Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

A key study for reproductive toxicity was performed with registered substance in rats by means of an OECD 422 study with a test substance containing >93% active ingredient at dose levels of 60, 120 and 300 mg/kg bw/day. NOAEL-levels were 60 mg/kg bw for paternal/maternal toxicity and 120 mg/kg bw for reproductive toxicity. Reproductive findings included decreased gestation index, No. of corpora lutea, implantation sites, pups born (alive and dead), and (life) birth index. In conclusion, general parental toxicity was higher than reproductive toxicity, and the reproductive findings were considered to be secondary to the paternal/maternal toxicity.

Read-across from CAS no. 577-11-7 (Docusate sodium) three-generation study according to OECD TG 416 and GLP showed decreased body weights in P, F1 and F2 generations at 0.5 and 1% dietary concentrations, however these were not considered adverse and were not associated with any other (reproductive) findings. In a second (supporting) two-generation study where adaptations were done to exclude influence of taste of milk, also showed that highest concentration of 1% in the diet corresponding with 750 mg act. ingr./kg bw was NOAEL. Based on the absence of reproductive findings in the repeated dose toxicity studies and the multigeneration studies, no further testing is needed.

Link to relevant study records

Referenceopen allclose all

Endpoint:
three-generation reproductive toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
see attached read-across justification
Reason / purpose:
read-across source
Positive control:
No
Clinical signs:
no effects observed
Description (incidence and severity):
There were no treatment-related clinical observations for F0 males and females. Alopecia was noted with similar frequency in the control and treated groups.A common occurence in rodents maintained on ground feed is malocclusion. No parental females (F0) aborted or had physical or behavioral abnormalities during F1 gestation and lactation.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Survival was 100% for all groups of males and females at termination of the F0 generation animals except for the high-dose males where it was 97%. One male in this group (Animal No. C25201) was sacrificed at Week 10 in moribund condition due to an apparent fracture of the nasal bones.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Dose levels of 0.5% and 1.0% caused a reduction in body weight for the F0 males and the values were statistically significant at Weeks 9, 10, 18 and 19 for the 1.0% dose level and at Week 17 for the 0.5% dose level. No effect on body weight was seen for females at any dose level during the premating phase or during gestation. Although body weights for females during lactation were slightly lower than those of controls, the differences were not statistically significant.
There were no consistent significant differences in body weight gains for males. Body weight gains for 1.0% dose level females during Gestation Days 14 to 20 and 0 to 20 were significantly lower than those of controls .
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Although food consumptions for treated males were slightly lower than those of controls throughout the premating phase, these differences were significant only during Week 4 for the 0.5% dose level and during Weeks 4 and 5 for the 1.0% dose level. Food consumptions for 1.0% dose level females were lower than those of controls throughout lactation and statistically significant for all intervals except Days 10 through 14 and Days 17 through 21.
Compound consumption across groups was approximately proportional to the level of DSS administered in the test diets. As one would expect, it was decreased during the premating growth phase for both sexes(although to a greater extent in males ), remained low during gestation, and then increased appreciably during lactation.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
There were no significant differences from the control group for male fertility indices during breeding for F1 litters. Fertility in males was defined by a female giving birth to a litter.
No significant differences from controls were observed for mating, fertility, or gestation indices, days required to mate, or the length of gestation. No parental females aborted or had physical or behavioral abnormalities during F1 gestation and lactation.
BODY WEIGHT (PARENTAL ANIMALS)

Table 1.Parental Body weights
Dose: 0 0.1% 0.5% 1%
Males Generation
Premating (age)
- Initial F0 (7 weeks) 232 238 241 238
F1 (6 weeks) 149 156 144 131*
F2 (7 weeks) 206 217 197 180
- Final F0 506 507 493 479
F1 510 512 492 447*
F2 531 536 492* 467*
Females
Premating
- Initial F0 (7 weeks) 165 163 165 166
F1 (6 weeks) 127 129 121 114*
F2 (7 weeks) 160 162 148 145
- Final F0 206 204 204 206
F1 281 296 271 255*
F2 285 290 271 269*
Gestation
- Initial F0 218 217 216 218
F1 278 294 267 258*
F2 288 291 277 270
- Final F0 361 365 360 350
F1 396 107 379 369*
F2 401 405 392 378*
Lactation
- Initial F0 280 280 275 267
F1 309 319 292 283*
F2 320 317 305 283*
- Final F0 288 294 290 273
F1 304 319 302 293
F2 313 316 314 300
* p < 0.05 (One-way ANOVA variance)
Dose descriptor:
NOAEC
Effect level:
0.1 other: %
Based on:
act. ingr.
Remarks:
DSS, in the diet
Sex:
male/female
Basis for effect level:
body weight and weight gain
Remarks on result:
other: Generation: maternal/paternal toxicity
Key result
Dose descriptor:
NOAEC
Effect level:
1 other: %
Based on:
act. ingr.
Remarks:
DSS, in the diet
Sex:
male/female
Basis for effect level:
other: no effects up to highest concentration
Remarks on result:
other: reproduction/offspring
Clinical signs:
no effects observed
Description (incidence and severity):
There were no treatment-related clinical observations for F1 males and females. Alopecia was noted with similar frequency in the control and treated groups .
As in the F0 generation, there were several instances of malocclusion and/or related signs. When necessary, the incisors of affected animals were clipped to alleviate the condition.
No parental females (F1) aborted or had physical or behavioral abnormalities during F2 gestation and lactation.

Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
Survival was 100% for all groups of males and females at termination of the F1 generation animals.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weights for F1 males and females at the 1.0% dose levels were significantly lower than those of controls throughout the premating phase, excluding Week 2 for females, and throughout the treatment period for males. Body weights for F1 males and females in the 0.5%. dose group were also low (although not statistically significant) during the entire premating phase.
Gestation body weights tor females were significantly lower than those of controls at the1.0% dose level throughout gestation. Body weights during lactation were significantly lower on days 0, 7, and 14 at the 1.0% dose level; body weights on Lactation Day 21 at the 1.0% dose level were also lower than those of controls, although this difference was not statistically significant.
Body weight gains were significantly lower than those of controls during Weeks 5 and 6 for males and during Weeks 3, 5, and 8 for females at the 1.0% dose level, and during Weeks 5 and 10 for females at the 0.5% dose level.

Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumptions were significantly lower than those of controls throughout the premating phase· (except f or Week 3) for males at the 1.0% dose level, and during Weeks 6 through 9 for males at the 0.5% dose level. Female food consumptions were significantly lower than those of controls during Weeks 7 and 8 at both the 0.5% and 1.0% dose levels.
Food consumptions during gestation were significantly greater than those of control only during Days 0 through 4 at the 0.1% dose level. Food consumptions during lactation were significantly lower during Days 7 through 10 at the 0.5% dose level, and during Days 4 through 7, 7 through 10, and 14 through 17 at the 1.0% dose level.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no treatment-related macroscopic observations in any animals examined in the study (F0, F1 and F2 adults and F3 weanlings).
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
There were no significant differences from the control group for male fertility indices during breeding for F2 litters .
No significant differences from controls were observed for mating, fertility, or gestation indices, days required to mate, or the length of gestation. No parental females aborted or had physical or behavioral abnormalities during F2 gestation and lactation.

Dose descriptor:
NOAEC
Effect level:
0.1 other: %
Based on:
act. ingr.
Remarks:
DSS, diet
Sex:
male/female
Basis for effect level:
body weight and weight gain
Remarks on result:
other: maternal/paternal toxicity
Key result
Dose descriptor:
NOAEC
Effect level:
1 other: %
Based on:
act. ingr.
Remarks:
DSS, diet
Sex:
male/female
Basis for effect level:
other: no effects up to highest concentration
Remarks on result:
other: reproduction/offspring
Clinical signs:
no effects observed
Description (incidence and severity):
No significant differences from controls were observed for the total and mean number of pups born alive, litter size, survivability, or sex ratio. No pups from F1 litters had external abnormalities.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
No significant differences from controls were observed for the total and mean number of pups born alive, litter size, survivability, or sex ratio.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Male and female pup weights on days 4, 7, 14, and 21 and pup weight gains from Days 0 through 4, 4 through 7, 7 through 14, and 14 through 21 at the 1.0% dose level were significantly lower than those of controls. Male and female pup weights on day 21 and weight gains from Days 7 through 14 (females only), and from days 14 through 21 (males and females) for the 0.5% dose level were significantly lower than those of controls.
Food consumption and compound intake (if feeding study):
not examined
Description (incidence and severity):
Pups were allowed to nurse for 21 days before weaning. Food consumption after weaning is described under the parental generation.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
No pups from F1 litters had external abnormalities.
Histopathological findings:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
BODY WEIGHT (OFFSPRING)

Table 2.Offspring Body weights
Dose: 0 0.1% 0.5% 1%
Males
- Day 0 F1 6.5 6.8 6.4 6.6
F2 6.5 6.7 6.4 6.3
F3 6.7 6.7 6.8 6.1*
- Day 21 F1 15.6 14.7 13.7* 11.5*
F2 17.7 17.8 14.8* 12.4*
F3 19.7 19.9 17.6 13.4*
Females
- Day 0 F1 6.2 6.4 6.1 6.2
F2 6.1 6.4 6.1 6.0
F3 6.4 6.4 6.4 5.8*
- Day 21 F1 15.4 14.74 13.1* 10.8*
F2 16.5 17.1 14.5 11.4*
F3 18.6 19.3 16.0* 12.9*
* p < 0.05 (One-way ANOVA variance)
Dose descriptor:
NOAEC
Generation:
F1
Effect level:
0.1 other: %
Based on:
act. ingr.
Remarks:
DSS, diet
Sex:
male/female
Basis for effect level:
body weight and weight gain
Clinical signs:
no effects observed
Description (incidence and severity):
No significant differences from controls were observed for the total and mean number of pups born alive, litter size, survivability, or sex ratio.
Alopecia, tremors, and rough hair coat were recorded on day 21 for three pups (two males and one female) from one F2 litter at the 0.5% dose level.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
No significant differences from controls were observed for the total and mean number of pups born alive, litter size, survivability, or sex ratio.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Beginning on day 7, male and female pup weights and pup weight gains at the 0.5% and 1.0% dose levels were consistently lower than those of controls; the differences were statistically significant for all values except day 7 female pup weight, male and female pup weight gains for the 0.5% dose level. On Day 4 of lactation no milk was observed in the abdomen of three pups from two litters in the control group, seven pups from five litters in the 0.1% dose group, 18 pups from 10 litters in the 0.5% dose group, and 10 pups from five litters in the 1.0% dose group.
Food consumption and compound intake (if feeding study):
not examined
Description (incidence and severity):
Pups were allowed to nurse for 21 days before weaning. Food consumption after weaning is described under the parental generation.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Alopecia, tremors, and rough hair coat were recorded on day 21 for three pups (two males and one female) from one litter at the 0.5% dose level.
Histopathological findings:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
See under Details on results (F1).
Dose descriptor:
NOAEC
Generation:
F2
Effect level:
0.1 other: %
Based on:
act. ingr.
Remarks:
DSS, diet
Sex:
male/female
Basis for effect level:
body weight and weight gain
Key result
Reproductive effects observed:
no
Lowest effective dose / conc.:
1 other: %

Extension on the F2 parents to generate an F3 litter generation:

Male fertility indices at the 0.5% and 1.0% dose levels during breeding for F3 litters were significantly higher than that of the control group.

No significant differences from controls were observed for mating, fertility, or gestation indices, days required to mate, or the length of gestation. No parental females aborted or had physical or behavioral abnormalities during F3 gestation and lactation.

Body weights for males were significantly lower than those of controls throughout the treatment period at the 1.0%; dose level, and from Week 3 through the duration of the treatment period for males at t he 0.5%; dose level. Body weights were significantly lower than those of controls for F2 females at the 1.0% dose level throughout the premating phase (except Weeks 0 and 2} , and during Weeks 1, 3 through 6, and 8 for females at the 0.5% dose level.

Gestation body weights were significantly lower than those of controls at the 1.0% dose level throughout gestation. Body weights during lactation were significantly lower on Days 0, 7, and 14 at the 1.0% dose level; mean body weight on lactation Day 21 at the 1.0% dose level also was lower than that of the control, although this difference was not statistically significant.

Body weight gains were significantly lower than these of controls during weeks 4, 5 through 8, 13, 15, 17 , 20, 23, and 24 formales at the 1.0% dose level , during Weeks 4, 6, 7, 13, 16, 23, and 24 for males at the 0.5% dose level, and during Week 3 for males at the 0.1% dose level. There were no consistent significant differences in weight gains for females during the treatment period.

Body weight gains during lactation were significantly higher than these of the control between Days 14 and 21 at the 1.0% dose level, and between Days 0 and 21 for the 0.5% and 1.0% dose levels.

Food consumptions were significantly lower than those of controls during Weeks 2, 5 through 7, and 10 for males at the 1.0% dose level, and during Weeks 5 through 7 for males at the 0.5% dose level, Female food consumptions were significantly lower than those of controls during Week 2 at both the 0.5% and 1.0% dose levels, and were significantly higher during Week 7 at the 0.1% dose level.

F3 Litter data:

No significant differences from controls were observed for the mean number of pups born alive, litter size, survivability, or sex ratio. The proportions of the total number of pups born alive and found dead at the 1.0% dose level were significantly lower and higher, respectively, than those of the controls. These differences may merely reflect the effect of a slight reduction in the number of litters in the control group, and a disproportionate influence of very few litters in the high-dose group; i.e. , two litters accounted for 13 (six plus seven) of the 17 pups found dead; there was one dead pup each in four other litters. Male and female pup weights and pup weight gains at the 1.0% dose level were significantly lower than these of controls throughout lactation. At the 0.5% dose level, pup weight gains for males and females from Days 4 through 7, for males from Days 7 through 14, and tor fema1es from Days 14 through 21 were significantly lower than these of the control, as were male pup weights on Day 14 and male and female pup weights on Day 21. One pup in the0.5% dose group and 17 pups from seven litters In the 1.0% dose group had no milk in the abdomen on Day 4 of lactation. One female pup in the control group was icteric on Day 4. Another control pup from a different litter was missing the left eye. Five males and five females from one litter at the 1.0% dose level had urine stains on Day 21.

Conclusions:
Read-across substance DSS administered in the diet to three successive generations of rats at levels of 0.5% and 1.0% caused a reduction in body weights for parental males of all generations and for F1 and F2 females. Pup weights at the 0.5% and 1.0% dose levels were lower than those of the control in all three generations. However, the reduced body weight did not interfere with growth and development or reproductive performance, and the test substance had no adverse effects at levels on the reproductive function of either sex in any generation up to 1% in the diet.
Based on the results of this study, when read-across substance DSS was administered in the diet to three succesive generations of rats, the no-observable-effect level (NOEL) for body weights of parental animals and offspring was 0.1%; the NOEL for reproductive parameters was 1.0% DSS.
Executive summary:

Read-across substance Docusate sodium was administered in the diet to three successive generations at levels of 0.1%, 0.5% and 1.0% in the diets of 30 males and 30 females per group, dosed for 10 and 2 weeks respectively. The dose levels of 0.5% and 1.0% caused a reduction in body weights for parental males of all generations and for F1 and F2 females. Pup weights at the 0.5% and 1.0% dose levels were lower than those of the control in all three generations. However, the reduced body weight did not interfere with growth and development or reproductive performance, and the test substance had no adverse effects at levels on the reproductive function of either sex in any generation up to 1.0% in the diet. There were no other effects on parental or reproductive parameters.

Based on the results of this study, when read-across substance DSS was administered in the diet to three succesive generations of rats, the no-observable-effect level (NOEL) for body weights of parental animals and offspring was 0.1%; the NOEL for reproductive parameters was 1.0% DSS.

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to GLP and valid methods and is considered relevant and reliable for classification.
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
adopted March 22, 1996
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At +10°C to +25°C
- Stability under test conditions: The measured concentrations of the test item in the test item vehicle mixtures were between 99.7% and 108.8% of the nominal concentrations. These values indicated correctly prepared test item vehicle mixtures, which were stable at room temperature for at least 24 hours.
- Solubility and stability of the test substance in the solvent/vehicle: The test item vehicle mixtures were stable at room temperature for at least 24 hours. No phase separation occured between the test item and the vehicle during the procedure of administration of the test item vehicle mixtures to the animals.
Species:
rat
Strain:
other: CD® /Crl:CD(SD)
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 dosing: Males: 59 days; Females: 69 days
- Weight at start of dosing: Males: 328.9 – 368.9 g; Females: 210.3 – 253.9 g
- Fasting period before study: the night before the day of blood withdrawal for Iaboratory examination
- Housing: With exception of the mating period, the animals were kept singly in MAKROLON cages (type III plus) with a basal surface of approx. 39 cm x 23 cm and a height of approx. 18 cm. Granulated textured wood (Granulat A2, J. Brandenburg, 49424 Goldenstedt/Arkeburg, Germany) was used as bedding material in these cages. The cages were cleaned and changed once a week.
- Diet (e.g. ad libitum): Commercial ssniff® R/Z V1324 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany), ad libitum with the exception of the night before the day of blood withdrawal for Iaboratory examination
- Water (e.g. ad libitum): Tap water was offered daily ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C ± 3 °C (maximum range)
- Humidity (%): 55% ± 15% (maximum range)
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: October 25, 2012 To: Males: December 20, 2012; Females: January 3, 2013
Route of administration:
oral: gavage
Vehicle:
other: tap water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: administration volume was 10 mL/kg bw/day.
The test item was dissolved in the vehicle tap water to concentrations of 6, 12 and 30 mg test item/mL tap water. The test item formulations were freshly prepared and adjusted to the animal's current body weight on each administration day.

VEHICLE: tap water
Details on mating procedure:
- M/F ratio per cage: 1/1 (1 male and 1 female animal were placed in one cage during the dark period).
- Length of cohabitation: The female was placed with the same male until pregnancy had occurred or 2 weeks had elapsed.
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: yes, this procedure was repeated until at least 8 pregnant dams were available for each group.
- After successful mating each pregnant female was caged (how): The animals were kept singly in MAKROLON cages (type III plus) with a basal surface of approx. 39 cm x 23 cm and a height of approx. 18 cm, on one side of the room with each dose group separated by an empty row.
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
Males: Beginning 2 weeks prior to mating lasting up to the day before sacrifice until a minimum dosing period of 28 days was completed.
Females: Beginning 2 weeks prior to mating continuing up to, and including, day 3 post partum or the day before sacrifice.



Frequency of treatment:
Once daily
Details on study schedule:
Screening study
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
active ingredient
Dose / conc.:
60 mg/kg bw/day (actual dose received)
Remarks:
active ingredient
Dose / conc.:
120 mg/kg bw/day (actual dose received)
Remarks:
active ingredient
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
active ingredient
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels have been selected in agreement with the Sponsor based on the results of a 14-day dose-range-finding study in rats dosed at 100, 300 and 1000 mg (active ingredient)/kg bw by oral gavage (LPT Study No. 28932).
Four of 5 males and all (5) females died prematurely. Oral treatment with 1000 mg/kg bw/day caused signs of systemic toxicity in farm of pilo-erection, reduced motility, pultaeous faeces/diarrhoea, salivation, increased drinking water consumption, ataxia or decreased body temperature in the male and/or female rats.
A decrease in body weight, body weight at autopsy and food consumption was noted for the male and female rats treated with the intermediate and the high dose of Disodium C12-18 alkyl sulfosuccinate. At necropsy, whitish deposits on the stomach mucosa were observed in the male rats treated orally with 300 mg/kg bw/day. The high dose of 1000 mg/kg bw/day led to further changes in the gastro-intestinal tract in both sexes such as inflation or discolourations. The examination of organ weights revealed a dose-related increase of liver weights.
Based on the results of this study, the dose levels selected are 60, 120 and 300 mg/kg bw.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes , cage side observations included skin/fur, eyes, mucous membranes, respiratory and circulatory systems, somatomotor activity and behaviour patterns. The onset, intensity and duration of any signs observed were recorded.
- Time schedule: Throughout the test period, each animal were observed for clinical signs at least once daily. Individual animals were observed before and after dosing at each time of dosing for any signs of behavioural changes, reaction to treatment or illness. Mortality was recorded twice daily. In addition, animals were checked regularly throughout the working day from 7:00 a.m. to 3:45 p.m. On Saturdays and Sundays animals were checked regularly from 7:00 a.m. to 11:00 a.m. with a final check performed at approximately 3:30 p.m.
- Cage side observations checked in table [No.2-1 to 2-14] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
Additionally, once before the first exposure (to allow for within-subject comparisons) and once a week thereafter, detailed clinical observations were made in all animals; in test week 4 these observations were performed prior to any laboratory investigations. These observations were made outside the home cage in a standard arena and at the same time, each time. Signs observed included changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, pilo-erection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereo-typies (e.g. excessive grooming, repetitive circling) or bizarre behaviour (e.g. self-mutilation, walking backwards) were also recorded.
- Detailed clinical observations checked in table [No.3-1 to 3-3] were included.

BODY WEIGHT: Yes.
- Time schedule for examinations:
Males and females were weighed on the first day of dosing, weekly thereafter and at termination. During gestation, females were weighed on days 0, 7, 14 and 20 and within 24 hours of parturition (day 1 post-partum) and day 4 post-partum. Body weights were recorded individually for each adult animal.
The pups were weighed within 24 hours of parturition (day 1 post-partum) and on day 4 post-partum.

FOOD CONSUMPTION:
The quantity of food left by individual animals was recorded on a weekly or daily basis throughout the experimental period with the exception of the mating period.
Food intake per rat (g/rat/week) was calculated using the total amount of food given to and left by each rat in each group upon completion of a treatment week. From these data the food consumption (in g/kg bw/day) was determined using the following formula:
Relative food consumption[g/kg b.w./day] = (Total food given [g] - Total food left [g])/ Number of animal days# x Body weight [kg]

# The term 'animal days' counts one animal day for each animal alive for a whole day; it is assumed that on the day of death an animal does not eat.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Water consumption was monitored daily by visual appraisal throughout the study.

HAEMATOLOGY: see Section 7.5.1

CLINICAL CHEMISTRY: See Section 7.5.1

NEUROLOGICAL OBSERVATIONS: see Section 7.5.1

REPRODUCTIVE PARAMETERS:
Number of pregnant females
Pre-coital time
Gestation length calculated from day 0 of pregnancy
Corpora lutea
lmplantation sites
Number of (viable) pups day0/4


REPRODUCTIVE INDICES:
Gestation Index
Fertility Index
Birth Index
Live Birth Index
Viability Index
Pre-implantation loss [%]
Post-implantation loss [%]

Oestrous cyclicity (parental animals):
Estrus cycle was evaluated during histolopathological examination of the uterus and vagina.
Sperm parameters (parental animals):
Parameters examined in male parental animals (P):
testis weight, epididymis weight
At the time of sacrifice or death during the study, the adult animals were examined macroscopically for any abnormalities or pathological changes. Special attention was paid to the organs of the reproductive system.
The following organs or parts of organs of all male adult animals were fixed in 7% formalin; testes and epididymides were fixed in Bouin' s fixative:
Epididymis (2), Gross lesions, Prostate, Seminal vesicle, Testicle (2).
Detailed histopathologic examination was performed on one testicle and one epididymis (with special emphasis on the qualitative stages of spermatogenesis and histopathology of interstitial testicular structure) of all adult males of groups 1 to 4 following H-E and PAS staining.
Litter observations:
STANDARDISATION OF LITTERS
- screening study: Dead pups and pups sacrificed at day 4 post-partum, or shortly thereafter, were carefully examined externally for gross abnormalities.

PARAMETERS EXAMINED
Number of pups absolute (total/live)
Number of pups per dam (total/live)
Number of male and female pups (total/live)
Number of stillbirths
Mean pup weight

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was not determined for pups born or found dead.
Postmortem examinations (parental animals):
SACRIFICE
- Paternal animals animals: All surviving animals: The male animals were sacrificed on test day 43.
- Maternal animals: All surviving animals: Dams with offspring were sacrificed on day 4 postpartum, or shortly thereafter. Females showing no evidence of copulation were sacrificed 24 days after the last day of the mating period.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

ORGAN WEIGHTS: Yes
-The following organs of all adult animals were weighed individually before fixation and identified as left or right:
Epididymis (2), Testicle (2)
- Determination of the organ weights of the following organs was only performed from 20 adult males and 20 adult females, which were randomly selected: Adrenal gland (2), Hear, Liver, Thymus, Brain, Kidney (2), Spleen. Adrenal glands and kidneys were weighed individually and identified as left or right.
- Animals Nos.:
Group 1: 1, 2, 4, 5, 8 11, 14, 18, 19, 20
Group 2: 22, 25, 27, 29, 30 31, 34, 35, 36, 40
Group 3: 41, 43, 44, 48, 49 51, 54, 56, 57,59
Group 4: 62, 65, 66, 68, 69 72, 73, 75, 76, 78

HISTOPATHOLOGY: Yes
- The following organs or parts of organs of all adult animals were fixed in 7% formalin; testes and epididymides were fixed in Bouin's fixative:
Epididymis (2), Gross lesions, Mammary gland, Ovary (2), Prostate, Seminal vesicle, Testicle (2), Uterus (incl. cervix and oviducts), Vagina.
Detailed histopathological examination was performed on one testicle and one epididymis with special emphasis of the qualitative stages of spermatogenesis and histopathology of interstitial testicular structure of the selected animals of group 1 and 4 following haematoxylin-eosin and PAS staining. - See Section 7.8.1. & 7.8.2
- The following organs or parts of organs of all adult animals were fixed in 7% formalin; testes and epididymides were fixed in Bouin's fixative:
Epididymis (2), Gross lesions, Mammary gland, Ovary (2), Prostate, Seminal vesicle, Testicle (2), Uterus (incl. cervix and oviducts), Vagina
-In addition, the following organs or parts of organs of the selected 20 adult males and 20 adult females (see section above) were fixed in 7% formalin:
Adrenal gland (2)
Bone marrow (os femoris)
Brain (cerebrum, cerebellum, brain stem)
Heart (left and right ventricle, septum)
Intestine, small (duodenum, jejunum, ileum, incl. Peyer's patches, Swiss roll method)
Intestine, large (colon, rectum)
Kidney and ureter (2)
Liver
Lungs (with mainstem bronchi and
bronchioles), preserved by inflation with
fixative and then immersion
Lymph node (1, cervical), Lymph node (1, mesenteric)
Nerve (sciatic)
Oesophagus
Spinal cord (3 sections)
Spleen
Stomach
Thyroid (incl. parathyroids)
Thymus
Tissue masses or tumours (incl. regional lymph nodes)
Tongue (incl. base)
Trachea (incl. larynx)
Urinary bladder
-Only the 10 selected animals from the control group and the high dose group (20 animals in total) were considered for histopathological evaluation.
Group 1: 1, 2, 4, 5, 8 11, 14, 18, 19, 20
Group 4: 62, 65, 66, 68, 69 72, 73, 75, 76, 78

Adrenal glands and kidneys were weighed individually and identified as left or right.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed at 4 days of age.
- These animals were subjected to postmortem macroscopic examinations as follows:
Dead pups and pups sacrificed at day 4 post-partum, or shortly thereafter, were carefully examined externally for gross abnormalities.
The animals were sacrificed under ether anaesthesia by cutting the aorta abdominalis, exsanguinated, weighed, dissected and inspected macroscopically. All superficial tissues were examined visually and by palpation and the cranial roof removed to allow observation of the brain, pituitary gland and cranial nerves. After ventral midline incision and skin reflection all subcutaneous tissues were examined. The condition of the thoracic viscera were noted with due attention to the thymus, lymph nodes and heart.
The abdominal viscera were examined before and after removal; the urinary bladder was examined externally and by palpation. The gastro-intestinal tract was examined as a whole and the stomach and caecum were incised and examined.
The lungs were removed and all pleural surfaces examined under suitable illumination.
The liver and the kidneys were examined. Any abnormalities in the appearance and size of the gonads, adrenals, uterus, intra-abdominal lymph nodes and accessory reproductive organs were recorded.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.
Statistics:
Toxicology and Pathology data were captured, whenever possible, using the departmental computerized systems (Provantis® Integrated preclinical software, Instem LSS Ltd.). Raw data not fully compatible with the computerized systems were maintained on paper according to appropriate SOPs.
The test item-treated groups (2- 4) were compared with the control group (1 ).
The following statistical methods are used:

STUDENT's t-test: All numerical functional tests (≤ 0.05 and p ≤0.01)

Multiple t-test based on DUNNETT, C. W .; New tables for multiple Comparisons with a control; Biometrics, 482-491 (Sept 1964): Body weight I Food consumption IHaematology I Clinical chemistry I Absolute and relative organ weights (≤0.05 and p ≤ 0.01)

For all numerical values (e.g. body weight, food consumption and organ weight data) homogeneity of variances was tested by using the BARTLETT chi-square test. lf the variances were homogeneous, the DUNNETT test (p ≤ 0.01) was used to compare the experimental groups with the control group.
In case of heterogeneity of variances, the STUDENT' s t-test was carried out; limit of significance was p≤0.01.

For the comparison of classification measurements (for example the fertility index) the FISHER's exact test, n < 100 or chi2-test with Yates' correction for continuity, n ≥100 (p ≤0.05 and p ≤ 0.01) were employed.

These statistical procedures were used for all data. Significantly different data were indicated in the tables of the report.
The mean values and standard deviations were calculated to the highest possible degree of accuracy and then rounded to the reported number of decimal places. Hence, deviations to the last decimal place of up to ± 1 may occur caused by rounding.
Reproductive indices:
Gestation Index
Fertility Index
Birth Index
Live Birth Index
Viability Index
Pre-implantation loss [%]
Post-implantation loss [%]
Offspring viability indices:
Gestation length calculated from day 0 of pregnancy
Corpora lutea
lmplantation sites
Number of pups absolute
Number of pups per dam
Number of male and female pups
Number of stillbirths
Number of pups with malformations
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Slight salivation was noted in 2 animals of the intermediate dose group (120 mg act. ingr./kg b.w./day) on one day each. In the high dose group (300 mg act. ingr./kg b.w./day) piloerection and slight to moderate salivation were noted for several animals during the whole study for 1 up to 13 test days. Breathing sounds were noted in animal no. 61 for 1 day.
In the intermediate dose group (120 mg act. ingr./kg b.w./day) piloerection was noted for 1 animal on 1 day during the mating period. During the gestation period moderate salivation was noted in 3 animals on one day each. In the high dose group (300 mg act. ingr./kg b.w./day) piloerection was noted for several animals on 3 up to 10 test days and slight to extreme salivation in all animals (2 up to 11 test days) during the pre-mating, mating and gestation period. A haemorrhagic vagina or nose was noted for animal no. 72 on gestation days 11 and 16. During the lactation period piloerection,
reduced motility and changes in the status of faeces were noted for the emaciated animal no. 75.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
1male and 1 female of the high dose group died on day 33 and day 26 respectively; slight signs of systemic toxicity were noted predominantly in form of pilo-erection and increased salivation in males and females dosed at 120 & 300 mg/kg bw.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
decreased at 120 and 300 mg/kg bw in male rats and at 300 mg/kg bw in female rats premating, mating, during gestation and during lactation
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
A statistically significant (p≤0.01) reduction in food consumption by 14.1% was noted in the intermediate dose group (120 mg act. ingr./kg b.w./day) during the first test week.
In the high dose group (300 mg act. ingr./kg b.w./day) the food consumption was statistically significantly (p≤0.01) reduced by 20.7% during the first and by 15.3% during the second test week..
At the intermediate dose group (120 mg act.ingr./kg b.w./day) a statistically significant reduction in food consumption was noted during the first week of gestation by 7.3% (p≤0.05)
and during the second week of gestation by 13.8% (p≤0.01). In the high dose group (300 mg act. ingr./kg b.w./day) a statistically significant (p≤0.01) reduction in food consumption was noted from
the first test week (by 26.3%) until the end of the gestation period (by 6.8%).
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
increased MCHC in males at 120 mg/kg bw; increased haemoglobin, red blood cells, haematocrit and MCHC value in males dosed 300 mg/kg bw; decrease aPTT time in females dosed 300 mg/kg bw
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
increased ALAT in males dosed at 120 mg/kg bw; increased ALAT, aP and ASAT and decreased cholesterol in males dosed at 300 mg/kg bw; increased ALAT and ASAT and decreased globulin, cholesterol, chloride, potassium in females dosed 300 mg/kg bw
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
hepatocellular hypertrophy and macrovesicular vacuolation in liver and squamous cell hyperplasia in the non-glandular stomach in males and females at 300 mg/kg bw; changes in the mammary glands, the uterus and vagina in females at 300 mg/kg bw
Histopathological findings: neoplastic:
not examined
Reproductive function: oestrous cycle:
effects observed, treatment-related
Description (incidence and severity):
Only 1/5 high dose females was in metestrus versus 4/5 of the controls, suggesting perturbation of the oestrus cycle in animals dosed at 300 mg/kg when compared to the controls.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Histopathological examination performed on one testicle and one epididymis (with special emphasis on the qualitative stages of spermatogenesis (proliferative, meiotic and spermiogenic phases)(1,2) and histopathology of the interstitial testicular structure), did not reveal any test item-related effects. No test item-related microscopic changes were seen in the reproductive organs for males.
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
reduced gestation index, No. implantation sites and No.(live) born pups per dam and birth index; increased implantation loss index at 300 mg/kg bw
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
One of 10 male animals (no. 64) of the high dose group (300 mg test item/kg bw/day) died on test day 33, showing piloerection and reduced motility before death.
One of 10 female animals (no. 71) of the high dose group (300 mg test item/kg bw/day) died on gestation day 9, showing piloerection and salivation on a few days during the premating, mating and gestation period.
Male animals
Slight salivation was noted in 2 animals of the intermediate dose group (120 mg test item/kg bw/day) on one day each.
In the high dose group (300 mg test item/kg bw/day) piloerection and slight to moderate salivation was noted for several animals during the whole study for 1 up to 13 test days. Breathing sounds were noted in animal no. 61 for 1 day.
Female animals
In the intermediate dose group (120 mg test item/kg bw/day) piloerection was noted for 1 animal on 1 day during the mating period. During the gestation period moderate salivation was noted in 3 animals on one day each.
In the high dose group (300 mg test item/kg bw/day) piloerection was noted for several animals on 3 up to 10 test days and slight to extreme salivation in all female animals (2 up to 11 test days) during the pre-mating, mating and gestation period. A haemorrhagic vagina or nose was noted for animal no. 72 on gestation days 11 and 16. During the lactation period piloerection, reduced motility and changes in the status of faeces were noted for the emaciated female animal no. 75.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Male animals
A reduction in body weight was noted in the intermediate dose group (120 mg test item/kg bw/day) from test day 8 by 6.7% until the end of the study by 6.2%, statistically significant (p≤0.05) on test days 8, 22, 29 and 42.
In the high dose group (300 mg test item/kg bw/day) the reduction in body weight was more pronounced, with 9.0% on test day 8 and 13.3% at the end of the study, statistically significant (p≤0.01) from test day 8 to the end of the study.
Accordingly, statistically significant (p≤0.05 or p≤0.01) reductions in body weight gain were noted in the intermediate and the high dose group (120 and 300 mg test item/kg bw/day).
Female animals
A decrease in body weight (300 mg test item/kg bw/day) was noted in the high dose group, starting at the end of the pre-mating period by 5.4%. Statistically significant (p≤0.01) reductions were noted during the gestation period from gestation day 7 (by 9.6%) to 20 (by 23.9%) and on lactation day 1 (by 22.0%) and 4 (by 23.8%).
Statistically significant reductions in body weight gain were noted on gestation day 14 (p≤0.01) and 20 (p≤0.05) in the intermediate dose group (120 mg test item/kg bw/day).
In the high dose group (300 mg test item/kg bw/day) statistically significant reductions in body weight gain were noted on test day 8 (p≤0.01) and during the gestation period from gestation day 7 (p≤0.05) to gestation day 20 (p≤0.01).
Male animals
A statistically significant (p≤0.01) reduction in food consumption by 14.1% was noted in the intermediate dose group (120 mg test item/kg bw/day) during the first test week.
In the high dose group (300 mg test item/kg bw/day) the food consumption was statistically significantly (p≤0.01) reduced by 20.7% during the first and by 15.3% during the second test week.
Female animals
At the intermediate dose group (120 mg test item/kg bw/day) a statistically significant reduction in food consumption was noted during the first week of gestation by 7.3% (p≤0.05) and during the second week of gestation by 13.8% (p≤0.01).
In the high dose group (300 mg test item/kg bw/day) a statistically significant (p≤0.01) reduction in food consumption was noted from the first test week (by 26.3%) until the end of the gestation period (by 6.8%).

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS): gavage

REPRODUCTIVE FUNCTION:
-Pre-coital time:
No test item-related influence was noted.
-Gestation length:
No test item-related influence was noted.
-Evaluation of reproduction parameters of the dams:
Only 3 of 8 pregnant dams of the high dose (300 mg test item/kg) group littered live pups, leading to a statistically significant (p≤0.01) reduced gestation index of 37.5%.
Five of 8 pregnant dams showed a total loss of implantation sites, leading to a statistically significant (p≤0.05) reduction in the mean number of implantation sites per dam and a statistically significantly (p≤0.01) increased post-implantation loss of 66.3%.
Accordingly, the whole number of born pups (alive and dead) per dam and the number of live born pups per dam were statistically significantly (p≤0.01) reduced, leading to a statistically significant (p≤0.01) reduction of the birth index to 39.5%.
The percentage of stillbirths in the high dose group was 14.7%, leading to a statistically significant (p≤0.05) reduction in the live birth index.
No test item related differences were noted for the fertility index and the preimplantation loss.

ORGAN WEIGHTS (PARENTAL ANIMALS)
Male animals
Starting at the intermediate dose group (120 mg test item/kg bw/day) a statistically significant (p≤0.05, right only) dose related decrease by 18.3% at maximum was noted for the absolute organ weight of the left + right epididymis.
In the high dose group (300 mg test item/kg) the following statistically significant changes were noted:
An increase by 34.0% (p≤0.01) of the relative liver weight and by 19.6% (non-significant) of the absolute liver weight.
The relative and absolute organ weight of the thymus was decreased by 42.5% (p≤0.05) and by 49.0% (p≤0.01).
The relative organ weights of the left and right gonads were reduced by 14.9% (p≤0.01) and by 13.7% (p≤0.05).
Female animals
In the intermediate dose group (120 mg test item/kg) a statistically significant (p≤0.01) increase was noted in the relative liver weight by 23%.
The absolute heart weight was statistically significantly decreased by 16.7%.
The relative organ weights of the left and right adrenal glands were statistically significantly (p≤0.01) increased by 26.2% and 24.4%.
In the high dose group (300 mg test item/kg) the relative organ weights of the left and right kidneys were statistically significantly increased by 17.2% (p≤0.05) and by 20.9% (p≤0.01).
The absolute organ weight of the heart was statistically significantly (p≤0.05) decreased by 33.1%.
The absolute organ weight of the right gonad was statistically significantly (p≤0.05) decreased by 37.5% and the absolute organ weight of the left gonad non-significantly by 25.5%.
Similar but not statistically significant increases as in the intermediate dose groups were noted for the relative liver weight and the left and right adrenal glands.

GROSS PATHOLOGY (PARENTAL ANIMALS)
Body weight at autopsy:
Male animals
In the high dose group (300 mg test item/kg bw/day) the body weight at autopsy was statistically significantly (p≤0.01) decreased by 13.5%.
Female animals
A statistically significant (p≤0.01) reduction in the body weight at autopsy by 22.2% was noted in the high dose group (300 mg test item/kg bw/day).
Macroscopic post mortem findings:
Male animals
Macroscopic changes were noted in the stomach of 3 animals of the high dose group (300 mg test item/kg bw/day) in form of a detachment of the mucosa, whitish thickenings and ulcers. The findings were considered to be test item-related.
Female animals
A test item-related detachment of the mucosa was noted in the stomach of one animal of the inter-mediate dose group (120 mg test item/kg bw/day)
In the high dose group (300 mg test item/kg bw/day) a test item-related detachment of the mucosa was noted in 2 animals.

HISTOPATHOLOGY (PARENTAL ANIMALS)(restricted to the control group and the high dose group):
Male and female animals
Test item related changes were noted in the liver (hepatocellular hypertrophy and macrovesicular vacuolation evoked by fatty change) and in the non-glandular stomach (squamous cell hyperplasia) in the animals of the high dose group (300 mg test item/kg).
Female animals
Test item related changes were noted in the mammary glands, the uterus and vagina in form of a decreased acinar development, stromal hyperplasia in the endometrium and metestrus in only 1 of 5 animals of the high dose group (300 mg test item/kg bw/day).

No microscopic changes were noted for the reproductive organs of the male and female rats of the high dose group (300 mg test item/kg bw/day) and no changes were noted on the stages of spermatogenesis.
Dose descriptor:
NOAEL
Effect level:
120 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: reproductive effects
Clinical signs:
not specified
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
reduced survival rate of the pups (75.9% versus control) in the high dose group of 300 mg test item/kg
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
reduced at 300 mg/kg bw on lactation day 1 by 17.1% (p≤0.05) and on lactation day 4 by 29.3% (p≤0.01)
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
VIABILITY (OFFSPRING)
The survival rate of the pups was statistically significantly reduced to 75.9% in the high dose group (300 mg test item/kg)

BODY WEIGHT (OFFSPRING)
The mean litter weight of the pups was statistically significantly reduced on lactation day 1 by 17.1% (p≤0.05) and on lactation day 4 by 29.3% (p≤0.01) in the high dose group (300 mg test item/kg).
The total litter weight per dam was also statistically significantly reduced on lactation day 1 by 42.4% (p≤0.05) and on lactation day 4 by 56.3% (p≤0.01) in the high dose group (300 mg test item/kg).

GROSS PATHOLOGY (OFFSPRING)
No visible gross abnormalities were noted in the control and the treatment groups.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
120 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
body weight and weight gain
Reproductive effects observed:
not specified

Table 1. Fertility and Reproductive parameters Parental generation

Parameter

Group 1

Control

Group 2

60 mg/kg

Group 3

120 mg/kg

Group 4

300 mg/kg

No. females evaluated

10

10

10

10

Mean precoital time (days)

3.8

3.4

2.9

3.8

Number of pregnant dams

10

10

10

8 +1#

Fertility index (%)

100

100

100

90

No. dams with pups (live + dead)

10

10

10

4

Gestation length (days)

22.7

22.9

22.9

23.3

No. dams with live pups

10

10

10

3

Gestation Index (%)

100

100

100

37.5**

No.Corpora lutea(total)

160

142

151

104

No.Corpora lutea(mean)

16.0

14.2

15.1

13.0

No. Implantation sites (total)

143

119

149

86

No. Implantation sites (mean)

14.3

11.9

14.9

10.8v

Number of pups at birth (total)

136

109

134

34vv

Number of pups at birth (mean)

13.6

10.9

13.4

4.3vv

Birth Index (mean %)

95.1

90.3

90.2

36.0

Birth Index (total %)

95.1

91.6

89.9

39.52

Number of stillbirths

3

0

4

5

No. of dams with stillborn pups

2

0

1

2

Number of live born pups (total)

133

109

130

29vv

Number of live born pups (mean)

13.3

10.9

13.0

3.6vv

Live birth index (mean %)

97.7

100.0

96.7

67.3

Live birth index (total %)

97.8

100.0

97.0

85.3b

Pre-implantation loss (mean %)

8.8

16.1

1.3

14.7

Pre-implantation loss (total %)

10.6

16.2

1.3b

17.3

Post-implantation loss (mean %)

7.9

9.7

12.6

68.5

Post-implantation loss (total %)

7.0

8.4

12.8

66.3b

1 p≤0.05 Chi2-test

2 p≤0.01 Chi2-test

*  p≤0.05 Fisher test

**p≤0.01 Fisher test

v p≤0.05 Dunnett test or Student’s t-test

vvp≤0.01 Dunnett test or Student’s t-test

ap< 0.05 Chi2-test

bp<0.01 Chi2-test

#: Animal no. 71 was excluded, because of its premature death on gestation day 9

Conclusions:
The following no-observed adverse-effect levels were established:
Paternal and Maternal toxicity: NOAEL= 60 mg/kg b.w./day, p.o.
Reproductive toxicity: NOAEL= 120 mg/kg b.w./day, p.o.
Executive summary:

The aim of the study was to obtain information on possible effects of the test item on general toxicity, reproduction and/or development according to OECD guideline 422. The test item was administered orally to rats at dose levels of 60, 120 or 300 mg test item/kg bw/day. The application started two weeks before mating on test day one and ended on the day or one day before sacrifice. Day of sacrifice was on test day 43 for the male rats and on lactation day 4 or shortly thereafter for the female rats.

Effects on the parental generation (general toxicity) :

One of 10 male and one of 10 female animals of the high dose group (300 mg test item/kg bw/day) died prematurely on test day 33 or on gestation day 9 (TD 26). Slight to moderate salivation was noted in a few male and female animals of the intermediate dose group (120 mg test item/kg bw/day) on 1 day each, which was regarded as test item-related. In the high dose group (300 mg test item/kg bw/day) piloerection and a slight to extreme salivation was noted for several to all male and female animals on several days and regarded as test item-related. A statistically significant reduction in body weight was noted for the male animals of the intermediate dose group (120 mg test item/kg bw/day) and for both sexes at the high dose group (300 mg test item/kg bw/day).

Statistically significant increases in the activity of ALAT and/or aP and ASAT and decreases in the globulin, cholesterol, chloride and potassium concentrations were noted for the male and/or female animals of the intermediate and/or the high dose group (120 and/or 300 mg test item/kg bw/day).

Statistically significant changes were noted for several organ weights of the male and female animals of the intermediate and the high dose group (120 and 300 mg test item/kg bw/day), most remarkable for the thymus and liver weights of the animals of the high dose group.

Macroscopic inspection at autopsy revealed test item-related changes in the stomachs of male and female animals of the intermediate and high dose group (120 and 300 mg test item/kg bw/day).

Histopathological examination of the organs from animals of the high dose group (300 mg test item/kg bw/day) revealed test item-related changes in the liver (hepatocellular hypertrophy and macrovesicular vacuolation evoked by fatty change) and the non-glandular stomach (squamous cell hyperplasia) of male and female animals.

Reproductive toxicity

The high number of 5 pregnant dams with a total loss of implantation sites in the high dose group (300 mg test item/kg bw/day) led to a statistically significant reduction in the gestation index, in the mean number of implantation sites per dam, in the mean number of born pups per dam and in the birth index. Accordingly, the implantation loss index was statistically significantly increased in the high dose group.

The high percentage of stillbirths led to a statistically significantly reduced live birth index in the high dose group (300 mg test item/kg bw/day). Test item related effects were also noted on the pups from the 3 remaining dams of the high dose group (300 mg test item/kg bw/day), expressed by a statistically significantly reduced survival rate during the lactation period, a statistically significant reduction in the mean litter weight and in the total litter weight per dam on lactation day 1 and 4.

The following no-observed adverse-effect levels were established:

Paternal and Maternal toxicity: NOAEL= 60 mg/kg b.w./day, p.o.

Reproductive toxicity: NOAEL=120 mg/kg b.w./day, p.o.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
120 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Klimisch 1
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Reproductive screening

A key study for reproductive screening was performed for the registered substance by means of an oral combined repeated dose and reproduction/development screening study according to OECD guideline 422 (Hansen, 2013a). The test item was a solid formulation (containing >93% active ingredient) which was administered as a watery solution orally by gavage to rats at dose levels of 60, 120 and 300 mg act. ingr./kg bw/day for for at least 28 days in male rats up to 54 days in female rats. Paternal/maternal toxicity was observed at 120 and 300 mg/kg bw in males. No test item-related influence was noted on the reproduction toxicity parameters at 60 and 120 mg/kg bw. The high number of 5 pregnant dams with a total loss of implantation sites in the high dose group (300 mg/kg b.w./day) led to a statistically significant reduction in the gestation index, in the mean number of implantation sites per dam, in the mean number of born pups per dam and in the birth index. Accordingly, the implantation loss index was statistically significantly increased in the high dose group. The high percentage of stillbirths led to a statistically significantly reduced live birth index in the high dose group (300 mg /kg b.w./day). Test item related effects were also noted on the pups from the 3 remaining dams of the high dose group (300 mg/kg b.w./day), expressed by a statistically significantly reduced survival rate during the lactation period, a statistically significant reduction in the mean litter weight and in the total litter weight per dam on lactation day 1 and 4.

In conclusion, general parental toxicity was higher than reproductive toxicity, and the reproductive findings were considered to be secondary to the paternal/maternal toxicity. NOAEL-levels were as follows: 60 mg/kg bw for paternal/maternal toxicity (based on clinical signs, reduced body weight, food consumption, organ weights and changes in biochemical parameters at the high dose levels) and 120 mg/kg bw for reproductive toxicity (based on reduced gestation index, birth index, live birth index and increased post implantation loss at the high dose levels).

 

Multigeneration studies

Further data on reproductive toxicity were available from read across substance Docusate sodium (CAS No. 577-11-7). Justification for read across with the category of Di-ester sulphosuccinates is documented in a separate document attached in Section 13. - A key 3-generation toxicity study at dietary dose levels of 0.1, 0.5 and 1.0% in the diet (MacKenzie, 1986) conducted according to OECD caused a reduction in body weights at the dose levels of 0.5 and 1% in the diet for parental males of all generations and for F1 and F2 females. Pup weights at the 0.5% and 1.0% dose levels were lower than those of the control in all three generations, however this did not interfere with growth and development or reproductive performance, and had no adverse effects at levels on the reproductive function of either sex in any generation up to 1%. There were no other effects on parental or reproductive parameters. The NOEL for body weights of parental animals and offspring was 0.1%; the NOEL for reproductive parameters was 1.0%, which was considered to correspond with approximately 750 mg/kg bw/day. - In a supporting 2-generation toxicity study in rats, 0.5 and 1% were given in the diet (Levinskas & Shaffer, 1970). In the first mating of the F0 generation and the second mating of the F2 generation, pups were weaned directly onto the diets which were being fed to their parents. In the other 3 matings of this study, dams were given a control diet on the day before delivery to avoid a bitter taste of the milk. Pups of all litters were examined for gross defects. Autopsies were performed, however, only on pups from the first mating of the F2 animals. Portions of all major organs were taken for histopathology processing and examination from one male and female from each litter. The other male and female were skinned and eviscerated, and the carcasses cleared, and the skeletons stained and examined for defects. In both the first mating of the F0 generation and the second mating of the F2 generation, the fertility and gestation indices were high and comparable. The viability index was good, albeit slightly down for the F3b pups, while the lactation index was depressed for both of these matings. In addition, the mean weight of the pups at weaning decreased with increasing concentrations of test material in the diet of the dams. In the second mating of the F0 animals, the viability and lactation indices and the mean weight of the test pups at weaning still showed decreases relative to the control values. However, in the 2 subsequent matings, all indices for the dosed animals were numerically high and compared favorably with the corresponding control values. Also, the mean weight of the pups at weaning was essentially similar for all groups. Consequently, it is concluded that diets containing 1% or less had no adverse effect on the reproduction and lactation performance of rats. The lowering of the survival rate and the mean body weight of the F1a and F3b pups is attributed to an impairment of nutrition as a result of the taste which is believed to have been secreted into the milk of the dams. Microscopic study of tissues showed findings which were similar in all groups. In processing the skeletons, the presence of an extra sternebrae in the sternum between the 5th and 6th sternebrae was not considered to parental exposure of test material. It is concluded that feeding of test material to rats from weaning through reproductive age for successive generations at levels of 1%, or less, did not produce lesions or anomalies in the offspring which could be attributed to the compound.

In conclusion, 2 multigeneration studies with read-across substance CAS no. 577 -11 -7 (Docusate sodium) showed that there were no reproductive findings.

 

Conclusion

An oral gavage reproductive screening study with registered substance showed a NOAEL of 60 mg/kg bw for paternal/maternal toxicity, whereas 120 mg/kg bw was NOAEL for reproductive and developmental toxicity. The latter was based on reduced gestation index, (life) birth index and No. of pups and increased post-implantation loss at 300 mg/kg bw. In conclusion, general parental toxicity was higher than reproductive toxicity, and the reproductive findings were considered to be secondary to the paternal/maternal toxicity. Multigeneration studies with read across substance Docusate sodium (CAS 577-11-7) showed slight maternal/paternal toxicity at 0.5 and 1% in the diet, however this was not confirmed in a second study. The test article was considered to have influenced the taste of the milk, for which adaptations were done in the second study. From both studies, it can be concluded that the substance up to 1% in the diet did not lead to effects on fertility or postnatal development; this concentration corresponded with 750 mg/kg bw/day, which is higher than NOAEL for paternal/maternal toxicity. Based on the secondary findings to parental toxicity in the screening study, and absence of reproductive findings in the repeated dose studies and the multigeneration studies with structurally similar substance, no further testing is needed.

 

 

Justification for selection of Effect on fertility via oral route: Key study for reproductive toxicity screening. The study was selected as it was done with registered substance and because it had a lower NOAEL than the multigeneration toxicity studies with read across substance.

Effects on developmental toxicity

Description of key information

A combined reproductive/developmental screening study according to OECD 422 which was performed with registered substance did not reveal external visible changes up to highest tested dose of 300 mg/kg bw. Decreased number of pups alive on day 4 of lactation and decreased viability index were observed, as well as decreased body weight of the pups both on day 0 and day 4. NOAEL for paternal/maternal toxicity was 60 mg/kg bw/day, whereas NOAEL for developmental toxicity was 120 mg/kg bw. The developmental findings were considered secondary to the paternal/maternal toxicity.

A combined reproduction-teratogenicity study with read across substance CAS No. 37294-49-8 (disodium C-isodecyl sulphonatosuccinate) in 2 generations resulted in a NOAEL for embryotoxicity of 750 mg/kg (1% in the diet) and a NOEL for teratogenicity of 3000 mg/kg (4% in the diet).

Prenatal developmental toxicity was tested by dietary administration of read across substance Docusate sodium in rats from day 6 to 15 of gestation. 1% in the diet was a maternal and developmental NOAEL, whereas at 2% in the diet visceral and skeletal anomalies were observed, which was secondary to maternal toxicity. This was confirmed in a similar study with Docusate calcium given at subtoxic and toxic dose levels, where the same could be observed. Based on the absence of developmental findings in the screening study and teratogenicity study, no further testing is needed.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
see attached read-across justification
Reason / purpose:
read-across source
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Among rats given dietary levels of 2.0% DSS, there were significant depressions in maternal weight-gains.
Rats fed diets containing 1.0% level of DSS showed no significant maternal effects on the various parameters.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not specified
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Details on results:
Rats fed diets containing 1.0% level of DSS) showed no significant maternal effects on the various parameters. Among rats given dietary levels of 2.0% DSS, there were significant depressions in maternal weight gains.
Number of abortions:
no effects observed
Pre- and post-implantation loss:
not specified
Total litter losses by resorption:
effects observed, treatment-related
Description (incidence and severity):
In the 2.0% DSS group 1 pregnancy with total resorptions was observed (No statistical significance). No pregnancy with total resorptions was observed in the control or 1.0% DSS group.
Early or late resorptions:
effects observed, treatment-related
Description (incidence and severity):
Among rats given dietary levels of 2.0% DSS, there were significant increases in the number of resorptions of 13.7% as compared to the control frequency of 5.6%.
Dead fetuses:
no effects observed
Description (incidence and severity):
0.5% occurrence of dead fetuses was seen in the 2.0% DSS group versus 0.7% in the control group. No dead fetuses were observed in the 1.0% DSS group.
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
not examined
Details on maternal toxic effects:
Maternal toxic effects:yes. Remark: 2.0% in the diet
Key result
Dose descriptor:
NOAEC
Effect level:
1 other: %
Based on:
act. ingr.
Remarks:
in the diet
Basis for effect level:
body weight and weight gain
early or late resorptions
Remarks on result:
other: DSS
Key result
Dose descriptor:
NOAEL
Effect level:
1 074 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Basis for effect level:
body weight and weight gain
early or late resorptions
Remarks on result:
other: DSS
Fetal body weight changes:
no effects observed
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
There is no significant reduction in viable fetuses in the dosed animals animals compared to control animals.
Changes in sex ratio:
not specified
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
Description (incidence and severity):
There is no postnatal evaluation in an OECD 414 study.
External malformations:
effects observed, treatment-related
Description (incidence and severity):
Near toxic or toxic dietary levels of 2.0% DSS produced significant incidences of gross abnormalities either among litters (25.0%) or fetal populations (20.2%) as compared to none in the controls. These abnormalities consisted of cranial buble, exencephaly, spina bifida (not significant), microphtalmia or anophtalmia (not significant).
Skeletal malformations:
no effects observed
Visceral malformations:
effects observed, treatment-related
Description (incidence and severity):
The visceral observations confirmed the significance of the exencephalous characteristics and anophtalmia for the group given dietary levels of 2.0% DSS.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
In the 2.0% DSS group, skeletal observations revealed a significant incidence of variations including incomplete ossification to absence of the various cranial bones, a curved or open vertebral column, and a variety of defects of the vertebrae and ribs.
Details on embryotoxic / teratogenic effects:
Details on embryotoxic / teratogenic effects:
See Table 1-4.
Key result
Dose descriptor:
NOAEC
Effect level:
1 other: %
Based on:
act. ingr.
Remarks:
diet
Sex:
male/female
Basis for effect level:
external malformations
visceral malformations
other:
Remarks on result:
other: secondary to high maternally toxic dose
Key result
Dose descriptor:
NOAEL
Effect level:
1 074 mg/kg bw/day
Based on:
act. ingr.
Remarks:
diet
Sex:
male/female
Basis for effect level:
external malformations
visceral malformations
other: skeletal variations
Abnormalities:
effects observed, treatment-related
Localisation:
external: cranium
skeletal: skull
skeletal: rib
visceral/soft tissue: central nervous system
visceral/soft tissue: eye
Description (incidence and severity):
only at 2.0% dietary level.
Developmental effects observed:
yes
Lowest effective dose / conc.:
2 other: %
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
no

Table 1. Maternal and fetal results of pregnant rats given various amounts if DSS in their diets during  gestational days 6 through 15.

Parameter

 Control        

1.0% DSS

2.0% DSS

Maternal

Group  (I-A)

(II-A)

(II-B)

No. of pregnant rats

43

22

20

No. of pregnancies with total resorptions

0

0

1

No. of pregnancies with viable fetuses

43

22

19

Average weight gain of dams with viable fetuses(g):

 

 

 

Days 6 to 15

78

86

52*

Days 15 to 21

66

67

77

Average, apparent food intake of dams with viable fetuses (g/rat/day):

 

 

 

Days 6 to 15

22.5

24.8

21.4

Days 15 to 21

28.6

32.1

33.4

Calculated compound consumed (mg/kg/day)

--

1074

1988

Litters

 

 

 

Total number of:

implantations

 

411

 

203

 

219

Resorptions

(% occurence)

23

(5.6)

8

(3.9)

30*a

(13.7)

Dead fetuses

(% occurrence)

3

(0.7)

0

1

(0.5)

Viable fetuses

(% occurrence)

385

(93.7)

195

(96.1)

188

(85.5)

Fetal weight (g)

4.6

5.2

4.7

Litters size (viable fetuses)

8.9

8.9

9.9

External major malformations1:

No. of litters affected

(% occurrence)

 

 

0

 

 

0

 

 

5*

(25.0)

No. of fetuses affected

(% occurrence)

 

0

 

0

36*a

(20.2)

* Significantly different from control (p< 0.05)

a Significance by Chi-square, but not Mann-Whitney U test

1 Primarily, exencephaly varying degrees and associated anomalies (See Table 2)

    

Table 2. Morphological observations of fetuses delivered from rats given DSS in their diets on gestational days 6 through 15.

Morphology

 Control

1.0% DSS

2.0% DSS

External observations1:

Group (I-A)

(II-A)

(II-B)

Total number examined

388a

195

189

Major anomalies:

  Adactyly

 

0

 

0

 

0

  Hemimelia

0

0

0

  Schistocelia

0

0

2

  Dome shaped head

0

0

0

  Cranial bubble (1-2mm)

0

0

9*

  Exencephaly

0

0

18*

  Exencephaly (cleft condition)

0

0

7*

  Anencephaly

0

0

0

  Spina bifida

0

0

6

  Macroglossia

0

0

0

  Micro- or anophtalmia

0

0

3

Defects:

  Hematoma (subcutaneous)

 

2

 

0

 

0

  Edamatous abdomen

0

0

0

  Tail short & curled

0

0

0

  Abducted fifth digit, left

   Rear foot

0

0

1

1 Fetuses may have more than one defect

a Fifty-four fetuses examined grossly only. (Shipment c valid as controls only)

      *Significantly different from control (p< 0.05) by Chi-square only

 

Table 3. Visceral observations of fetuses delivered from rats given DSS in their diets on gestation days  6 through 15.

Visceral observations

Dose:      Control

1.0 % DSS

2.0% DSS

Groups:       (I-A)

(II-A)

(II-B)

Total number of fetuses examined

165a

98

91

Defects1:

  Exencephalous   characteristics                     

 

0

 

0

 

11*

  Dilated lateral ventricles

1

3

5

  Microphtalmia

0

1

0

  Anolphtalmia

0

0

23*

  Retinal foldings

0

0

0

  Anotia or microtia

0

0

0

  Cleft palate

0

0

1

  Situs transversus – aorta, esophagus

  & stomach

1

0

0

  Intestinal agenesis

0

0

0

  Arch of aorta absent or right sided

0

0

0

  Diaphragmic hernia

0

0

1

  Dilated renal pelves

2

0

3

  Ectopic kidneys(s) &/or variation in size

1

0

0

  Renal agenesis

0

0

2

  Dilated ureters

6

0

3

  Adrenal agenesis

0

0

1

  Testes – ectopic or enlarged

1

0

1

  Hermaphroditism

0

0

3

1Fetuses may have more than one defect

aExcludes 1 fetus lost

*Significantly different from control (p<0.05) by Chi-square only

Table 4. Skeletal observations of fetuses delivered from rats given DSS in their diets on gestation days  6 through 15.

 

Skeletal observations

Dose:      Control

1.0 % DSS

2.0% DSS

Group  (I-A)

(II-A)

(II-B)

Total number of fetuses examined

167a

97

98

Defects1:

  Cranial bones,

  incomplete to lack of ossification :

   Nasal                    

 

 

 

0

 

 

 

0

 

 

 

4

   Frontal

1

0

20*

   Parietal

1

1

19*

   Interparietal

1

2

18*

   Supraoccipital

0

0

15*

   Exoccipital

0

0

2

   Atlas

0

0

1

   Zygomatic

0

0

1

   Premaxilla

0

0

1

   Tympanic bullae

0

0

5

   Mandibles

0

0

1

   Hyoid

0

0

3

  Eye orbit, reduction

0

0

0

  Exoccipital, fused to atlas

0

0

0

  Vertebrla column, curved &/or open

0

0

5

  Vertebrae:

 

 

 

   misshapened &/or retarded 

   development

0

0

5

   thoracic, bipartite centra

2

1

5

   lumbar, bipartite centra

0

0

2

  Sternebrae:

 

 

 

   fused

0

0

0

   hypoplastic to absent

0

0

1

   one or two absent

1

0

0

   staircase

0

0

3

   bipartite

0

0

2

  Rib(s):

 

 

 

   accesory

6

5

5

   Absent or less developed

0

0

7*

   wavy

2

2

0

   fused

0

0

2

  Pelvic, hypoplastic to absent

0

0

0

  Brachydactyly

0

0

0

  Syndactyly

0

0

0

  Adactyly

0

0

0

  Hemimelia & small scapula

0

0

 0

1Fetuses may have more than one defect

aExcludes 1 fetus destroyed during cleaning process

*Significantly different from control (p<0.05) by Chi-square only

 

Conclusions:
Subtoxic dietary levels of 1.0% read-across substance docusate sodium ingested on gestational days 6 through 15 showed no adverse effects on the various maternal or fetal parameters. Near toxic or toxic dietary levels of 2.0% DSS produced significant incidences of resorptions (13.7%) and gross abnormalities either among litters (25.0%) or fetal populations (20.2%) as compared to controls. Interpretation of the results of the present experiments, in which only maternally toxic dose levels induce teratogenicity, indicates no real hazard with the recommended human use of these surfactants.
Executive summary:

Prenatal developmental toxicity was studied in rats dosed from day 6 to day 15 of gestation by dietary administration of read-across substance docusate sodium at dose levels of 1.0 and 2.0 % in the diet. Subtoxic dietary levels of 1.0% showed no adverse effects on the various maternal or fetal parameters. Near toxic or toxic dietary levels of 2.0% docusate sodium produced significant depressions in maternal weight-gains and increased incidences of resorptions (13.7%) and gross abnormalities either among litters (25.0%) or fetal populations (20.2%) as compared the controls. These abnormalities consisted primarily of exencephaly of varying degrees with, at times, spina bifida, anophtalmia and associated skeletal defects. The visceral observations confirmed the significance of the exencephalous characteristics and anophtalmia for the group given dietary levels of 2.0%. In this group, skeletal observations revealed a significant incidence of incomplete ossification to absence of the various cranial bones, a curved or open vertebral column, and a variety of defects of the vertebrae and ribs. There were significant depressions in maternal weight gains in the 2.0% DSS-group. Interpretation of the results of the present experiment, in which only maternally toxic dose levels induce teratogenicity, indicates no real hazard with the recommended human use of these surfactants.

The concentration of 1% in the diet is considered as maternal and developmental NOAEL. This dose level corresponded with 1074 mg/kg body weight, as calculated in the study.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 074 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Klimisch 2
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

Supporting data for absence of teratogenicity and developmental toxicity was available from following studies:

- A combined oral repeated dose and reproduction/development screening study was conducted with registered substance according to OECD guideline 422 (Hansen, 2013a). The test item was a solid formulation (containing >93% active ingredient) which was administered as a watery solution orally by gavage to rats at dose levels of 60, 120 and 300 mg act. ingr./kg bw/day for for at least 28 days in male rats up to 54 days in female rats. Paternal/maternal toxicity was observed at 120 and 300 mg/kg bw in males.No test item-related influence was noted on the reproduction toxicity parameters at 60 and 120 mg/kg bw. In the high dose group (300 mg/kg b.w./day)the total litter weight per dam of the 3 dams with live born pups was reduced on lactation day 1 and on lactation day 4 by 42 and 56%, respectively. A statistically significantly (p≤0.01) decreased viability index between day 4 and day1 was also noted in this dose group.The external examinations of the pups revealed no test item-related external visible changes in any of the treatment groups, except for ‘no milk in the stomach’ in pups which were found dead during the lactation period.

NOAEL-levels were as follows: 60 mg/kg bw for paternal/maternal toxicity (based on clinical signs, reduced body weight, food consumption, organ weights and changes in biochemical parameters at the high dose levels) and 120 mg/kg bw for developmental toxicity (based on a reduced viability index at the high dose levels).

- A combined reproduction-teratogenicity study was available for read across substance CAS No. 37294 -49 -8 (disodium C-isodecyl sulphonatosuccinate) in 2 generations (Tegeris and Underwood, 1975). The test item containing +-50% active ingredient was fed to Charles River CD Sprague-Dawley rats at 1% and 4% in the diet, while the control group received normal diet. The original females were allowed to deliver their first 2 litters (F1a & F1b), while the third litters were used for teratological evaluation and partial histology on 5 pairs per group (F1c). Females were allowed to rest 20 days between weaning and their next breeding. All litters were standardized to 8 newborn to equalize the maternal stress.

- F1a pups were examined to calculate Fertility Index (FI), Viability Index (VI) and Lactation Index (LI); they were discarded at weaning.

- F1b pups were examined to calculate Fertility Index (FI), Viability Index (VI) and Lactation Index (LI), with part of them that were studied postmortem (autopsy and 5 pairs per group for histology) and the other part were selected for second generation breeding, leading to F2a (discarded at weaning) and F2b (teratological examination and partial histology on 5 pairs per group).

- F1c were used for teratological evaluation an d partial histology on 5 pairs per group.

Although data suggest that the test material under the conditions of this experiment is not teratogenic in the rat it does, however, appear to depress the rate of body weight gain in the pups at 4% in the diet. The number of live born pups , and related to this Fertility Index (FI), Viability Index (VI) and Lactation Index (LI) also decreased at 4%. There were no histological effects on the gonads. NOEL for teratogenicity is 3000 mg/kg (4% in the diet); NOAEL for embryotoxicity is 750 mg/kg (1% in the diet).

 

In conclusion, both a combined reproductive/developmental screening study with registered substance and a combined reproduction-teratogenicity study with read across substance CAS No. 37294 -49 -8 did not indicate potential for developmental toxicity.

 

Teratogenicity testing

Further data on prenatal developmental toxicity were available from read across substance Docusate sodium (CAS No. 577-11-7). Justification for read across with the category of Di-ester sulphosuccinates is documented in a separate document attached in Section 13.

- A key study for prenatal developmental toxicity was performed in rats dosed from day 6-15 of gestation with read across substance Docusate sodium dosed at dietary dose levels of 1.0 and 2.0 % in the diet (Roell et al., 1976). The study was conducted according to OECD 414 guideline, and was considered to be reliable, adequate and relevant. Subtoxic dietary levels of 1.0% showed no adverse effects on the various maternal or fetal parameters. Toxic dietary levels of 2.0% Docusate sodium produced significant depressions in maternal weight-gains and increased incidences of resorptions (13.7%) and gross abnormalities either among litters (25.0%) or fetal populations (20.2%) as compared to controls. These abnormalities consisted primarily of exencephaly of varying degrees with, at times, spina bifida, anophthalmia and associated skeletal defects. The visceral observations confirmed the significance of the exencephalous characteristics and anophthalmia for the group given dietary levels of 2.0%. In this group, skeletal observations revealed a significant incidence of incomplete ossification to absence of the various cranial bones, a curved or open vertebral column, and a variety of defects of the vertebrae and ribs. Interpretation of the results of the present experiment, in which only maternally toxic doses induce teratogenicity, indicates no real hazard with the recommended human use of these surfactants. The concentration of 1% in the diet is considered as maternal and developmental NOAEL. This dose level corresponded with a test article intake of 1074 mg/kg body weight, as calculated in the study.

- As supporting information, prenatal developmental toxicity was also studied in rats by dietary administration of Docusate 'calcium' (DCS) at dose levels of 0.5, 1.0, 1.5 and 2.0 % in the diet as well as by oral gavage at 250, 500, 750 and 1000 mg/kg bw (Roell et all., 1976). Subtoxic dietary levels of 0.5 and 1.0% Docusate calcium ingested on gestational days 6 through 15 showed no adverse effects on the various maternal or fetal parameters. Near toxic or toxic dietary levels of 1.5 and 2.0% DCS produced significant incidences of resorptions and gross abnormalities consisting primarily of exencephaly of varying degrees with spina bifida, anophthalmia and associated skeletal defects. However, dietary levels of 2% of DCS fed to pregnant rats for 3 days (days 6-8, 8-10 or 10-12) did not produce teratogenic response. Also, DCS given to pregnant rats by oral intubation at maternally subtoxic doses (250-750 mg/kg) and a slightly toxic dose (1000 mg/kg) did not lead to malformations, however the incidence of resorptions was increased at the 2 toxic doses. Likewise doses of 500 and 750 mg/kg given by gavage from day 6-15 produced an increase in resorptions at the highest dose without a teratogenic effect. Since only maternally toxic doses fed on gestational day 6-15 produced embryotoxic and teratogenic effects, it is concluded that no real hazard exists. 

In conclusion, both a prenatal developmental toxicity with read across substance Docusate sodium and with read across substance Docusate calcium were negative for teratogenicity at non-toxic dose levels.

 

Conclusion

An oral gavage reproductive screening study with registered substance showed NOAEL of 60 mg/kg bw for paternal/maternal toxicity, whereas 120 mg/kg bw was NOAEL for reproductive and developmental toxicity. The latter was based on reduced gestation index, (life) birth index and No. of pups and increased post-implantation loss at 300 mg/kg bw.

A combined reproduction-teratogenicity study with read across substance CAS No. 37294-49-8 (disodium C-isodecyl sulphonatosuccinate) in 2 generations resulted in a NOAEL for embryotoxicity of 750 mg/kg (1% in the diet) and a NOEL for teratogenicity of 3000 mg/kg (4% in the diet).

Prenatal developmental toxicity was tested by dietary administration of read across substance CAS No. 577-11-7 (Docusate sodium) in rats from day 6 to 15 of gestation. 1% in the diet was a maternal and developmental NOAEL corresponding to 1074 mg/kg bw, whereas at 2% in the diet visceral and skeletal anomalies were observed, which were seen at systemic toxic dose level and considered secondary to maternal toxicity. This was confirmed in a similar study with Docusate calcium given at subtoxic and toxic dose levels, where the same could be observed.

Based on the absence of developmental findings in the teratogenicity study, and taking into account that same metabolic and toxicological behavior can be expected for structural similar substances, no further testing is needed.

 

Justification for classification or non-classification

Based on these results and according to the CLP (No. 1272/2008 of 16 December 2008), the test substance does not have to be classified and has no obligatory labelling requirement for reproductive and developmental toxicity.