Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
read-across based on grouping of substances (category approach)
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:
Data has been read across from a structurally identical substance forming a salt with sodium instead of potassium. This is not expected to chenge the toxicological properties in a significant way. See further discussion in the read across justification attached.
Cross-reference
Reason / purpose:
read-across: supporting information

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
adopted July 29, 2016
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
dated May 30, 2008.
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
other: mammalian erythrocyte micronucleus test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: Rolls of white wax-like plastic sheets

Test animals

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 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

Administration / exposure

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
Doses / concentrationsopen allclose all
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

Examinations

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.


Results and discussion

Test resultsopen allclose all
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

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.