Butanedioic acid, 2-sulfo-, 1(or 4)-[2-(C12-18 and C18-unsatd. amido)ethyl] esters, disodium salts

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

A key study for reproductive screening toxicity was performed in rats by means of an OECD 422 study with a liquid formulation containing 41.5% active ingredient given by oral gavage at dose levels of 100, 300 and 1000 mg act. ingr./kg bw/day. No reproductive toxicity effects were observed up to 1000 mg/kg bw. At the dose of 1000 mg/kg bw, decrease body weight and other systemic effects were observed, therefore NOAEL for systemic toxicity was 300 mg/kg bw/day, whereas NOAEL for reproductive toxicity was 1000 mg/kg bw/day.

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 (the latter corresponding with 750 mg/kg bw), however these were not considered adverse and were not associated with any other (reproductive) findings.

In a second (supporting) two-generation study the highest concentration of 1% in the diet corresponding with 750 mg act. ingr./kg bw was NOAEL.

Link to relevant study records

Reference

Reference 1

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

other: CD® / Crl:CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Source: Charles River Laboratories Research, Models and Services Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age at start of dosing : Males 55 days; Females: 48 days
- Weight range at start of dosing: Males: 281.0 to 308.0 g; Females: 68.9 to 195.7 g
- Fasting period before study: Ad libitum with exception of the night before the day of blood withdrawal for laboratory 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) is used as bedding material in these cages. The cages were cleaned and changed once a week.
- Diet (e.g. ad libitum): ssniff® R/Z V1324 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany, ad libitum with exception of the night before the day of blood withdrawal for laboratory examination.
- Water (e.g. ad libitum): Tap water was offered daily ad libitum.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C ± 3°C (maximum range)
- Humidity (%): 55% ± 15% (maximum range)
- Air changes (per hr): Not provided
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
Males From: August 27, 2012 To: October 2, 2012
Females From: August 27, 2012 To: October 19, 2012

Route of administration:

oral: gavage

Vehicle:

other: tap water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Application volume: 5 mL/kg bw/day. The test item was dissolved in the vehicle tap water to concentrations of 20, 60 and 200 mg test item /mL tap water and was administered orally at a constant volume once daily. The amount of the test item was adjusted to the animal's current body weight daily. The test item-vehicle mixture was freshly prepared every day.

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 occurred or 2 weeks had elapsed.
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- After approx. 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: yes. This mating-procedure was repeated until at least 8 pregnant dams were available for each group.
- After successful mating each pregnant female was caged (how): 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 the other side of the animal room than the males with each dose group separated by an empty row.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For the analysis of the test item-vehicle mixtures samples of approx. 2 x 5 mL were taken at the following time points and stored at ≤ -20°C until analysis at LPT.
*Start of treatment period: Analysis of stability and concentration: Immediately after preparation of the test item-vehicle mixtures as well as 8 and 24 hours after storage of the test item preparations at room temperature: 3 samples/dose level group = 9 samples
*End of treatment period: Concentration: During treatment with the test item always before administration to the last animal/dose level group: 3 samples
The samples were labelled with the study number, test item, test species, type of sample, aliquot number, concentration, test day, sampling time and date.
The validation of the analytical method is part of LPT study No. 28344 (14-day dose-range-finding).
The measured actual concentrations of the test item in the test item vehicle mixtures were between 99.99% and 102.96% of the nominal concentrations (table 26).

Duration of treatment / exposure:

Males: 2 weeks prior to mating, during the mating period and approx. 2 weeks post mating at least until the minimum total dosing period of 28 days has been completed (up to and including the day before sacrifice).
Females: 2 weeks prior to mating and continuing up to, and including, day 3 post-partum or the day before sacrifice.

Frequency of treatment:

daily

Details on study schedule:

- Age at mating of the mated animals in the study: 10 weeks

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

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 act. ingr./kg bw by oral gavage (LPT Study No. 28344). None of the animals died prematurely. None of the male and female rats treated orally with 100 or 300 mg act. ingr./kg bw/day revealed any changes in behaviour, external appearance or faeces. Salivation was noted for 2 of 5 male animals treated at 1000 mg/kg bw/day on 1 or 3 test days starting on test day 9 and increased faeces was noted for 3 of 5 male and 2 of 5 female high dosed animals on 6 or 9 test days starting on test day 5. No test item-related changes on body weight and body weight gain were noted for the male and female rats up to 1000 mg act. ingr./kg bw/day. No test item-related changes on food consumption were noted for the male and female rats treated orally with 100 or 300 mg act. ingr./kg bw/day. The food consumption of the male and female animals treated with 1000 mg act. ingr./kg bw/day was slightly increased by 9% for the males and by 10% for the females in test week 2 (statistically significant at p ≤ 0.01 for both sexes). No test item-related influence was noted for the drinking water consumption at any of the tested dose levels. None of the male and female rats treated orally with 100, 300 or 1000 mg act. ingr ./kg bw/day revealed changes at macroscopic inspection at necropsy or organ weights.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Throughout the test period, each animal was observed for clinical signs at least once daily. 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.
- Individual animals were observed before and after dosing at each time of dosing for any signs of behavioural changes, reaction to treatment or illness. Cageside 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.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Additionally, once before the first exposure (to allow within-subject comparisons) and once a week thereafter, detailed clinical observations were made in all animals outside the home cage in a standard arena and at the same time, each time preferably by observers unaware of the treatment. Signs noted included changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, pilo-erection, pupil size, and unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), difficult or prolonged parturition or bizarre behaviour (e.g. self-mutilation, walking backwards) were also recorded.

BODY WEIGHT: Yes. Body weights were recorded individually for each adult animal.
- 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 0 or 1 post-partum) and day 4 post-partum.

FOOD CONSUMPTION: Yes
The quantity of food left by individual animals was recorded on a weekly basis throughout the experimental period with the execution 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 on completion of a treatment week.

WATER CONSUMPTION:
- Time schedule for examinations: Water consumption was monitored daily by visual appraisal throughout the study.

OTHER:
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 [%]

Sperm parameters (parental animals):

Parameters examined in P male parental generations:
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
- Male animals: All surviving animals: The male animals were sacrificed after a minimum total dosing period of 28 days if no longer needed for further mating.
- 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.Special attention was paid to the organs of the reproductive system.Apparently non-pregnant uteri were placed in a 10% aqueous solution of ammonium sulfide for about 10 minutes
to stain possible implantation sites in the endometrium according to SALEWSKI.
The numbers of corpora lutea and implantation sites were recorded in the female adult animals and reported.

HISTOPATHOLOGY / ORGAN WEIGHTS
-Determination of organ weights was performed before fixation of the organs.
Microscopic examination and organ weights of all adult male animals and identified as left or right: Epididymis (2) ,Testicle (2)
Determination of the organ weights of the following organs was only performed from 5 adult animals/sex/group, which were randomly selected:
Adrenal gland (2), Brain, Heart, Kidney (2), Liver, Spleen, Thymus.
Adrenal glands and kidneys were weighed individually and identified as left or right.
-The following organs or parts of organs of all adult animals were fixed in 7% formalin; testes and epi didymides were fixed in Bouin' s fixative:
Epididymis (2), Gross lesions, Mammary gland, Ovary (2), Prostate, Seminal vesicle, Testicle (2), Ute
rus (incl. cervix and oviducts), Vagina.
In addition to the organs listed in before, the following organs or parts of organs of the afore-mentioned selected animals (5 male and 5 female adult animals per group) were fixed in 7% formalin:
Adrenal gland (2), Bone marrow (os femoris), Brain (cerebrum, cerebellum, brain stem), Heart (right and left 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 (including regional lymph nodes),Tongue (including base), Trachea (including larynx), Urinary bladder.
Any other organs displaying macroscopic changes were also preserved.
-Only the selected animals from the control group and the high dose group were considered for histopathological evaluation.
Histopathological examinations were performed on haematoxylin and eosin stained paraffin sections of the following organs and tissues:
Adrenal gland (2), Bone marrow (os femoris), Brain (cerebrum, cerebellum, brain stem), Epididymis (1) #, Gross lesions, 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), Mammary gland, Nerve (sciatic), Oesophagus, Ovary (2), Prostate, Seminal vesicle, Spinal cord (3 sections), Spleen, Stomach, Testicle (1) #, Thyroid (incl. parathyroids), Thymus , Tissue masses or tumours (including regional lymph nodes), Tongue (incl. base), Trachea (incl. larynx), Urinary bladder, 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.
Parathyroids cannot always be identified macroscopically during autopsy. They were examined microscopically if in the plane of section. Missing parathyroids are mentioned among the pathology data of the individual animals.
In addition to the paraffin sections, frozen sections of the heart, liver and one kidney were prepared and stained with Oil Red O.
In addition, further slides of the lungs of all high dosed females were prepared from the wet material, as in the first run 4 of 5 lungs were collapsed, and could not evaluated microscopically

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were sacrificed at 4 days of age.
- These animals were subjected to postmortem examinations macroscopic examination) as follows:
Dead pups and pups sacrificed at day 4 post-partum, or shortly thereafter, were carefully examined externally for gross abnormalities.

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®8 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 were used:
STUDENT' s t-test All numerical functional tests (p ≤0.01)
Multiple t-test based on Body weight I Food consumption I
DUNNETT, C. W. Haematology I Clinical chemistry I
New tables for multiple Absolute and relative organ weights
Comparisons with a control (p ≤ 0.05 and ≤0.01)
Biometrics, 482-491 (Sept 1 964)

For all numerical values (e.g. body weight, food consumption and organ weight data) homogeneity of variances was tested by using the BARTLETT chi2-test. lf the variances are 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 is p ≤ 0.01.
Exact test of R. A. FISHER Histopathology, if applicable (p≤0.05)
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
Pre-implantation loss [%]
Post-implantation loss [%]

Offspring viability indices:

Birth Index
Live Birth Index
Survival Index

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Slightly increased salivationwas noted in one male rat dosed at 1000 mg/kg bw/day.
No signs of clinical toxicity were noted in all female treatment groups.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No premature deaths were noted in the male and female rats treated with 100, 300 or 1000 mg/kg bw/ day.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Slight reduction of body weight and body weight gain was noted in male and female rats dosed at 1000 mg/kg bw/day.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

oral gavage study
Slight reduction in food consumption was noted in male and female rats dosed at 1000 mg/kg bw/day

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Increased number of eosinophils in intermediate dose females on test day 15 lacking dose dependence (p≤0.05)

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

In the high dose group (1000 mg/kg bw/day) a statistically significant (p≤0.01) increase (males: +66%; females: +46%)) was noted for the plasma activity of ALAT.
In the male high dose group (1000 mg/kg bw/day) a decrease in albumin on test day 15 (p≤0.01) was noted but not considered test-item related (slight alteration in comparison to control animals without biological relevance).
In the female intermediate dose group (300 mg/kg bw/day) an increase in bile acids on test day 15 (p≤0.05) was noted but not considered test-item related (lacking dose dependence).

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related influence was noted for the fore- and hindlimb grip strength in any male/female treatment group (100, 300 or 1000 mg/kg bw/day).
No test item-related influence was noted on the spontaneous motility of the rats in any of the treatment groups.
In the male low dose group (100 mg/kg bw/day) a decrease in hindlimb grip strength on test day 36 (p≤0.05) was noted but not considered test-item related (lacking dose dependence).

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related influence in observational screening was noted for any treatment group (100, 300 or 1000 mg/kg bw/day).
In the high dose males a statistically significant decreased mean body temperature was noticed in comparison to the control group. This was considered to be not test item related.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

No test item-related microscopic changes were seen in the reproductive organs for both males and females.
A test item-related squamous cell hyperplasia was noted in the forestomachs from 5/5 male and 5/5 female animals of the high dose group (1000 mg/kg bw/day). These test item-related stomach changes were localized in the zone adjoining the glandular stomach mucosa (forestomach or
non-glandular mucosa) and attained statistical significance for both sexes (p≤0.01). Occasionally the squamous cell hyperplasia with subsequent hyperkeratinization was associated with acute inflammation of the submucosa in the non-glandular stomach (for 2/5 males and 1/5 females).
Examination of the stomachs from the animals (5 per group) of the low- and intermediate dose groups (100 and 300 mg/kg bw/day) did not reveal any test item-related changes. As humans lack a forestomach, the relevance of these changes for humans is questionable.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

effects observed, non-treatment-related

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

No statistically significant differences were noted in the length of the pre-coital time between the control group and the treatment groups.
There were no statistically significant differences for the female fertility rates between the control and the treatment groups.
No test item-related influence was noted on the gestation length of the females in any of the treatment groups compared to the control group.
No statistically significant differences were noted in the number of corpora lutea between the control and the treatment groups of P0 dams.
No statistically significant differences were noted in the number of implantation sites between the control and the treatment groups of P0 dams.
No statistically significant differences were noted in the total number of born pups (alive and dead) or in the number of pups born alive between the control group and the treatment groups of P0 dams.
No test item-related differences were noted between the control group and the treatment groups of P0 dams with respect to the reproduction indices which were calculated from the above mentioned parameters (Fertility index, Gestation index, Birth index, Live birth index, Pre-implantaion losss and Post-implantation loss).
Parameters with statistically significant differences in comparison to the control group which were considered as to be not test item related:
-Increased Birth Index in low and intermediate dose females
-Increased Pre-implantation loss in low dose females
-Decreased Post-implantation loss in low and intermediate dose females

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No premature deaths were noted in the male and female rats treated with 100, 300 and 1000 mg/kg bw/day.
No signs of clinical toxicity were noted for the male and female rats of the low and intermediate dose groups (100 and 300 mg/kg bw/day).
A slightly increased salivation was noted in one male rat, no further signs of clinical toxicity were noted for the male and female rats of the high dose group (1000 mg/kg bw/day). No test item related influence was noted for the male and female rats of all treatment groups (100, 300 and 1000 mg/kg bw/day) during the observational and functional (grip strength and spontaneous motility) neurological
screenings.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
A slight reduction in body weight was noted for the male and female rats of the high dose group (1000 mg/kg bw/day). For the male rats the reduction in body weight was noted from test day 8 (4.4%) until test day 36 (5.5%) , statistically significant (p≤0.05) in comparison to the control group on test day 8. Body weight gain was accordingly reduced in the high dose group during the whole study, mostly pronounced and statistically significant at the end of the first test week on test day 8 (p≤0.01), with a reduction in body weight gain by 35.4% in comparison to the control group. During the further course of the study the differences in body weight gain between the high dose group and the control group declined, leading to a body weight gain at the end of the study of 42.1%, the control group revealed a body weight gain of 50.5%.
For the high dose female rats a slight, not statistically significant, reduction in body weight was noted of 3.7% and 3.4% was noted on test days 8 and 15 (pre-mating). Body weight gain was 6.6% on test day 8 and 10.6% on test day 15 in the high dose group in comparison to 10.7% (test day 8) and 14.6% (test day 15) in the control group. Body weight was decreased during the whole gestation period between 7.6% on gestation day 0 and 6.5% on gestation day 20, statistically significant (p≤0.05) on gestation days 7 and 14 with 9.7% and 7.9% decrease, respectively. A small reduction in body weight gain was noted in the high dose group on gestation day 0 (15% in comparison to 18%. On gestation day 20 body weight gain was nearly identical in the high dose group (59.7%) and the control group (59.4%). During lactation a slight but not significant reduction in body weight was noted in the intermediate dose female rats by 4.4 % on lactation day 0 and 5.6% on lactation day 4. In the high dose group the body weight was statistically significantly (p≤0.05) reduced on lactation days 1 and 4 by 9.1% and 9.5%. No noticeable differences were noted in body weight gain during the lactation period with 5.6% in the high dose group and 6.1% in the control group.

A slightly statistically significant (p ≤ 0.01) increase in relative food consumption by 10.3% was noted in the high dose males during the 2nd test week. This was caused by the reduced body weight of the rats of the high dose group.
A slightly statistically significant (p≤0.05) decrease in relative food consumption by 7.4% was noted in the high dose females during the first test week.

HAEMATOLOGICAL FINDINGS
No test item-related influences were noted between the control group and the treatment groups for the haematological parameters, i.e. the haemoglobin content, the number of erythrocytes, leucocytes, reticulocytes and platelets, the haematocrit value, the thromboplastin time (TPT, aPTT), the mean corpuscular volume (MCV), the mean corpuscular haemoglobin (MCH) and the mean corpuscular haemoglobin concentration (MCHC). No test item-related changes were noted in the relative and absolute differential blood counts.
Statistically significant increased number of eosinophils (p≤0.05) was noted in the intermediate dose females on test day 15. This was considered to be not test item related ( lacking dose dependence).

CLINICAL BIOCHEMISTRY
In the high dose male and female group (1000 mg/kg bw/day) a statistically significant (p≤0.01) increase was noted for the plasma activity of ALAT.
In males no test item related influence was noted for the plasma levels of globulin, the albumin/globulin ratio, bilirubin (total), cholesterol (total), creatinine, glucose, urea in blood, sodium, potassium,calcium, chloride, the activity of the alkaline phosphatise (aP), the activity of ASAT and the serum levels of the bile acids. Decreased albumin in the high dose males (p≤0.01) was considered to be not test item related (slight alteration in comparison to control animals without biological relevance and 4/5 individual values within range of LPT Background Data))
In females no test item related influence was noted for the plasma levels of albumin, of globulin, the albumin/globulin ratio, bilirubin (total), cholesterol (total), creatinine, glucose, protein (total), urera in blood, sodium, potassium, calcium, chloride, the activity of the alkaline phosphatase (aP) and the
serum levels of the bile acids. Increased bile acids in the intermediate dose females (p≤0.05) was considered to be not test item related (lacking dose dependence).

BEHAVIOUR (FUNCTIONAL FINDINGS)
The functional neurological screenings were performed on test day 36 for the male rats and between test days 40-50 for the female rats, 2 hours after dosing.
No test item-related influence was noted for the fore- and hindlimb grip strength in any male/female treatment group (100, 300 or 1000 mg/kg bw/day).
Hinglimb grip strength in low dose males was statistically significant decreased on test day 36 (p≤0.05) but lacking dose dependence and was considered to be not test item related.
No test item-related influence was noted on the spontaneous motility of the rats in any of the treatment groups. No test item-related influence on relative and absolute organ weights was noted for the rats treated with 100, 300 or 1000 mg/kg bw/day in comparison to the control group.
Statistically significant differences in the relative organ weights compared to the control, which are not considered to be test item-related were:
- increased relative weight of left gonads in low and high dose males (p≤0.05)
- increased relative weight of right kidney in high dose females (p≤0.05)
- increased relative weight of right adrenal in high dose males (p≤0.05)
Statistically significant differences in the absolute organ weights compared to the control, which are not considered to be test item-related were:
-decreased absolute brain weight in high dose males (p≤0.05)
-decreased absolute heart weight by 16.6% in high dose females (p≤0.05)

GROSS PATHOLOGY (PARENTAL ANIMALS)
Macroscopic inspection at autopsy for the males was performed on test day 37.
No test-item related findings were noted in the low and intermediate dose group (100 and 300 mg/kg bw/day).
However, one animal (no. 3) with a reduction in the size of the testes was noted in the control group.
At the low dose group (100 mg/kg bw/day) animal no. 21 showed a partly reddened thymus and animal no. 29 a thickened right prostate.
In the intermediate dose group (300 mg/kg bw/day) one animal (no. 49) with macroscopic changes in the stomach (yellowish contents, detachment of mucosa) was found.
All changes are considered to be not test item-related but spontaneous due to the low number of occurrence.
In the high dose group (1000 mg/kg bw./day) 2 animals with macroscopic changes in the stomach were noted: Animal no. 68 showed a whitish thickening in the cardia part of the stomach and in the stomach of animal no. 69 a yellowish content was noted.
These findings were considered to be test item-related, because they were associated with microscopic changes in the forestomach from the animals of the high dose group.
Macroscopic inspection at autopsy for the females was performed between test days 43 and 54.
No test-item related macroscopic changes were noted in any treatment group (100, 300 and 1000 mg/kg bw/day) in females.
In 2 of the 3 non-pregnant rats (nos 16 and 54) from the control and the intermediate dose group (300mg/kg bw/day) a thickened uterus was noted.
These findings were considered to be not test item-related but spontaneous due to the low number ofoccurrence.

NEUROPATHOLOGY (OBSERVATIONAL FINDINGS)
The observational screenings were performed on test day 36 for the male rats and between test days 40-50 for the female rats, 2 hours after dosing.
No test item-related influence was noted for any treatment group (100, 300 or 1000 mg/kg bw/day).
A decrease in mean body temperature (p≤0.05) in the high dose males on test day 36 was considered to be not test item related. The slight alteration in comparison to control animals was without biological relevance (only 0.4°C difference to controls)

HISTOPATHOLOGY (PARENTAL ANIMALS)
Histopathological examination performed on one testicle and one epididymis with special emphasis on the qualitative stages of spermatogenesis (proliferative, meiotic and spermiogenic phases) and histopathology of the interstitial testicular structure of all adult male animals of the highest dose group and the control group following H & E and PAS staining, did not reveal any test item-related effects.
No test item-related microscopic changes were seen in the reproductive organs for both males and females.
A pulmonary congestion was noted in the lungs from 4/5 male (control 0/5) and 5/5 female rats (control 4/5) of the high dose group (1000 mg/kg bw/day). As pulmonary congestion is occasionally seen in rats as a background finding, this change was not considered to be test item-related.
A test item-related squamous cell hyperplasia was noted in the fore-stomachs from 5/5 male and 5/5 female animals of the high dose group (1000 mg/kg bw/day). These test item-related stomach changes were localized in the zone adjoining the glandular stomach mucosa (forestomach or
13 the squamous cell hyperplasia with subsequent hyperkeratinization was associated with acute inflammation of the submucosa in the non-glandular stomach (for 2/5 males and 1/5 females).
Examination of the stomachs from the animals (5 per group) of the low- and intermediate dose groups (100 and 300 mg/kg bw/day) did not reveal any test item-related changes. As humans lack a fore-stomach, the relevance of these changes for humans is questionable.
All other microscopic changes observed were either coincidental, or lie within the normal background alterations which may be seen in untreated rats of this age and strain.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
All animals were successfully mated as determinated by positive sperm detection. No statistically significant differences were noted in the length of the pre-coital time between the control group and the treatment groups.
There were no statistically significant differences for the female fertility rates between the control and the treatment groups.
No test item-related influence was noted on the gestation length of the females in any of the treatment groups compared to the control group.
No statistically significant differences were noted in the number of corpora lutea between the control and the treatment groups of P0 dams.
No statistically significant differences were noted in the number of implantation sites between the control and the treatment groups of P0 dams.
No statistically significant differences were noted in the total number of born pups (alive and dead) or in the number of pups born alive between the control group and the treatment groups of P0 dams.
No test item-related differences were noted between the control group and the treatment groups of P0 dams with respect to the reproduction indices which were calculated from the above mentioned parameters (Fertility index, Gestation index, Birth index, Live birth index, Pre-implantation loss and Post-implantation loss).
Parameters with statistically significant differences in comparison to the control group which were considered as to be not test item related:
-Increased Birth Index in low and intermediate dose females (p≤0.05)
-Increased Pre-implantation loss in low dose females (p≤0.05)
-Decreased Post-implantation loss in low and intermediate dose females (p≤0.05).

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Effect level:

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

Based on:

act. ingr.

Sex:

male/female

Remarks on result:

other: highest dose tested

Dose descriptor:

NOAEL

Remarks:

general toxicity

Effect level:

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

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

clinical signs

body weight and weight gain

food consumption and compound intake

clinical biochemistry

gross pathology

histopathology: non-neoplastic

Details on results:

No P1 generation: screening study

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

No test item-related differences were noted between the survival index of the control group and the treatment groups.
No live born pup from the control group and the low and intermediate dose groups died between lactation day 1 and 4, leading to a survival index of 100% for these groups.
In the high dose group (1000 mg/kg bw/day) 9 of 10 dams revealed a survival index of 100%, whereas all 3 live born pups from dam no. 77 were found dead without milk on lactation day 2, leading to a survival index of 0.0% for dam no. 77 and a survival index of 97.6% for the pups of the high dose group. This small decrease in the survival index of the pups of the high dose group was regarded as to be spontaneous.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No test item-related influence was noted on the mean litter weight of the pups in all treatment groups.
No test item-related influence was noted on the total litter weight of the pups in all treatment groups.

Food consumption and compound intake (if feeding study):

not examined

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

Description (incidence and severity):

Pups were sacrificed on day 4 of lactation.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

The external examinations of the pups at sacrifice on day 4 of lactation revealed no test item-related external visible changes or gross abnormalities after treatment of the parental animals with 100, 300 or 1000 mg/kg bw/day. No milk was noted in the stomachs of the 3 dead pups from dam no. 77. This observation was spontaneous and considered as not test-item related.

Histopathological findings:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

VIABILITY (OFFSPRING)
No test item-related influence was noted on the survival rate of the pups.
No live born pup from the control group and the low and intermediate dose groups died between lactation day 1 and 4, leading to a survival index of 100% for these groups.
In the high dose group (1000 mg/kg bw/day) 9 of 10 dams revealed a survival index of 100%, whereas all 3 live born pups from dam no. 77 were found dead without milk on lactation day 2, leading to a survival index of 0.0% for dam no. 77 and a survival index of 97.6% for the pups of the high dose group. This small decrease in the survival index of the pups of the high dose group was regarded as to be spontaneous.

BODY WEIGHT (OFFSPRING)
No test item-related influence was noted on the mean litter weight of the pups in all treatment groups.
No test item-related influence was noted on the total litter weight of the pups in all treatment groups.

GROSS PATHOLOGY (OFFSPRING)
The external examinations of the pups at sacrifice on day 4 of lactation revealed no test item-related external visible changes or gross abnormalities after treatment of the parental animals with 100, 300 or 1000 mg/kg bw/day. No milk was noted in the stomachs of the 3 dead pups from dam no. 77. This observation was spontaneous and considered as not test-item related.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: development of F1 offspring

Remarks on result:

other: highest dose tested

No F2 generation: screening study

Reproductive effects observed:

not specified

Table 1. Fertility and Reproductive parameters Parental generation

Parameter	Group 1 Control	Group 2 100 mg/kg	Group 3 300 mg/kg	Group 4 1000 mg/kg
No. of females evaluated for pre-coital time	10	10	10	10
Mean precoital interval (days)	4.5	3.4	3.9	2.6
No. of females evaluated for fertility	10	10	10	10
Number of pregnant dams	8	9	9	10
Fertility index (%)	80	90	90	100
No. of females evaluated for gestation length	8	9	9	10
Gestation length (days)	22.0	22.2	22.2	22.1
Number of dams with live pups	8	9	9	10
Gestation Index (%)	100	100	100	100
Corpora lutea(total)	110	137	129	142
Corpora lutea(mean)	13.8	15.2	14.3	14.2
Implantation sites (total)	110	131	128	139
Implantation sites (mean)	13.8	14.6	14.2	13.9
Number of pups at birth (total)	96	125	123	127
Number of pups at birth (mean)	12.0	13.9	13.7	12.7
Birth Index (mean %)	90.4	95.5	95.9	90.1
Birth Index (total^# %)	87	95^1	96^1	90.1
Number of stillbirths	0	0	1	0
No. of dams with stillborn pups	0	0	1	0
Number of live born pups (total)	96	125	123	126
Number of live born pups (mean)	12.0	13.9	13.7	12.6
Live birth index (mean %)	100.0	100.0	100.0	97.5
Live birth index (total^#1 %)	100	100	100	99
Pre-implantation loss (mean %)	0.0	4.4	0.7	1.9
Pre-implantation loss (total^#2 %)	0.0	4.4^2	0.8	2.1
Post-implantation loss (mean %)	9.6	4.5	4.1	11.2
Post-implantation loss (total^#3 %)	12.7	4.6^2	3.9^2	9.4
Number of runts	0	0	0	0
Number of malformed pups	0	0	0	0

^# based on the total No. of implantation sites and total No. of pups at birth (alive and dead)

^#1 based on the total No. live born pups and total No. of pups at birth (alive and dead)

^#2 based on the total No. corpora lutea and total No. of implantation sites

^#3 based on the total No. implantation sites and toal number of live born pups

¹ p≤0.05 Chi²-test

² p<0.05 Chi²-test

Conclusions:

NOAEL (no-observed-adverse-effect level) for reproductive toxicity: >= 1000 mg/kg bw/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 by gavage to rats at dose levels of 100, 300 and 1000 mg active ingredient/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 37 for the male rats and between lactation day 4 and 7 for the female rats.

Effects on the parental generation (general toxicity)
No test item-related premature death was noted in any treatment group (100, 300 and 1000 mg test item/kg bw/day).

No signs of clinical toxicity were noted for the male and female rats of the low and intermediate dose groups (100 and 300 mg test item/kg bw/day). A slightly increased salivation was noted in one male rat, no further signs of clinical toxicity were noted for the male and female rats of the high dose group (1000 mg test item/kg bw/day). A slight reduction in body weight was noted for the male and female rats of the high dose group (1000 mg test item/kg bw/day). For the male rats the reduction in body weight was noted from test day 8 (4.4%) until test day 36 (5.5%) and for the female rats from gestation day 0 (7.6%) until lactation day 4 (9.5%). The body weight at autopsy was reduced accordingly.

Effects on reproduction parameters and organs

No test item-related influence was noted on the reproduction parameters in any treatment group (100, 300 and1000 mg/kg bw/day).

Microscopic examination revealed no changes in the reproductive organs from the male and female rats of the high dose group (1000 mg/kg bw/day).

Effects on the F₀-generation

NOAEL (no-observed-adverse-effect level): 300 mg/kg bw/day, p.o.

Effects on reproductive toxicity

NOAEL (no-observed-adverse-effect level): >=1000 mg/kg bw/day, p.o.

 

Reference 2

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 for cross-reference:

read-across source

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

Remarks:

DSS

Effect level:

0.1 other: %

Based on:

act. ingr.

Remarks:

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

Remarks:

DSS

Effect level:

1 other: %

Based on:

act. ingr.

Remarks:

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

Remarks:

DSS

Effect level:

0.1 other: %

Based on:

act. ingr.

Remarks:

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

Remarks:

DSS

Effect level:

1 other: %

Based on:

act. ingr.

Remarks:

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

Remarks:

DSS

Generation:

F1

Effect level:

0.1 other: %

Based on:

act. ingr.

Remarks:

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

Remarks:

DSS

Generation:

F2

Effect level:

0.1 other: %

Based on:

act. ingr.

Remarks:

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 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.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

High quality (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 with registered substance was performed by means of an oral combined repeated dose and reproduction/development screening study according to OECD guideline 422 (Hansen, 2013d). The test item (liquid formulation) containing 41.5% active ingredient was administered orally by gavage to rats at dose levels of 100, 300 and 1000 mg act. ingr./kg bw/day for for at least 28 days in male rats and at least 39 days in females. No test item-related premature death was noted in any treatment group. No signs of clinical toxicity were noted for the male and female rats of the low and intermediate dose groups (100 and 300 mg/kg bw/day), whereas slightly increased salivation was noted in one male rat as the only finding at 1000 mg/kg bw/day. A slight reduction in body weight was noted for the male and female rats dosed at 1000 mg/kg bw/day. Other parameters such as neurological observations, haematology and serum chemistry, as well as gross and histopathology are discussed in the repeated dose toxicity section 7.5. No test item-related influence was noted on the reproductive parameters in any treatment group (100, 300 and1000 mg/kg bw/day). Microscopic examination revealed no changes in the reproductive organs from the male and female rats of the high dose group (1000 mg/kg bw/day). NOAEL for paternal/maternal toxicity was 300 mg/kg bw/day, whereas NOAEL for reproductive toxicity was >= 1000 mg/kg bw.

 

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 subgroup of Di-ester sulphosuccinates is documented in a separate read-across justification 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.

 

Conclusion

An oral gavage reproductive screening study with registered substance showed a NOAEL of 300 mg/kg bw for paternal/maternal systemic toxicity, whereas 1000 mg/kg bw was NOAEL for reproductive and developmental toxicity as well as for neurotoxicity. Multigeneration studies with read across substance Docusate sodium (CAS No. 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. 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 corresponds with 750 mg/kg bw/day, which is higher than NOAEL for paternal/maternal toxicity. Based on the absence of reproductive findings in the screening study and the repeated dose toxicity studies and availability of multigeneration studies, no further testing is needed.

Effects on developmental toxicity

Description of key information

Developmental toxicity was not observed in the oral combined repeated dose and reproduction/development screening study according to OECD guideline 422, in which rats were dosed at 100, 300 and 1000 mg act. ingr./kg bw/day with the registered test substance. No test item related influence was noted on the survival rate and the mean and total body weights of the pups. External examination of the pups revealed no visible changes related to the test item. NOAEL for systemic toxicity was 300 mg/kg bw/day, whereas NOAEL for reproductive and developmental toxicity was >= 1000 mg/kg bw.

A key prenatal developmental toxicity study in female rats at dose levels of 100, 300 or 1000 mg/kg b.w./day of the registered test substance. Maternal changes at 1000 mg/kg bw/day included a reduction of the body weight, body weight gain, the food consumption (transient), carcass weight, and pathologic changes in the forestomach, of which the latter is not considered relevant for humans. There were no changes in reproductive parameters or fetal findings. The NOAEL was 300 mg act. ingr./kg b.w./day for the dams and above 1000 mg act. ingr./kg b.w./day for the fetuses.

Prenatal developmental toxicity was also 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:

experimental study

Adequacy of study:

key study

Study period:

8 January 2019 -

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted 25 June 2018

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Version / remarks:

Commission Regulation (EU) No. 260/2014 adopted 24 January 2014, published in the Official Journal of the European Union L81, dated 19 March 2014

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

Test material contains 41.2% solid content, therefore a correction factor 2.43 was applied.
See confidential information.

Species:

rat

Strain:

other: CD®/ Crl:CD (SD)

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 on day 0 of pregnancy: 62 - 70 days
- Weight on day 0 of pregnancy: 197.5 g - 275.6 g
- Fasting period before study: not reported
- Housing: 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 released for animal bedding (Granulat A2, J. Brandenburg, 49424 Goldenstedt/Arkeburg, Germany) was used as bedding material in the cages. The cages were cleaned and changed once a week. The animals received one piece of wood (certified for animal use) to gnaw on once weekly at change of the cages. Octagon-shaped red-tinted huts (polycarbonate) were placed in the cages to offer the animals a resting and hiding place.
- Diet (e.g. ad libitum): Commercial diet ssniff® R/Z V1324 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany, served as food. This food was offered daily ad libitum.
- Water (e.g. ad libitum): Drinking water (in drinking bottles) was offered ad libitum.
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C ± 3°C (maximum range)
- Humidity (%):55% ± 10% (maximum range)
- Air changes (per hr): between fifteen to twenty air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
From start of mating: 14 January 2019
To termination of in-life part: 13 February 2019

Route of administration:

oral: gavage

Vehicle:

other: tap water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test item formulations were freshly prepared every day.
The test item was diluted in the vehicle to the appropriate concentrations and was administered orally at a constant volume/kg bw once daily from the 6th to the 20th day of gestation.
The amount of the test item was adjusted to the animal's current body weight daily. The control animals received the vehicle at the same administration volume daily in the same way.
In addition, the homogeneity and concentration of the test item mixture were monitored.
The male rats for mating remained untreated.
Administration volume: 5 mL/kg bw/day

VEHICLE
- Concentration in vehicle: The test item was diluted in the vehicle to the appropriate concentrations and was administered orally at a constant volume/kg bw once daily from the 6th to the 20th day of gestation.
The amount of the test item was adjusted to the animal's current body weight daily. The control animals received the vehicle at the same administration volume daily in the same way.
- Amount of vehicle (if gavage): Administration volume: 5 mL/kg bw/day

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For the analysis of the test item-vehicle formulations, samples of approximately 10 mL were taken at the following times and stored at -20 °C ± 10 % until analysis at LPT:
-At start of dosing:
Analysis of concentration: During treatment always before administration to the last animal of the group (1 sample/test item group).
Number of samples:1 x 3 = 3 (Sampling date: 22 January 2019)
Homogeneity: At the start of treatment, during (middle) administration and before administration to the last animal of the test item group. (3 samples/test item group)
Number of samples: 3 x 3 = 9 (Sampling date: 22 January 2019)
- At the end of the dosing period, at a time when the majority of the animals was dosed:
Analysis of concentration: During treatment always before administration to the last animal of the group (1 sample/test item group).
Number of samples:1 x 3 = 3 (Sampling date: 04 February 2019)
Sum of all samples:=15
The samples were labelled with the study number, species, type of sample, concentration, sampling time, date and test day.
The analytical method was validated in LPT Study No. 36867. The samples were analysed at LPT.
-Results:
Concentration before administration of the last animal of each dose group at start of dosing and at a time when the majority of animals was dosed: 99.2% - 101.6% nominal concentration.
Homogeneity at the start of administration, during administration and before administration to the last animal of each dose group: 98.5% - 100.2% nominal concentration.
The measured actual concentrations of the test item in the test item vehicle-mixtures were between 98.5% and 101.6% of the nominal concentrations, indicating correctly prepared and homogenous formulations.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1
- Length of cohabitation: 1 male and 1 female animal were placed together in one cage during the dark period. Each morning a vaginal smear was taken to check for the presence of sperm. If findings were negative, mating was repeated with the same partner. This procedure was repeated until 25 mated dams were available for each groups.
- Any non-pregnant rats would have been excluded from the analysis of the results and replaced with the designated spare animals of each group treated and mated together with the other animals. A post-mortem negative staining according to SALEWSKI was carried out in the replaced animals in order to confirm the non-pregnancy status.
- Verification of same strain and source of both sexes: Sexually mature ('proved') male rats of the same breed served as partners. The female breeding partners were randomly chosen.
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

15 administration days from gestation days 6 to 20

Frequency of treatment:

once daily

Duration of test:

5 adaptation days, 1 mating day, 15 administration days from gestation days 6 to 20, laparotomy on gestation day 21= 22 days

Dose / conc.:

100 other: mg act. ingr./kg bw/day actual ingested

Dose / conc.:

300 other: mg act. ingr./kg bw/day actual ingested

Dose / conc.:

1 000 other: mg act. ingr./kg bw/day actual ingested

No. of animals per sex per dose:

25 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels for this study had been selected to be 100, 300 and 1000 mg act. ingr./kg bw/day by the Sponsor based on available toxicological data of an OECD 422 study (LPT study report no. 28640). The dose of 1000 mg/kg b. was considered to be a maximum tolerated dose.
In this OECD 422 study, the test item was administered orally to rats at dose levels of 100, 300 and 1000 mg act. ingr./kg bw/day. The administration started two weeks before mating on test day one and ended the day before sacrifice. Males were dosed up to test day 36 and females were dosed up to test day 54 (day 3 post-partum).
- Rationale for animal assignment (if not random): Four (4) groups of pregnant rats were established, each obtained from matings which were carried out on a daily basis. Vaginal smears were taken each morning. Day 0 of pregnancy was the day on which sperm was found in the vaginal smear. When positive, the animals were assigned to the test groups by mating day using a Provantis® -generated randomisation based on the body weight of the animals.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes.
- Time schedule: In addition to detailed clinical observations, animals were checked regularly throughout the working day from 7.00 a.m. to 3.45 p.m.
On Saturdays and Sundays, the animals were checked regularly starting from 7.00 a.m. to 11.00 a.m. with a final check performed at approximately 3.30 p.m.
Further checks were made early in the morning and again in the afternoon of each working day to look for dead or moribund animals. This allowed post mortem examinations to be carried out during the working period of that day. On Saturdays and Sundays, a similar procedure was followed except that the final check was carried out at approximately midday.
Animals showing signs of abortion or premature delivery would have been sacrificed on the same day. Fetuses obtained this way were examined for abnormal development, whenever possible. No abortion or premature delivery occurred in the study.

DETAILED CLINICAL OBSERVATIONS: Yes. Individual animals were observed daily for behavioural changes, reaction to treatment, or illness.
- Time schedule: Immediately after administration, any signs of illness or reaction to treatment were recorded. In case of changes, the animals were observed until the symptoms disappeared.
Dated and signed records of appearance, change and disappearance of clinical signs were maintained on clinical history sheets for individual animals.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each rat was recorded on day 0 of gestation (the day of detection of a positive mating sign), followed by daily weighing - always at the same time of the day.

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes. The quantity of food consumed by each rat was recorded daily. Food intake per rat (g/rat/day) was calculated using the total amount of food given to and left by each rat in each group on completion of a treatment day.
The relative food consumption (g/kg bw/day) was calculated using the following formula:
Daily food consumption [g/kg bw/day]= Total food intake in g / Body weight in kg

WATER CONSUMPTION: Visual
- Time schedule for examinations: Daily monitoring by visual appraisal of the drinking water bottles was maintained throughout the study. Dehydration of the dams was avoided.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 21. The rats were laparotomised under CO2 narcosis
- Organs examined: . The thyroids (including parathyroids) and the gravid uterus (in toto) of the dams were removed and weighed.
In order to check for possible test item effects, a dissection with macroscopic examination of the internal organs and placentae of the dams was carried out on the day of sacrifice or on the day on which the animals were found dead. The thyroids and any organs with macroscopic findings of all dams (including deceased or prematurely sacrificed animals) were fixed in 7% neutral buffered formalin.
- The thyroids of all evaluated dams were examined histopathologically after preparation of haematoxylin-eosin stained paraffin sections.

OTHER:
BODY WEIGHT GAIN: The body weight gain was calculated in intervals (i.e. gestation day 0-3, 3 6, 6-9, 9-12, 12 15, 15-18 and 18-21), for the whole study (gestation day 0 - 21) and for the period after the start of dosing (gestation day 6 to gestation day 21). Furthermore the carcass weight and the net weight gain from day 6 is given. These values are stated in the report.
These measurements were also used for calculating the daily amount of test item to be administered.

THYROID HORMONE (T3, T4, TSH) DETERMINATION: In order to obtain approximately 2 x 150 µL serum for each endocrine endpoint (T3, T4, TSH), a sufficient volume of blood was taken from the retrobulbar ve-nous plexus under isoflurane anaesthesia from animals fasted overnight following a randomisation scheme. Blood samples were taken always at the same time of day (approximately from 7:00 a.m. to 10:00 a.m.). The samples were divided into aliquots and stored at -20°C ± 10% at LPT until analysis using ELISA.
The T3, T4 and TSH ELISA (commercial kits: IBL International GmbH , instrument: Tecan Sunrise ) was conducted at LPT.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: Weight of placentae

Fetal examinations:

- External examinations: Yes: all per litter ((dead and alive)
- Soft tissue examinations: Yes: The remaining 50% of the number of fetuses (cfr skeletal examinations) in each litter were examined for soft tissue anomalies.
- Skeletal examinations: Yes: 50% of the number of fetuses in each litter
- Head examinations: No

Weight of fetuses
- individual data per fetus (alive and dead)
- mean per litter
- mean per sex and litter
- mean per group
- mean per sex and group

Fetuses
- number of evaluated dams with viable fetuses
- number per dam (alive)
- number per dam (dead)
- number of fetuses (alive + dead) per sex and dam
- number of fetuses (alive + dead) per sex and group
- distribution in the uterine horns
- absolute number of fetuses alive per group
- mean number of fetuses alive per group per dam
- mean % of fetuses alive per group per dam
- male/female ratio (alive + dead) per litter
- male/female ratio (alive + dead) per group

Runts
- number per dam
- number per group

Statistics:

-Parametrical data:
The statistical evaluation of the parametrical values was done by Provantis using the following settings:
Homogeneity of variances and normality of distribution were tested using the BARTLETT's and SHAPIRO-WILK's test. In case of heterogeneity and/or non-normality of distribution, stepwise transformation of the values into logarithmic or rank values was performed prior to ANOVA. If the ANOVA yielded a significant effect (p ≤ 0.05), intergroup comparisons with the control group were made by the DUNNETT’s test (p ≤ 0.01 and p ≤ 0.05).
-Non-parametrical data:
The statistical evaluation of non-parametrical values was done using the FISHER or Chi2 test: FISHERs exact test, n < 100; (p ≤ 0.05 and p ≤ 0.01) or Chi2 test, n ≥ 100 (p ≤ 0.05 and p ≤ 0.01).
The respective calculations for the FISHER and Chi2 test were performed using Provantis (maternal macroscopic findings at necropsy or findings during the external or internal macroscopic examination of the fetuses) or an internal computer program (e.g. findings during the fetal skeletal or soft tissue examination).
The statistical evaluation of the pre- and post-implantation index per group using the number of corpora lutea, implantation sites and/ or fetuses per was done using StatXact 4.0.1 software.
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.

Indices:

Total malformation rate (%) = (malformed fetuses per group / fetuses per group) x 100
Total variation rate (%) = (fetuses per group with variations / fetuses per group) x 100
Total retardation rate (%) = (fetuses per group with retardations / fetuses per group) x 100
Pre-implantation loss (%) = ((Corpora lutea (per group) – implantations (per group)) / Corpora lutea (per group)) x 100
Post-implantation loss (%) = ((Implantations (per group) – living fetuses (per group)) / Implantations (per group)) x 100
Litter indices:
Pre-implantation loss [%] =Sum of pre-implantation losses per litter in a group [%] / Number of litters in a group
Post-implantation loss [%] = Sum of post-implantation losses per litter in a group [%] / Number of litters in a group

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

(No toxicologically relevant effects). No adverse influences on the behaviour, external appearance or faeces were noted for the treatment groups (100, 300 or 1000 mg act. ingr./kg bw/day). However, at 1000 mg act. ingr./kg bw/day, salivation was noted for 16 of 20 females. This was not considered to be an adverse effect.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No test item-related premature deaths were noted in the dose groups (100, 300 or 1000 mg act. ingr./kg bw/day).
In the intermediate dose group (300 mg act. ingr./kg bw/day), female no. 67 was prematurely sacrificed on its gestation day 12 due to animal welfare reasons. Female no. 67 was noted with clinical observations in form of piloerection and slightly reduced motility on GD 11. Also, a reduced food consumption from GD 7 onwards and a reduced body weight from GD 8 onwards were noted. Necropsy revealed macroscopic changes in form of a haemorrhagic nose and canthus of the left eye, dark red discoloured lungs and a stomach ulcus. The death of the animal was regarded to be due to a misgavage.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 1000 mg act. ingr./kg bw/day, a reduced body weight and a reduced body weight gain were noted from GD 16 until study termination on GD 21 (at maximum 6.1% below the value of the control group for the body weight and 22% below the value of the control group from GD 6 to GD 21 for the body weight gain).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

At 1000 mg act. Ingr./kg bw/day, a test item-related transient decrease in food consumption was noted between GD 6 and GD 10.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Description (incidence and severity):

No test item-related differences were noted.

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Thyroid hormone concentration: No toxicologically relevant changes for serum T3, T4 and TSH levels were noted.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Uterus and carcass weights: No test item-related differences were noted for the gravid uterus weight. In the high dose group (1000 mg act. ingr./kg bw/day), a reduction was noted for the carcass weight (7.0% below the value of the control group, p ≤ 0.01).
Thyroid weights: No test item-related differences were noted.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Macroscopic inspection of the dams at necropsy revealed no adverse effects at 100 and 300 mg act. ingr./kg bw/day, but test item-related changes in the cardiac region of the stomach in form of a (partly) whitish thickening or white deposits for 18 of 20 high dose animals at 1000 mg act. ingr./kg bw/day. However, due to the absence of a forestomach in humans this finding is of no noteworthy relevance for humans.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Histopathologic examination of the thyroids revealed no test item-related changes.

Histopathological findings: neoplastic:

no effects observed

Details on results:

- Behaviour, external appearance, faeces:
No changes in behaviour, the external appearance or the faeces were noted in the control group and the low dose group (100 mg act. ingr./kg bw/day) and no toxicologically relevant changes were noted for the intermediate and high dose groups (300 or 1000 mg act. ingr./kg bw/day).
In the intermediate dose group female no. 67 was prematurely sacrificed on its gestation day 12 due to animal welfare reasons; two further animals (nos. 59 and 60) were noted with clinical observations. However, as only 3 of 22 animals including prematurely sacrificed animal no. 67 were affected and the clinical signs were noted only for a few days , the clinical signs were considered to be not toxicologically relevant. In addition, animal no. 59 was identified with 19 dead fetuses at laparotomy. However, it is unknown whether the fetuses died first and were the cause for the observations noted for animal no. 59 or the death of all fetuses was caused by the poor health of the dam. Nevertheless, this animal can be considered as an outlier.
In the high dose group (1000 mg act. ingr./kg b.w./day), clinical observations were noted in form of piloerection, salivation and increased water consumption. As piloerection and increased water consumption were noted only for a few group 4 females, these two clinical observations were not considered to be an adverse effect. Salivation was noted for 16 of 20 animals. However, as animals were affected not more than two days each and salivation lasted only shortly, the observations of salivation are regarded to be test item-related but not considered to be an adverse effect.
- Mortality:
No test item-related premature deaths were noted in the dose groups (100, 300 or 1000 mg act. ingr./kg bw/day). In the intermediate dose group (300 mg act. ingr./kg bw/day), female no. 67 was prematurely sacrificed on its gestation day 12 due to animal welfare reasons. Female no. 67 was noted with clinical observations in form of piloerection and slightly reduced motility on GD 11. Also, a reduced food consumption from GD 7 onwards and a reduced body weight from GD 8 onwards were noted. Necropsy revealed macroscopic changes in form of a haemorrhagic nose and canthus of the left eye, dark red discoloured lungs and a stomach ulcus. The death of the animal was regarded to be due to a misgavage.
- Body weight and body weight gain:
No test item-related differences in body weight were noted between the dams of the control group and low and intermediate dose groups (100 or 300 mg act. ingr./kg bw/day).
In the intermediate dose group (300 mg act. ingr./kg bw/day), a reduced but not statistically significant body weight was noted from GD 18 until GD 21 (at maximum 5.9% below the value of the control group). However, as this was mainly due to the two females nos. 59 (animal with 19 dead fetuses) and female no. 60 that were in a poor health conditions; the reduced body weight was considered to be not test item-related. In the high dose group (1000 mg act. ingr. /kg bw/day), a decreased body weight was noted from GD 16 until study termination on GD 21 (at maximum 6.1% below the value of the control group on GD 21, statistically significant for GD 20 and GD 21 at p ≤ 0.05 or 0.01) for nearly all animals. This constantly decreased body weight was considered to be test item-related.
No test item-related difference between the control group and the animals treated with 100 or 300 act. ingr./kg bw/day was noted for the body weight gain from GD 0 to 21. At 300 mg act. ingr./kg bw/day, a slight reduction was noted for the body weight gain (statistically significant for the body weight gain of the treatment period from GD 6 to GD 21, p ≤ 0.01). However, this was mainly due to only two females (nos. 59 and 60). Animal no. 59 delivered 19 dead fetuses with distinctly reduced body weights and also the fetuses of animal no. 60 had a lower body weight due to the poor health of this dam. The decreased body weights of the fetuses led to decreased maternal body weights and therefore, the reduced body weight gain was considered to be not test item-related. At 1000 mg act. ingr./kg bw/day, a statistically significant reduction was noted for the body weight gain for the whole study period (GD 0 to GD 21) and for the treatment period (GD 6 to GD 21) at p ≤ 0.01 for nearly all animals. As this was in accordance with the test item-related reduction in the body weight, also the reduced body weight gain was considered to be test item-related.
There were no test item-related influences on the gravid uterus weight. Therefore, the gravid uterus weight had the same influence on the body weight gain for all groups
- Food and drinking water consumption:
No test item-related difference was noted between the control group and the treatment groups (100, 300 or 1000 mg act. ingr./kg bw/day). In the high dose group, a statistically significantly (at p ≤ 0.05 or 0.01) decreased food consumption was noted between GD 6 to GD 10 (at maximum 25.8% below the value of the control group on GD 7 to GD 8). It reflects the decreased body weight and body weight gain observed in this group. As the food consumption recovered thereafter, this transient reduction in food consumption was considered to be test item-related. Furthermore, a statistically significant increase in food consumption was noted for the high dose group (GD 2 to GD 3) and a statistically significant decrease was noted for the low dose group (GD 12 to GD 13). However, as these differences were either before start of treatment (high dose group) or lasted for only one day (low dose group) these differences were considered to be spontaneous.
Although female no. 86 of the high dose group was noted with increased water consumption on gestation day 20 and 21, no test item-related changes in drinking water consumption were noted between the dams of the control group and the dams of the treatment groups by visual appraisal.
- Thyroid hormone concentration:
No test item-related differences were noted for the serum concentration of T4 in all dose groups (100, 300 or 1000 mg act. ingr./kg bw/day) and for T3 and TSH in the low dose group.
In the intermediate and high dose group (300 or 1000 mg act. ingr./kg bw/day), a dose-related increase was noted for the serum concentration of T3 (64.4% and 92.2% above the value of the control group, both statistically significant at p < 0.01) and TSH (8.8% and 19.8% above the value of the control group, not statistically significant). However, no difference was noted for the thyroid weights and the histopathological examination of the thyroids revealed no pathologic changes. Therefore, as it is known that histopathological examination of the thyroids is usually more sensitive than hormone levels (Beekhuijzen et al., 2016) the increases in serum levels of T3 and TSH were considered to be of no toxicological relevance.
- Necropsy findings:
No test item-related observations were noted for the dams of the low and intermediate dose groups (100 or 300 mg act. ingr./kg bw/day) during the macroscopic inspection of the organs and tissues.
In the intermediate dose group, macroscopic changes were noted for three females: No. 59 that delivered only dead fetuses revealed changes in form of a haemorrhagic nose and snout, rough fur , an anus that was soiled with faeces and inflated intestines filled with yellow liquid. Female no. 60 was noted with rough fur11 and few haemorrhagic foci in the stomach and the prematurely sacrificed female no. 67 was noted with a nose and the canthus of the left eye that was haemorrhagic, dark-red discoloured lungs and a stomach ulcus.
All findings were regarded to be spontaneous (animals no. 59 and 60) or related to the misgavage (animal no. 67). It is unknown whether the dead fetuses of animal no. 59 were the cause or the result of the bad health condition of the dam. Although the occurrence of dead fetuses is a very rare incident, this incident with all fetuses being dead is considered to be not test item-related as it was observed in only 1 animal of the intermediate dose group and therefore regarded to be an outlier.
In the high dose group (1000 mg act. ingr./kg bw/day), necropsy revealed for single animals changes in the stomach in form of ulcers (no. 80) and detachment of the mucosa (no. 93). Furthermore, a (partly) whitish thickening of the cardiac region of the stomach was noted for 18 of 20 examined animals.
Due to the high incidence of stomach observations, the changes related to the cardiac region of the stomach observed in the high dose animals were considered to be test item-related. However, due to the absence of a forestomach in humans this finding is of no noteworthy relevance for humans.
- Histopathology:
No test item-related morphological lesions were noted during histopathologic examination of the thyroids of the dose groups (100, 300 or 1000 mg act. ingr./kg bw/day).
In all groups, histopathologic examination revealed microfollicular structures with cells in the follicular lumen of the thyroids and squamous cell cysts of the thyroid follicle. However, these findings were considered to be coincidental and not test item-related.
- Thyroid weights
No test item-related differences to the control group were noted for the thyroid weights of the dose groups (100, 300 or 1000 mg act. ingr./kg bw/day).
- Gravid uterus weight, carcass weight and body weight gain from day 6:
No test item-related differences were noted between the gravid uterus weight of the control dams and the dams of the treatment groups (100, 300 or 1000 mg act. ingr./kg bw/day).
For the carcass weight, a statistically significant decrease was noted for the high dose animals (7.0% below the value of the control group, p ≤ 0.01). As this was in accordance with the lower body weight, the reduced carcass weight was considered to be test item-related.
No test item-related differences were noted for the net body weight gain from GD 6 to 21 between the dams of the control group and the dams of the low and intermediate dose groups (100 or 300 mg act. ingr./kg bw/day).
In the intermediate dose group, a statistically significant decrease (p ≤ 0.01) was noted for the net body weight gain. However, as also the low net body weight gain was mainly due to the animals nos. 59 and 60, the reduction was considered to be not test item-related.
At 1000 mg act. ingr./kg bw/day, the distinctly decreased net body weight gain (9.2 g compared to 37.0 g in the control group, p ≤ 0.01) was considered to be test item-related as it was in accordance with the reduced carcass weight and the reduced weight of the whole body (gravid uterus weight + carcass weight).

Number of abortions:

no effects observed

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

In the intermediate dose group, a statistically significantly increased value was noted for the post-implantation loss (p ≤ 0.01). As this was mainly due to one animal that delivered only dead fetuses (animal no. 59), the increased post-implantation loss was considered to be spontaneous and not test item-related.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

effects observed, non-treatment-related

Description (incidence and severity):

In the intermediate dose group, a statistically significantly increased value was noted for the post-implantation loss (p ≤ 0.01). As this was mainly due to one animal that delivered only dead fetuses (animal no. 59), the increased post-implantation loss was considered to be spontaneous and not test item-related.

Dead fetuses:

effects observed, non-treatment-related

Description (incidence and severity):

In the intermediate dose group, one animal that delivered only dead fetuses (animal no. 59), which was considered to be spontaneous and not test item-related.

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

no effects observed

Other effects:

no effects observed

Details on maternal toxic effects:

No test item-related influence on the reproductive parameters (number of implantation sites, fetuses, resorptions and the index of pre- and post-implantation loss) was noted between the dams of the control group and the dams of the treatment groups (100, 300 or 1000 mg act. ingr./kg bw/day).
In the intermediate dose group, a statistically significantly increased value was noted for the post-implantation loss (p ≤ 0.01). As this was mainly due to one animal that delivered only dead fetuses (animal no. 59), the increased post-implantation loss was considered to be spontaneous and not test item-related.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

act. ingr.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

organ weights and organ / body weight ratios

Abnormalities:

effects observed, treatment-related

Localisation:

not specified

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Body weight of the fetuses and the placentae: No test item-related differences were noted between the control group and the dose groups.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

No dead fetus was noted in the control group and in the low and high dose group (100 or 1000 mg act. ingr./kg bw/day).
In the intermediate dose group (300 mg act. ingr./kg bw/day), dam no. 59 delivered only dead fetuses (11 male and 8 female fetuses). External inspection revealed one female fetus (no. 59-08) with a malformation in form of an anencephaly. However, as no other influences on the reproduction were noted and no dead fetuses were observed in the high dose group, the occurrence of one litter with only dead fetuses was considered to be not test item-related.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

No test item-related differences between the ratio of male and female fetuses were noted between the control group and the dose groups (100, 300 or 1000 mg act. ingr./kg bw/day).

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

No test item-related malformations were noted during the macroscopic examinations at laparotomy (external inspection and inspection of the organs and tissues for gross lesions).

Skeletal malformations:

no effects observed

Description (incidence and severity):

No test item-related malformations were noted during the skeletal examination according to DAWSON.

Visceral malformations:

no effects observed

Description (incidence and severity):

No test item-related malformations were noted during the soft tissue examination according to WILSON.

Other effects:

no effects observed

Description (incidence and severity):

Mortality: No test item-related death of fetuses was noted.
Variations: The macroscopic examinations at laparotomy, the skeletal examination according to DAWSON and the soft tissue examination according to WILSON revealed no test item-related variations
Retardations: No test item-related retardations (delays in ossification) were noted during the skeletal examination according to DAWSON.

Details on embryotoxic / teratogenic effects:

- Mortality:
No dead fetus was noted in the control group and in the low and high dose group (100 or 1000 mg act. ingr./kg bw/day).
In the intermediate dose group (300 mg act. ingr./kg bw/day), dam no. 59 delivered only dead fetuses (11 male and 8 female fetuses). External inspection revealed one female fetus (no. 59-08) with a malformation in form of an anencephaly. However, as no other influences on the reproduction were noted and no dead fetuses were observed in the high dose group, the occurrence of one litter with only dead fetuses was considered to be not test item-related.
- Sex distribution of the fetuses:
No test item-related differences between the ratio of male and female fetuses were noted between the control group and the dose groups (100, 300 or 1000 mg act. ingr./kg bw/day).
- Weight of placentae and fetuses:
The placental and fetal weights showed no test item-related differences between the control group and the treatment groups (100, 300 or 1000 mg act. ingr./kg bw/day).
- Number of runts:
No runts were noted for the control group and the intermediate and high dose groups (300 or 1000 mg act. ingr./kg bw/day).
In the low dose group (100 mg act. ingr./kg bw/day), two runts (nos. 26-15 and 36-11) were noted. The occurrence of two runts in the low dose group is within the normal range of biological variability and therefore, was considered to be not test item-related.
- Ano-genital distance:
No test item-related differences to the control group were noted for the fetal ano-genital distance of the dose groups (100, 300 or 1000 mg act. ingr./kg bw/day).
- Macroscopic inspection of the fetuses at laparotomy:
1) External inspection at laparotomy
No macroscopically visible external malformations were noted for the fetuses of the low and high dose group (100 or 1000 mg act. ingr./kg bw/day) during the external inspection at laparotomy.
In the intermediate dose group (300 mg act. ingr./kg bw/day), one fetus (no. 59 08, dead at birth) was noted with a malformation in form of an anencephaly and one fetus (no. 54-19) was noted with multiple malformations in form of a bifurcated tail, supernumerary extremities at the back (presumably a conjoined twin), a cleft palate and an anencephaly. The occurrence of two fetuses with malformations was considered to be spontaneous and not test item-related.
2) Gross inspection of the organs and tissues at laparotomy
The macroscopic inspection of the organs and tissues for gross alterations at laparotomy revealed no malformations or variations for the fetuses of the control group and the fetuses of the dose groups (100, 300 or 1000 mg act. ingr./kg bw/day).
3) Testicular development
No cryptorchidism and no testicular malposition was noted during assessment of the testicular development of the male fetuses of the control group and the dose groups (100, 300 or 1000 mg act. ingr./kg bw/day).
- Skeletal examination according to DAWSON:
1) Skeletal malformations
No skeletal malformations were noted for the fetuses of the control group and the test item-treated groups (100, 300 or 1000 mg act. ingr./kg bw/day) during the skeletal examination according to DAWSON.
2) Skeletal variations
Skeletal variations were noted for the ribs (ribs isolated, ribs short or ribs wavy) and the sternum (bipartite or misaligned to a slight degree).
No test item-related difference in the incidence of the observed skeletal variations in comparison to the control group was noted for the fetuses of the treatment groups (100, 300 or 1000 mg act. ingr./kg bw/day).
3) Skeletal retardations
Retardations (delayed ossifications) were related to the skull (incomplete ossification of frontal, parietal, interparietal and/or supraoccipital areas), the hyoid (unossified), the sternum (sternebra(e) incompletely ossified, reduced in size or unossified), the thoracic vertebral bodies (bipartite, reduced in size or dumbbell-shaped), the caudal vertebral bodies (only one body ossified or all bodies unossified), the lumbar vertebral bodies (unossified), the sacral vertebral bodies (unossified), the lumbar vertebral arches (incompletely ossified), the os ischii (unossified or incompletely ossified), the os pubis (unossidied or incompletely ossified) and the metacarpalia/metatarsalia (absence of ossification in metacarpalia/metatarsalia 2 to 5).
No test item-related increase in the incidence of skeletal retardations at 100, 300 or 1000 mg act. ingr./kg bw/day was noted during skeletal examination according to DAWSON.
In the intermediate dose group, statistically significantly increased incidences were noted for skeletal retardations that were outside the range of the LPT background data (all caudal vertebral bodies unossified, os ischii unossified, os pubis unossified and sacral vertebral bodies unossified). However, the majority of the mentioned retardations were noted for dam no. 59 that littered only dead fetuses. Therefore, the retardations were considered to be not test item-related.
- Soft tissue examination according to WILSON:
1) Malformations
No malformations were noted for the fetuses of the control group and the fetuses of the low and high dose groups (100 or 1000 mg act. ingr./kg bw/day) during the soft tissue examination according to WILSON.
In the intermediate dose group (300 mg act. ingr./kg bw/day), one fetus (no. 59 8) was noted an anencephaly and one fetus (no. 54 19) was a conjoined twin with two spinal cords, extremities in the back region, a bifurcated tail, anencephaly, an open eye, a cleft palate and a missing anus.
However, as no malformations were noted in the high dose group, the occurrence of two fetuses with malformations in the intermediate dose group was considered to be spontaneous and not test item-related.
2) Variations
During the examination of the organs and tissues according to WILSON, variations were noted for the brain (dilatation of the 4th cerebral ventricle), the kidneys (uni- or bilateral dilatation of the renal pelvis or malpositioned) and the liver (haemorrhagic focus/foci).
No test item-related differences and no statistically significant differences in the incidences of the observed variations were noted between the control group and the treatment groups (100, 300 or 1000 mg act. ingr./kg bw/day).
3) Unclassified observations
No unclassified observations were noted for the control group and for the low and high dose group (100 or 1000 mg act. ingr./kg bw/day).
An unclassified observation in form of a thoracic cavity filled with blood was noted for two fetuses (nos. 60-1 and 60-2) of the intermediate dose group (300 mg act. ingr./kg bw/day). This observation was considered to be a preparation-induced artefact and not test item-related.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

act. ingr.

Sex:

male/female

Remarks on result:

other: highest tested dose

Key result

Developmental effects observed:

no

Conclusions:

Under the present test conditions, the no-observed-adverse-effect level (NOAEL) was 300 mg act. ingr./kg bw/day for the dams.
The no-observed-adverse-effect level (NOAEL) for the fetal organism was above 1000 mg act. ingr./kg bw/day.
Under the conditions of the study, Butanedioic acid, 2(or 3)-sulfo-,4-[2-[(1-oxo(C12-C8 (even numbered) and C18 (unsaturated)alkyl) amino]ethyl]esters, disodium salts did not show any teratogenic potential in rats

Executive summary:

The aim of the study was to obtain information of the influence of the test item on the pregnant rat and the fetus when administered orally during the critical period of organogenesis and the fetal development (6th to 20th day of gestation) according to OECD Guideline 414. The test item was administered orally by gavage to rats at dose levels of 100, 300 and 1000 mg active ingredient/kg bw/day starting from the 6th and lasting until the 20th day of pregnancy. Their development was observed during the gestation period (day of mating is day 0 of pregnancy). On gestation day 21 (one day before the calculated date of parturition), the dams were laparotomised and examined for corpora lutea, implantation sites and resorptions in the uterus and for the condition of the fetuses.

 

Examination of the dams:

No test item-related premature deaths were noted in any of the test groups.

No adverse influences on the behaviour, external appearance or faeces were noted for the treatment groups (100, 300 or 1000 mg act. ingr./kg bw/day). However, at 1000 mg act. ingr./kg bw/day, salivation was noted for 16 of 20 females.

At 1000 mg act. ingr./kg bw/day, a reduced body weight and a reduced body weight gain were noted from GD 16 until study termination on GD 21 (at maximum 6.1% below the value of the control group for the body weight and 22% below the value of the control group from GD 6 to GD 21 for the body weight gain).

At 1000 mg act. ingr./kg bw/day, a test item-related transient decrease in food consumption was noted between GD 6 and GD 10.

No test item-related differences were noted in drinking water consumption.

Macroscopic inspection of the dams at necropsy revealed no adverse effects at 100 and 300 mg act. ingr./kg bw/day, but test item-related changes in the cardiac region of the stomach in form of a (partly) whitish thickening or white deposits for 18 of 20 high dose animals at 1000 mg act. ingr./kg bw/day. However, due to the absence of a forestomach in humans this finding is of no noteworthy relevance for humans.

No test item-related differences were noted in thyroid weights.

No toxicologically relevant changes for serum T3, T4 and TSH levels were noted.

Histopathologic examination of the thyroids revealed no test item-related changes.

No test item-related differences were noted for the gravid uterus weight.

In the high dose group (1000 mg act. ingr./kg bw/day), a reduction was noted for the carcass weight (7.0% below the value of the control group, p ≤ 0.01).

No test item-related influence was noted on the reproductive parameters (number of implantation sites, resorptions and fetuses).

 

Examination of the fetus:

No test item-related death of fetuses was noted.

No test item-related differences in body weight of the fetuses and the placentae were noted between the control group and the dose groups.

 

Fetal alterations:

No test item-related malformations were noted during the macroscopic examinations at laparotomy (external inspection and inspection of the organs and tissues for gross lesions), and no malformations were noted during the skeletal examination according to DAWSON and the soft tissue examination according to WILSON.

The macroscopic examinations at laparotomy, the skeletal examination according to DAWSON and the soft tissue examination according to WILSON revealed no test item-related variations.

No test item-related retardations (delays in ossification) were noted during the skeletal examination according to DAWSON.

 

Analysis of test item formulations:

The measured actual concentrations of the test item in the test item vehicle mixtures were between 98.5% and 101.6% of the nominal concentrations, indicating correctly prepared and homogeneous formulations.

 

Conclusion

Under the present test conditions, the no-observed-adverse-effect level (NOAEL) was 300 mg act. ingr./kg bw/day for the dams.

The no-observed-adverse-effect level (NOAEL) for the fetal organism was above 1000 mg act. ingr./kg bw/day.

Under the conditions of the study, Butanedioic acid, 2(or 3)-sulfo-,4-[2-[(1-oxo(C12-C8 (even numbered) and C18 (unsaturated)alkyl) amino]ethyl]esters, disodium salts did not show any teratogenic potential in rats.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

High (Klimisch 1)

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

Developmental screening

Supporting data for absence of developmental toxicity were available from an oral combined repeated dose and reproduction/development screening study according to OECD guideline 422 (Hansen, 2013d) with the registered test item. The test item was administered orally by gavage to rats with a formulation containing 41.5% active ingredient at dose levels of 100, 300 and 1000 mg act. ingr./kg bw/day for for at least 28 days in male rats and at least 39 days in females. No test item-related premature death was noted in any treatment group. No signs of clinical toxicity were noted for the male and female rats of the low and intermediate dose groups (100 and 300 mg/kg bw/day), whereas slightly increased salivation was noted in one male rat as the only finding at 1000 mg/kg bw/day. A slight reduction in body weight was noted for the male and female rats dosed at 1000 mg/kg bw/day. Other parameters such as neurological observations, haematology and serum chemistry are discussed in the repeated dose toxicity section.

No test item-related influence was noted on the developmental toxicity parameters in any treatment group (100, 300 and 1000 mg/kg bw/day). Microscopic examination revealed no changes in the reproductive organs from the male and female rats of the high dose group (1000 mg/kg bw/day). No test item related influence was noted on the survival rate and the mean and total body weights of the pups. External examination of the pups revealed no visible changes related to the test item. NOAEL for paternal/maternal toxicity was 300 mg/kg bw/day, whereas NOAEL for developmental toxicity was >= 1000 mg/kg bw.

 

Developmental toxicity and teratogenicity testing

A key prenatal developmental toxicity study with the registered substance Butanedioic acid, 2(or 3)-sulfo-,4-[2-[(1-oxo(C12-C8 (even numbered) and C18 (unsaturated)alkyl) amino]ethyl]esters, disodium salts was performed female rats at dose levels of 100,300 or 1000 mg/kg bw/day from the 6th to 20th day of pregnancy (Hansen, 2020b).

No test item-related premature death was noted for any dose group. No test item-related changes in behaviour, external appearance, or faeces were noted for the treatment groups except for salivation in 16 of 20 animals dosed with 1000 mg act. ingr./kg bw/day. However, this was not considered to be an adverse effect. No toxicological relevant changes in the serum levels of thyroid hormones were noted for any dose group. Furthermore, no difference was noted for the thyroid weights, and histopathological examination of the thyroids revealed no pathological changes.

A test item-related reduction of the body weight, the body weight gain, the food consumption (transient) and the carcass weight was noted for the females dosed with 1000 mg/kg bw/day. In the high dose group (1000 mg./kg bw/day), pathologic changes were noted for the cardiac region of the stomach (non-glandular stomach, i.e. the forestomach). However, due to the absence of a forestomach in humans this finding is of no noteworthy relevance for humans.

The reproductive parameters (number of implantation sites, number of resorptions and number of fetuses) were not influenced by the test item.

No test item-related deaths of fetuses and no test item-related malformations, variations or retardations were noted.

Under the present test conditions, the NOAEL was 300 mg/kg bw/day for the dams. The NOAEL for the fetal organism was above 1000 mg/kg bw/day.

Further supporting 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 supporing 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.  

 

Conclusion

An oral gavage reproductive screening study with registered substance showed NOAEL of 300 mg/kg bw for paternal/maternal systemic toxicity, whereas 1000 mg/kg bw was NOAEL for reproductive and developmental toxicity.

Prenatal developmental toxicity was tested by dietary administration of registered substance in rats from day 6 -20 of gestation by oral gavage up to 1000 mg/kg bw and from read across substance CAS No. 577 -11 -7 (Docusate sodium) in rats from day 6 to 15 of gestation.Maternal NOAEL was 300 mg/kg bw, whereas the NOAEL in the fetuses was above 1000 mg/kg bw wiht registered substance. Read-across subststance Docusate sodium at 1% in the diet showed 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 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 the results and according to CLP regulation (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.