicity of the test item. No substantial and reproducible dose dependent increase in mutant colony numbers was observed in both main experiments. No relevant shift of the ratio of small versus large colonies was observed up to the maximum concentration of the test item. In conclusion it can be stated that during the mutagenicity test described and under the experimental conditions reported the test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation.

 

Weight of evidence was available for chromosome aberration potential from read across substances in the same category:

- Docusate sodium (CAS No. 577-11-7) was tested in V79 CHO cells (Cytec, Marshall 1993); the study was conducted according to OECD 473 and GLP guidelines, and was considered to be reliable, adequate and relevant. The highest dose level used, 470 µg/mL was close to the solubility limit of Docusate sodium in culture medium. Treatment of cultures with Docusate sodium in the absence of S9 resulted in frequencies of cells with aberration which were similar to and not significantly different from those in concurrent negative controls. Numbers of aberrant cells in all treated cultures fell within (or very close to) the historical negative control range. Cultures treated with Docusate sodium in the presence of S9 in Experiment 1 had significantly increased frequencies of cells with aberrations at the highest dose level chosen for analysis (120 µg/mL) which fell outside the historical negative control range. In contrast, cultures treated and sampled under these conditions in Experiment 2 had normal frequencies of aberrant cells at all dose levels analysed, however the highest concentration failed to induce 50% mitotic inhibition. This part of the assay was therefore repeated on 2 further occasions using a smaller interval, however, it was not possible to achieve 50-75% mitotic inhibition. The highest scorable dose in the second trial was 130 µg/mL and slides from these cultures were analyzed. Due to applicant and author, small but statistically significant increases in cells with aberrations were seen at both 125 and 130 µg/mL, however the effect was considered to be of marginal biological significance because numbers of aberrant cells fell outside the normal range in only a single replicate at the highest dose. Author further mentioned that most likely aberration induction involved an indirect mechanism. Therefore induction of chromosome aberration is not concluded based on the opinion of the applicant. This is underlined by a read across chromosome aberration studies below.

-Sodium dihexylsulfosuccinate (CAS No. 2373-38-8) was tested according to OECD 473 and GLP guidelines covering a broad range of doses, separated by narrow intervals, were performed both in the absence and presence of metabolic activation by a rat liver post-mitochondrial fraction (Cognis, Schulz 2003). A pre-test for cytotoxicity testing was performed up to 5000 µg/mL, resulting in reduced cell numbers after 4h treatment with 625 µg/mL and above in the absence and the presence of S-9 mix. Considering the toxicity data of the pre-test, 800 µg/mL (without S9) and 1000 µg/mL (with S-9) were chosen as top concentrations in the main experiment I. Dose selection of experiment II was also influenced by test item toxicity. In the range finding experiment clearly reduced cell numbers were observed after 24h exposure with 312,5 µg/mL and above. Therefore 600 µg/mL was chosen as top treatment concentration for continuous exposure in the absence of S-9 and 800 µg/mL in the presence of S-9. In both experiments, no biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed after treatment with the test item. No increase in frequencies of polyploid metaphases was found after treatment with the test item as compared to the frequencies of the controls. Appropriate positive controls induced statistically significant increases in cells with structural chromosome aberrations. In conclusion, the test material was considered to be non clastogenic in this chromosome aberration test with and without S9 mix when tested up to cytotoxic concentrations.

-Sodium bis (C11 -14 -isoalkyl, C12-rich) sulfosuccinate (CAS No. 848588-96-5) was tested in a Micronucleus test using human peripheral lymphocytes both in the presence and absence of metabolic activation by a rat liver post-mitochondrial fraction (S9 mix) from Aroclor 1254 induced animals (Flügge, 2013). The test was carried out employing 2 exposure times without S9 mix: (4 hours and 20 hours) and one exposure time with S9 mix (repeated). The harvesting time was 24 hours after the end of exposure. Each treatment was conducted in duplicate. The test item was completely dissolved in aqua ad iniectabilia, which also served as the vehicle control. Based on a preliminary experiment, cytotoxicity was noted starting at a concentration of 250 µg test item/mL in the experiment without and with metabolic activation. Hence, 250 µg/mL were employed as the top concentration for the main test without and with metabolic activation in two independent experiments, each (4-hour and 20-hour exposure). There was no increase in micronuclei up to the cytotoxic concentration when compared to control both with and without metabolic activation. Under the present test conditions, the test item tested up to cytotoxic concentrations, in the absence and in the presence of metabolic activation employing two exposure times (without S9) and one exposure time (with S9) revealed no indications of any chromosomal damage in the in vitro micronucleus test. In the same test, Mitomycin C and cyclophosphamide induced significant damage.

 

Read across with category members/structural analogues was performed for genotoxicity (chromosome aberration) but also for other endpoints (e.g. sensitisation, repeated dose toxicity), demonstrating a similar toxicological profile for Docusate sodium and Sodium dihexylsulfosuccinate and other analogues.

 

Summarising the results, no positive genotoxicity was observed with Docusate sodium and category member Sodium dihexylsulfosuccinate (CAS 2373-38-8 and CAS 845888-96 -5). As indicated in section 7.7, there is also information from a 24 -month carcinogenicity test in rats with Docusate sodium, which was negative for increased tumor incidence.

 

An in vivo cytogenetics assay was requested based on ECHA Communication number CCH-D-2114321066-61-01/D. Docusate sodium was used as read across substance for the registration of Potassium 1,2-bis(2-ethylhexyloxycarbonyl)ethanesulphonate (CAS 7491-09-0). Based on the results of the in vitro chromosomal aberration (OECD 473 -) study with Docusate sodium, which showed increases in the proportion of cells with structural aberrations, ECHA requested an in vivo cytogenetics assay (mammalian erythrocyte micronucleus test, mammalian bone marrow chromosomal aberration test or mammalian alkaline comet assay). The new study with Docusate sodium was conducted for both the registrations of both Potassium 1,2-bis(2-ethylhexyloxycarbonyl)ethanesulphonate and Docusate sodium, and is described below.

 

A key in vivo bone marrow micronucleus test was performed with Docusate sodium in the rat for the detection of damage to the chromosomes or the mitotic apparatus following repeated oral administration of the test item on two days at a 24-hour interval. Blood samples and bone marrow samples were taken for possible bioanalysis.

In a preliminary dose-range-finding study at dose levels of 500, 1000 and 2000 mg/kg bw, the maximum tolerated dose (MTD) leading to a reduction in the proportion of immature erythrocytes among total erythrocytes was determined to be 2000 mg/kg bw/day. No signs of systemic toxicity were noted up to the highest reasonable dose level of 2000 mg per kg bw/day. No animal died prematurely.

In the main study, dose levels of 500, 1000 and 2000 mg/bw/day and the vehicle (tap water) were administered two times at 0 and 24 hours. Females were compared to males for the vehicle control and high dose group due to differences in toxicity observed between male and female rats in the 2-week dietary toxicity study, where males showed increased liver and kidney weights whereas this was not marked in females. Hence, in this study each group consisted of 5 male rats and, in addition, 5 females each were employed for the vehicle control and high dose groups (1000 and 2000 mg/kg bw/day). The administration volume was 20 mL/kg bw.

The male animals treated with 500 or 1000 mg/kg bw/day revealed soft faeces on Test days 2 and 3, respectively. 1000 mg/kg bw/day caused slightly reduced motility, slight ataxia and slight dyspnoea in the males 6 hours after the first administration until Test day 3. The high dosed male and female animals with 2000 mg Docusate Sodium per kg bw/day revealed soft, mucous and evil-smelling faeces, slightly to moderately reduced motility, slight to moderate ataxia and slight to moderate dyspnoea 6 hours after administration until Test day 3 in all 5 of 5 male and 5 of 5 female animals. Four of 5 female animals died prematurely within three days and were not suitable for the micronucleus assay. Hence, 1000 mg/kg bw/day were administered to 5 female rats as the maximum tolerated dose level for females. 1000 mg/kg bw/day caused pilo-erection and soft faeces on Test days 2 and 3 in all 5 female rats. All high dosed animals (males: 2000 mg per kg bw/day and females: 1000 mg/kg) and, in addition, 3 of 5 male animals dosed with 1000 mg per kg bw/day lost body weight within 3 days.

Bone marrow was sampled on Test day 3 (24 hours after the last dose for the vehicle control and test item-treated animals and 48 hours after single administration for the positive reference item). The PCE/NCE ratios of the high dosed male (2000 mg/kg bw/day) and female animals (1000 mg/kg bw/day) were statistically significantly decreased (at p < 0.01) and of the males treated with 1000 mg/kg bw/day significantly decreased at p < 0.05 compared to the control animals which demonstrate cytotoxicity and, hence, bone marrow exposure to the test item. Therefore no further bioanalysis was necessary.

Docusate sodium did not increase the incidence of micronucleated polychromatic erythrocytes (PCE) at any of the three tested dose levels of 500, 1000 or 2000 mg test item/kg bw/day. The incidence of micronucleated PCE was 0.9 or 1.0 per 1000 PCEs for the high dosed samples for males (2000 mg/kg bw/day) and females (1000 mg/kg bw/day). The corresponding micronucleus frequency of the vehicle control (negative reference) was 0.9 or 1.0 per 1000 PCEs (males and females, respectively). Administration of cyclophosphamide (positive reference item) significantly increased the number of micronucleated PCE to 29.8 per 1000 PCEs.

In conclusion, under the present test conditions, Docusate sodium tested up to the maximum tolerated dose level of 2000 or 1000 mg/kg bw/day in males and females, respectively, following repeated oral administration showed no genotoxic properties in the rat bone marrow micronucleus test at a sampling time of 24 hours after the last dosing.

In the same system, cyclophosphamide (positive reference item) induced significant damage.

 

Based on the combination of in vitro and in vivo studies, Docusate sodium is not considered to have genetic toxicity potential.

Justification for classification or non-classification

As the mutagenicity experiments did not indicate genotoxicity potential, classification is not warranted.