Butanedioic acid, sulfo-, 1-C12-18-alkyl esters, disodium salts

Endpoint summary

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.

Link to relevant study records

Reference

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 for cross-reference:

reference to same study

Qualifier:

according to guideline

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. QA statement)

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 or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Research, Models and Services Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age at start of 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	10.8^v
Number of pups at birth (total)	136	109	134	34^vv
Number of pups at birth (mean)	13.6	10.9	13.4	4.3^vv
Birth Index (mean %)	95.1	90.3	90.2	36.0
Birth Index (total %)	95.1	91.6	89.9	39.5^2
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	29^vv
Number of live born pups (mean)	13.3	10.9	13.0	3.6^vv
Live birth index (mean %)	97.7	100.0	96.7	67.3
Live birth index (total %)	97.8	100.0	97.0	85.3^b
Pre-implantation loss (mean %)	8.8	16.1	1.3	14.7
Pre-implantation loss (total %)	10.6	16.2	1.3^b	17.3
Post-implantation loss (mean %)	7.9	9.7	12.6	68.5
Post-implantation loss (total %)	7.0	8.4	12.8	66.3^b

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).

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.

 

Effects on developmental toxicity

Description of key information

A new Prenatal developmental toxicity study with the registered substance is planned and will be updated later when results are available (currently waived in the dossier).

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 corresponding to 1074 mg/kg bw, 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 for cross-reference:

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 malformations^1: 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 observations^1:	Group (I-A)	(II-A)	(II-B)
Total number examined	388^a	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	165^a	98	91
Defects^1:   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	167^a	97	98
Defects^1:   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

- 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, 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

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.

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.