Registration Dossier

Administrative data

Description of key information

A combined repeated dose and reproductive/developmental screening study which was performed with registered substance according to OECD guideline 422 showed NOAEL-levels of 60 mg/kg bw for paternal/maternal toxicity, 60 mg/kg bw for reproductive toxicity and 120 mg/kg bw for developmental toxicity.

A 90 -day study was available for the read across substance CAS No. 37294 -49 -8 (disodium C-isodecyl sulphonatosuccinate) in the mono-ester subgroup, given to rats at 0.25, 1 and 4% in the diet. This study showed a NOAEL of 1% in the diet, corresponding with 750 mg/kg bw. For risk assessment, the lowest NOAEL of 60 mg/kg bw with registered substance tested in the OECD 422 study was selected as this approach was considered most conservative.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to GLP and valid testing guidelines, therefore it is considered relevant, adequate and reliable for classification.
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
adopted March 22, 1996
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At +10°C to +25°C
- Stability under test conditions: The measured concentrations of the test item in the test item vehicle mixtures were between 99.7% and 108.8% of the nominal concentrations. These values indicated correctly prepared test item vehicle mixtures, which were stable at room temperature for at least 24 hours.
- Solubility and stability of the test substance in the solvent/vehicle: The test item vehicle mixtures were stable at room temperature for at least 24 hours. No phase separation occured between the test item and the vehicle during the procedure of administration of the test item vehicle mixtures to the animals.
Species:
rat
Strain:
other: CD® / Crl:CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Research, Models and Services Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age at start of dosing: Males: 59 days; Females: 69 days
- Weight at start of dosing: Males: 328.9 – 368.9 g; Females: 210.3 – 253.9 g
- Fasting period before study: the night before the day of blood withdrawal for Iaboratory examination
- Housing: With exception of the mating period, the animals were kept singly in MAKROLON cages (type III plus) with a basal surface of approx. 39 cm x 23 cm and a height of approx. 18 cm. Granulated textured wood (Granulat A2, J. Brandenburg, 49424 Goldenstedt/Arkeburg, Germany) was used as bedding material in these cages. The cages were cleaned and changed once a week.
- Diet (e.g. ad libitum): Commercial ssniff® R/Z V1324 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany), ad libitum with the exception of the night before the day of blood withdrawal for Iaboratory examination
- Water (e.g. ad libitum): Tap water was offered daily ad libitum
- Acclimation period: 14 days

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

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

VEHICLE: tap water

Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
Males: Beginning 2 weeks prior to mating lasting up to the day before sacrifice until a minimum dosing period of 28 days was completed.
Females: Beginning 2 weeks prior to mating continuing up to, and including, day 3 post partum or the day before sacrifice.

Frequency of treatment:
Once daily
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
active ingredient
Dose / conc.:
60 mg/kg bw/day (actual dose received)
Remarks:
active ingredient
Dose / conc.:
120 mg/kg bw/day (actual dose received)
Remarks:
active ingredient
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
active ingredient
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels have been selected in agreement with the Sponsor based on the results of a 14-day dose-range-finding study in rats dosed at 100, 300 and 1000 mg (active ingredient)/kg bw by oral gavage (LPT Study No. 28932).
Four of 5 males and all (5) females died prematurely. Oral treatment with 1000 mg/kg bw/day caused signs of systemic toxicity in form of pilo-erection, reduced motility, pultaeous faeces/diarrhoea, salivation, increased drinking water consumption, ataxia or decreased body temperature in the male and/or female rats.
A decrease in body weight, body weight at autopsy and food consumption was noted for the male and female rats treated with the intermediate and the high dose of Disodium C12-18 alkyl sulfosuccinate. At necropsy, whitish deposits on the stomach mucosa were observed in the male rats treated orally with 300 mg/kg bw/day. The high dose of 1000 mg/kg bw/day led to further changes in the gastro-intestinal tract in both sexes such as inflation or discolourations. The examination of organ weights revealed a dose-related increase of liver weights.
Based on the results of this study, the dose levels selected are 60, 120 and 300 mg/kg bw.

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Cage side observations included skin/fur, eyes, mucous membranes, respiratory and circulatory systems, somatomotor activity and behaviour patterns. The onset, intensity and duration of any signs observed were recorded.
- Time schedule: Throughout the test period, each animal were observed for clinical signs at least once daily. Individual animals were observed before and after dosing at each time of dosing for any signs of behavioural changes, reaction to treatment or illness. Mortality was recorded twice daily. In addition, animals were checked regularly throughout the working day from 7:00 a.m. to 3:45 p.m. On Saturdays and Sundays animals were checked regularly from 7:00 a.m. to 11:00 a.m. with a final check performed at approximately 3:30 p.m.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before the first exposure (to allow for within-subject comparisons) and once a week thereafter, detailed clinical observations were made in all animals; in test week 4 these observations were performed prior to any laboratory investigations.
- These observations were made outside the home cage in a standard arena and at the same time, each time.
- Signs observed included changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, pilo-erection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereo-typies (e.g. excessive grooming, repetitive circling) or bizarre behaviour (e.g. self-mutilation, walking backwards) were also recorded.

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

FOOD CONSUMPTION: Yes
- The quantity of food left by individual animals was recorded on a weekly or daily basis throughout the experimental period with the exception of the mating period.
- Food intake per rat (g/rat/week) was calculated using the total amount of food given to and left by each rat in each group upon completion of a treatment week. From these data the food consumption (in g/kg bw/day) was determined using the following formula:
Relative food consumption [g/kg b.w./day] = (Total food given [g] - Total food left [g]) /Number of animal days# x Body weight [kg]
# The term 'animal days' counts one animal day for each animal alive for a whole day; it is assumed that on the day of death an animal does not eat.

FOOD EFFICIENCY: No

WATER CONSUMPTION: No
- Water consumption was monitored daily by visual appraisal throughout the study.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the pre-mating period
- Anaesthetic used for blood collection: Yes, ether anaesthesia
- Animals fasted: Yes , overnight
- How many animals: 5 male and 5 female animals randomly selected from each group.
- Parameters examined:
Haemoglobin content
Erythrocytes
Leucocytes
Differential blood count (relative and aboslute)
Reticulocytes
Platelets
Haematocrit value
Thromboplastin time
Activated partial thromboplastin time
Mean corpuscular volume (MCV)
Mean corpuscular haemoglobin (MCH)
Mean corpuscular haemoglobin concentration (MCHC)


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of the pre-mating period
- Animals fasted: Yes, overnight
- How many animals: 5 male and 5 female animals randomly selected from each group.
- Parameters examined:
Sodium
Potassium
Calcium
Chloride
Albumin
Globulin
Albumin/globulin ratio
Total bilirubin
Total cholesterol
Creatinine
Glucose
Total protein
Blood urea
Alanine amino- transferase (ALAT)
Alkaline phosphatase (aP)
Aspartate aminotransferase (ASAT)
Bile acids

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Screening of sensory reactivity to stimuli of different types (e.g. auditory, visual and proprioceptive stimuli) (based on Gad), as well as the assessment of grip strength (Meyer ) and motor activity assessment were conducted in five males and five females randomly selected from each group.
The screening was conducted two hours after dosing.
- Dose groups that were examined: five males and five females randomly selected from each group.
5 males per group: Shortly before scheduled sacrifice on test day(TD) 41
5 females per group: During lactation, shortly before scheduled kill on TD 40-55
- Battery of functions tested:
*Observational screenings (Righting reflex, Body temperature, Salivation, Startle response, Respiration, Mouth breathing, Urination, Convulsions, Pilo-erection, Diarrhoea, Pupil size, Pupil response, Lacrimation, lmpaired gait, Stereotypy, Toepinch, Tail pinch, Wire maneuver, Hind leg splay, Positional passivity, Tremors, Positive geotropism, Limb rotation, Auditory function)
*Functional screenings (Grip strength, Locomotor activity)

OTHER: Reproductive & developmental performance : See Section 7.8.1 & 7.8..2

Sacrifice and pathology:
GROSS PATHOLGY: Yes
-The male animals were sacrificed on test day 43. Dams with offspring were sacrificed on day 4 post-partum, or shortly thereafter. Females showing no evidence of copulation were sacrificed 24 days after the last day of the mating period.
-At the time of sacrifice, or premature 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- See Section 7.8.1 & 7.8.2.
-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.
-Dead pups and pups sacrificed at day 4 post-partum, or shortly thereafter, were carefully examined externally for gross abnormalities.

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

HISTOPATHOLOGY: Yes
- The following organs or parts of organs of all adult animals were fixed in 7% formalin; testes and epididymides were fixed in Bouin's fixative:
Epididymis (2), Gross lesions, Mammary gland, Ovary (2), Prostate, Seminal vesicle, Testicle (2), Uterus (incl. cervix and oviducts), Vagina.
Detailed histopathological examination was performed on one testicle and one epididymis with special emphasis of the qualitative stages of spermatogenesis and histopathology of interstitial testicular structure of the selected animals of group 1 and 4 following haematoxylin-eosin and PAS staining. - See Section 7.8.1. & 7.8.2
- The following organs or parts of organs of all adult animals were fixed in 7% formalin; testes and epididymides were fixed in Bouin's fixative:
Epididymis (2), Gross lesions, Mammary gland, Ovary (2), Prostate, Seminal vesicle, Testicle (2), Uterus (incl. cervix and oviducts), Vagina
-In addition, the following organs or parts of organs of the selected 20 adult males and 20 adult females (see section above) were fixed in 7% formalin:
Adrenal gland (2)
Bone marrow (os femoris)
Brain (cerebrum, cerebellum, brain stem)
Heart (left and right ventricle, septum)
Intestine, small (duodenum, jejunum, ileum, incl. Peyer's patches, Swiss roll method)
Intestine, large (colon, rectum)
Kidney and ureter (2)
Liver
Lungs (with mainstem bronchi and
bronchioles), preserved by inflation with
fixative and then immersion
Lymph node (1, cervical), Lymph node (1, mesenteric)
Nerve (sciatic)
Oesophagus
Spinal cord (3 sections)
Spleen
Stomach
Thyroid (incl. parathyroids)
Thymus
Tissue masses or tumours (incl. regional lymph nodes)
Tongue (incl. base)
Trachea (incl. larynx)
Urinary bladder
-Only the 10 selected animals from the control group and the high dose group (20 animals in total) were considered for histopathological evaluation.
Group 1: 1, 2, 4, 5, 8 11, 14, 18, 19, 20
Group 4: 62, 65, 66, 68, 69 72, 73, 75, 76, 78
Statistics:
Toxicology and Pathology data were captured, whenever possible, using the departmental computerized systems (Provantis® Integrated preclinical software, Instem LSS Ltd.). Raw data not fully compatible with the computerized systems were maintained on paper according to appropriate SOPs.
The test item-treated groups (2- 4) were compared with the control group (1 ).
The following statistical methods are used:

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

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

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

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

These statistical procedures were used for all data. Significantly different data were indicated in the tables of the report.
The mean values and standard deviations were calculated to the highest possible degree of accuracy and then rounded to the reported number of decimal places. Hence, deviations to the last decimal place of up to ± 1 may occur caused by rounding.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Slight salivation was noted in 2 animals of the intermediate dose group (120 mg act. ingr./kg b.w./day) on one day each. In the high dose group (300 mg act. ingr./kg b.w./day) piloerection and slight to moderate salivation were noted for several animals during the whole study for 1 up to 13 test days. Breathing sounds were noted in animal no. 61 for 1 day.
In the intermediate dose group (120 mg act. ingr./kg b.w./day) piloerection was noted for 1 animal on 1 day during the mating period. During the gestation period moderate salivation was noted in 3 animals on one day each. In the high dose group (300 mg act. ingr./kg b.w./day) piloerection was noted for several animals on 3 up to 10 test days and slight to extreme salivation in all animals (2 up to 11 test days) during the pre-mating, mating and gestation period. A haemorrhagic vagina or nose was noted for animal no. 72 on gestation days 11 and 16. During the lactation period piloerection,
reduced motility and changes in the status of faeces were noted for the emaciated animal no. 75.
Mortality:
mortality observed, treatment-related
Description (incidence):
1 male and 1 female of the high dose group died on day 33 and day 26 respectively; slight signs of systemic toxicity were noted predominantly in form of pilo-erection and increased salivation in males and females dosed at 120 & 300 mg/kg bw
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
decreased at 120 and 300 mg/kg bw in male rats and at 300 mg/kg bw in female rats premating, mating, during gestation and during lactation
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
A statistically significant (p≤0.01) reduction in food consumption by 14.1% was noted in the intermediate dose group (120 mg act. ingr./kg b.w./day) during the first test week.
In the high dose group (300 mg act. ingr./kg b.w./day) the food consumption was statistically significantly (p≤0.01) reduced by 20.7% during the first and by 15.3% during the second test week..
At the intermediate dose group (120 mg act.ingr./kg b.w./day) a statistically significant reduction in food consumption was noted during the first week of gestation by 7.3% (p≤0.05)
and during the second week of gestation by 13.8% (p≤0.01). In the high dose group (300 mg act. ingr./kg b.w./day) a statistically significant (p≤0.01) reduction in food consumption was noted from
the first test week (by 26.3%) until the end of the gestation period (by 6.8%).
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
increased MCHC in males at 120 mg/kg bw; increased haemoglobin, red blood cells, haematocrit and MCHC value in males dosed 300 mg/kg bw; decrease aPTT time in females dosed 300 mg/kg bw
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
increased ALAT in males dosed at 120 mg/kg bw; increased ALAT, aP and ASAT and decreased cholesterol in males dosed at 300 mg/kg bw; increased ALAT and ASAT and decreased globulin, cholesterol, chloride, potassium in females dosed 300 mg/kg bw
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
decr. epididymis weights in M at 120 mg/kg; incr. liver and decreased thymus/testes weights in M at 300 mg/kg; incr. liver/adrenal and decr. heart weights in F at 120 mg/kg; incr. kidney/adrenal weights and decr. heart/ovary weight in F at 300 mg/kg
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
detachment of stomach mucosa, whitish thickenings and ulcers in males dosed 300 mg/kg bw; detachment of stomach mucosa in females dosed at 120 and 300 mg/kg bw
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
hepatocellular hypertrophy and macrovesicular vacuolation in liver and squamous cell hyperplasia in the non-glandular stomach in males and females at 300 mg/kg bw; changes in the mammary glands, the uterus and vagina in females at 300 mg/kg bw
Histopathological findings: neoplastic:
no effects observed
Details on results:
MORTALITY:
One of 10 male animals (no. 64) of the high dose group (300 mg test item/kg bw/day) died on test day 33, showing piloerection and reduced motility before death.
One of 10 female animals (no. 71) of the high dose group (300 mg test item/kg b.w./day) died on gestation day 9, showing piloerection and salivation on a few days during the premating, mating and gestation period.

CLINICAL SIGNS:
Male animals
Slight salivation was noted in 2 animals of the intermediate dose group (120 mg test item/kg b.w./day) on one day each.
In the high dose group (300 mg test item/kg bw/day) piloerection and slight to moderate salivation was noted for several animals during the whole study for 1 up to 13 test days. Breathing sounds were noted in animal no. 61 for 1 day.
Female animals
In the intermediate dose group (120 mg test item/kg bw/day) piloerection was noted for 1 animal on 1 day during the mating period. During the gestation period moderate salivation was noted in 3 animals on one day each.
In the high dose group (300 mg test item/kg bw/day) piloerection was noted for several animals on 3 up to 10 test days and slight to extreme salivation in all female animals (2 up to 11 test days) during the pre-mating, mating and gestation period. A haemorrhagic vagina or nose was noted for animal no. 72 on gestation days 11 and 16. During the lactation period piloerection, reduced motility and changes in the status of faeces were noted for the emaciated female animal no. 75.

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

FOOD CONSUMPTION:
Male animals
A statistically significant (p≤0.01) reduction in food consumption by 14.1% was noted in the interme-diate dose group (120 mg test item/kg bw/day) during the first test week.
In the high dose group (300 mg test item/kg bw/day) the food consumption was statistically significantly (p≤0.01) reduced by 20.7% during the first and by 15.3% during the second test week.
Female animals
At the intermediate dose group (120 mg test item/kg bw/day) a statistically significant reduction in food consumption was noted during the first week of gestation by 7.3% (p≤0.05) and during the second week of gestation by 13.8% (p≤0.01).
In the high dose group (300 mg test item/kg bw/day) a statistically significant (p≤0.01) reduction in food consumption was noted from the first test week (by 26.3%) until the end of the gestation period (by 6.8%).

HAEMATOLOGY
Male animals
At 120 mg test item/kg bw/day the MCHC value was slightly but statistically significantly (p≤0.01) increased by 2.6%.
In the high dose group (300 mg test item/kg bw/day) a statistically significant (p≤0.01) increase was noted for the concentration of haemoglobin (+11.8%), the number of red blood cells (+11.1%), the haematocrit value (+9.1%) and the MCHC value (+2.6%).
Female animals
A statistically significant (p≤0.01) decrease in the aPTT time by 8.4% was noted in the high dose group (300 mg test item/kg bw/day).

CLINICAL CHEMISTRY
Male animals
In the intermediate dose group (120 mg test item/kg bw/day) statistically significant (p≤0.01) increases were noted for the enzyme activity of ALAT (+158%) and aP (+175%).
In the high dose group (300 mg test item/kg bw/day) statistically significant (p≤0.01) increases were noted for the activity of ALAT (+688.3%), aP (+306%), ASAT (91%).
A statistically significant (p≤0.05) decrease by 33% was noted in the high dose group for the cholesterol concentration.
Female animals
In the high dose group (300 mg test item/kg bw/day) statistically significant (p≤0.01) increases were noted in the activity of ALAT (+674%) and ASAT (+104%).
The concentrations of globulin (by 8.8%), cholesterol (by 62%), chloride (by 3.1%) and potassium (by18%) were statistically significantly (p≤0.01) decreased in the high dose group (300 mg test item/kg bw/day).

NEUROBEHAVIOUR
No test item-related influence was noted for observational and functional screening and for spontaneous motility.

ORGAN WEIGHTS
Male animals
In the high dose group (300 mg test item/kg bw/day) the body weight at autopsy was statistically significantly (p≤0.01) decreased by 13.5%.
Starting at the intermediate dose group (120 mg test item/kg bw/day) a statistically significant (p≤0.05, right only) dose related decrease by 18.3% at maximum was noted for the absolute organ weight of the left + right epididymis.
In the high dose group (300 mg test item/kg) the following statistically significant changes were noted:
An increase by 34.0% (p≤0.01) of the relative liver weight and by 19.6% (non-significant) of the abso-lute liver weight.
The relative and absolute organ weight of the thymus was decreased by 42.5% (p≤0.05) and by 49.0% (p≤0.01).
The relative organ weights of the left and right gonads were reduced by 14.9% (p≤0.01) and by 13.7% (p≤0.05).
Female animals
A statistically significant (p≤0.01) reduction in the body weight at autopsy by 22.2% was noted in the high dose group (300 mg test item/kg bw/day).
In the intermediate dose group (120 mg test item/kg) a statistically significant (p≤0.01) increase was noted in the relative liver weight by 23%.
The absolute heart weight was statistically significantly decreased by 16.7%.
The relative organ weights of the left and right adrenal glands were statistically significantly (p≤0.01) increased by 26.2% and 24.4%.
In the high dose group (300 mg test item/kg) the relative organ weights of the left and right kidneys were statistically significantly increased by 17.2% (p≤0.05) and by 20.9% (p≤0.01).
The absolute organ weight of the heart was statistically significantly (p≤0.05) decreased by 33.1%.
The absolute organ weight of the right gonad was statistically significantly (p≤0.05) decreased by 37.5% and the absolute organ weight of the left gonad non-significantly by 25.5%.
Similar but not statistically significant increases as in the intermediate dose groups were noted for the relative liver weight and the left and right adrenal glands.

GROSS PATHOLOGY
Male animals
Macroscopic changes were noted in the stomach of 3 animals of the high dose group (300 mg test item/kg bw/day) in form of a detachment of the mucosa, whitish thickenings and ulcers. The find-ings were considered to be test item-related.
Female animals
A test item-related detachment of the mucosa was noted in the stomach of one animal of the inter-mediate dose group (120 mg test item/kg bw/day)
In the high dose group (300 mg test item/kg bw/day) a test item-related detachment of the mucosa was noted in 2 animals.

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

No microscopic changes were noted for the reproductive organs of the male and female rats of the high dose group (300 mg test item/kg bw/day) and no changes were noted on the stages of spermatogenesis.



Dose descriptor:
NOAEL
Effect level:
60 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: paternal/maternal effects
Dose descriptor:
NOAEL
Effect level:
120 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: reproductive/developmental effects
Critical effects observed:
not specified

Table 1. Mean body weight male rats

Sex: Male

Day(s) Relative to Start Date

1

8vv

15v

22vv

29vv

36vv

42vv

Group 1: control

351.76

382.89

384.38

417.44

438.70

452.95

466.48

Group2: 60mg/kg

347.77

376.01

378.72

416.27

438.79

453.18

454.34

Group3:

120 mg/kg

347.15

357.20*

360.33

392.72*

407.73*

427.61

437.78*

Group 4:

300 mg/kg

347.69

348.30**

344.28**

366.48**

366.91**

389.61**

404.56**

v Group Factor Dunnett’s Test Anova: Statistical Test: Analysis of Variance p<0.05

vv Group Factor Dunnett’s Test Anova Statistical Test: Analysis of Variance p<0.01

*Statistical Test Dunnett 2 Sided: p < 0.05

**Statistical Test Dunnett 2 Sided: p < 0.01

 

 Table 2. Mean body weight female rats

Sex: Female

Day(s) Relative
 to Start Date

Day(s) Relative
 to Mating (L)

Day(s) Relative to Littering (A)

1

8

15

0

7v

14vv

20vv

1vv

4vv

Group 1: control

233.93

246.30

234.90

254.51

287.30

320.92

393.10

307.38

323.92

Group2: 60mg/kg

233.89

240.31

233.37

247.96

278.33

300.66

361.13

287.00

301.92

Group3:

120 mg/kg

234.95

246.07

238.78

253.23

275.22

296.82

364.65

281.60

301.66

Group 4:

300 mg/kg

237.37

231.22

222.11

242.82

289.58**

265.88**

277.20**

239.68**

246.93**

Statistical Test Dunnett’s Test (Anova)

Group Factor Dunnett’s Test (Anova): v –Statistical Test: Analysis of Variance p<0.05

                                                                vv- Statistical Test: Analysis of Variance p<0.01

**-Statistical Test: Dunnett 2 Sided p<0.01

 

 

Table 3. Mean haematological parameters male and female rats

Day 15 Relative to Start Date

Sex: Male

HGB

(mmol/L)

RBC

(x10E6/µL)

HCT

(%)

MCHC

(g/L)

Sex: Female

aPTT

(seconds)

Group 1: control

9.64

8.032

47.64

326.228

Group 1:

control

15.06

Group2: 60mg/kg

10.10

8.408

49.08

331.157

Group2:

60mg/kg

14.22

Group3:

120 mg/kg

10.20

8.676

49.04

334.830**

Group3:

120 mg/kg

14.18

Group 4:

300 mg/kg

10.78**

8.924*

51.96**

334.605**

Group 4:

300 mg/kg

13.80*

Dunnett:

* 5% significance level

** 1% significance level

 

Table 4. Mean biochemical parameters male rats with changes

Day 15 Relative to Start Date

Sex: Male

Cholesterol (total)

(mmol/L)

ALAT

(U/L)

aP

(U/L)

ASAT

(U/L)

Group 1: control

1.054

35.8

157.4

81.4

Group2: 60mg/kg

0.862

38.4

245.0

81.6

Group3:

120 mg/kg

0.730

92.4**

433.2**

93.2

Group 4:

300 mg/kg

0.702*

282.2**

639.4**

155.4**

Dunnett:

* 5% significance level

** 1% significance level

 

Table 5. Mean biochemical parameters female rats with changes

Day 15 Relative to Start Date

Sex: Female

Globulin

(g/L)

Cholesterol (total)

(mmol/L)

Chloride

(mmol/L)

Potassium

(mmol/L)

ALAT

(U/L)

ASAT

(U/L)

Group 1: control

28.86

1.760

102.2

3.542

34.4

77.2

Group2: 60mg/kg

27.58

1.266**

 

101.2

3.430

38.6

77.0

Group3:

120 mg/kg

27.24

1.400

101.2

3.230

60.4

93.4

Group 4:

300 mg/kg

26.32*

0.672**

99.0**

2.916**

266.4**

157.8**

Dunnett:

* 5% significance level

** 1% significance level

 

Table 6. Absolute organ weights in male and female rats (g) with changes

 

Gonads

Epididymis

Kidney

Heart

Thymus

Males

left

right

left

right

left

right

Group 1

1.804

1.824

0.722

0.767

1.586

1.560

1.382

0.486

Group 2

1.793

1.784

0.717

0.706

1.598

1.648

1.374

0.406

Group 3

1.750

1.751

0.679

0.676*

1.344*

1.336*

1.296

0.362

Group 4

7.789

1.789

0.651

0.627**

1.500

1.464

1.158

0.248**

Females

 

 

 

 

 

 

 

 

Group 1

0.055

0.064

 

 

0.942

0.964

1.080

0.296

Group 2

0.048

0.056

 

 

0.996

1.016

0.990

0.220

Group 3

0.056

0.061

 

 

0.982

0.982

0.900*

0.208

Group 4

0.041

0.040*

 

 

0.813

0.853

0.723*

0.168*

**( p≤0.01),

*(p≤ 0.05), Dunett test or Student’s t-test

 

Table 7. Relative organ weights in male and female rats (g) wiht changes

 

Adrenals

Gonads

Kidney

Liver

Thymus

Males

left

right

left

right

left

right

Group 1

0.078

0.077

4.086

4.130

3.658

3.600

31.48

1.114

Group 2

0.075

0.076

4.158

4.135

3.708

3.824

34.02

0.948

Group 3

0.082

0.083

4.230

4.233

3.268

3.254

33.80

0.880

Group 4

0.090

0.093

4.693**

4.696*

3.872

3.782

42.18**

0.640*

Females

 

 

 

 

 

 

 

 

Group 1

0.122

0.127

0.186

0.219

3.230

3.308

41.44

1.022

Group 2

0.138

0.126

0.170

0.201

3.508

3.578

45.16*

0.780

Group 3

0.154**

0.158**

0.204

0.220

3.526

3.534

50.98**

0.748

Group 4

0.199

0.178

0.193

0.193

3.785*

3.998**

50.13

0.735

**( p≤0.01),

*(p≤ 0.05), Dunett test or Student’s t-test

 

Table 8. Histopathology males and females

Sex

Male

Female

Group

Gr.1

Gr.2

Gr.3

Gr.4

Gr.1

Gr.2

Gr.3

Gr.4

No. animals

10

10 

 10

10

10

 10

 10

10

Examinded

5

 

 

5

5

 

 

5

Liver

Fatty change

0

 

 

3

0

 

 

4*

Hypertrophy, hepatocellular

0

 

 

5**

0

 

 

4*

Mammary gland

Marked acinar development

 

 

 

 

5

 

 

1*

Stomach

Marginal non-glandular; squamous cell hyperplasia

 

 

0

 

 

 

 

3

 

 

0

 

 

 

 

4*

Uterus (incl. cervix and oviducts)

Endometrium; stromal hyperplasia

 

 

 

 

5

 

 

1*

Vagina

Metestrus

 

 

 

 

4

 

 

1

Fisher’s Two-Tailed Exact Test Performed:

*= 5% Significance

**= 1% Significance

Conclusions:
The following no-observed adverse-effect (NOAEL) levels were established:
Paternal/ Maternal toxicity: NOAEL= 60 mg/kg bw/day, p.o.
Reproductive/Development toxicity: NOAEL=120 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 to rats at dose levels of 60, 120 or 300 mg test item/kg bw/day. The application started two weeks before mating on test day one and ended on the day or one day before sacrifice. Day of sacrifice was on test day 43 for the male rats and on lactation day 4 or shortly thereafter for the female rats.

Effects on the parental generation (general toxicity)

One of 10 male and one of 10 female animals of the high dose group (300 mg test item/kg bw/day) died prematurely on test day 33 or on gestation day 9 (TD 26).

Slight to moderate salivation was noted in a few male and female animals of the inter-mediate dose group (120 mg test item/kg bw/day) on 1 day each, which was regarded as test item-related.

In the high dose group (300 mg test item/kg bw/day) piloerection and a slight to extreme salivation was noted for several to all male and female animals on several days and regarded as test item-related.

A statistically significant reduction in body weight was noted for the male animals of the intermediate dose group (120 mg test item/kg bw/day) and for both sexes at the high dose group (300 mg test item/kg bw/day).

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

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

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

Histopathological examination of the organs from animals of the high dose group (300 mg test item/kg bw/day) revealed test item-related changes in the liver (hepatocellular hypertrophy and macrovesicular vacuolation evoked by fatty change) and the non-glandular stomach (squamous cell hyperplasia) of male and female animals. These latter changes are considered to be related to a local activity of the test item. As humans lack a forestomach, the relevance of these changes for humans is questionable.

Reproductive toxicity

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

The high percentage of stillbirths led to a statistically significantly reduced live birth index in the high dose group (300 mg test item/kg bw/day).

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

Effects on the development of the F1offsprings (pups)

In the high dose group (300 mgtest item/kg b.w./day)the total litter weight per dam of the 3 dams with live born pups was reduced on lactation day 1 and on lactation day 4 by 42 and 56%, respectively.

The external examinations of the pups revealed no testitem-related external visible changes in any of the treatment groups, except for ‘no milk in the stomach’ in pups which were found dead during the lactation period.

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

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

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

Endpoint:
sub-chronic toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
See attached read-across justification
Reason / purpose:
read-across source
Limit test:
no
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
All rats except one survived the duration of the experiment. A female rat from the high dose group, died at the end of the fifth week of the experiment. Histopathological examination of this rat revealed the presence of mild, chronic renal disease.
Mortality:
mortality observed, treatment-related
Description (incidence):
All rats except one survived the duration of the experiment. A female rat from the high dose group, died at the end of the fifth week of the experiment. Histopathological examination of this rat revealed the presence of mild, chronic renal disease.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
It is obvious that the intermediate and high level groups, both male and female, did not gain as much as the control and low dose groups did.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
The feed consumption for the high dose (4.00%) female group was higher than the feed consumption of the other female groups.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
The feed efficiency for the high dose (4.00%) group of rats both male and female, was less than the feed efficiency of the other groups.
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
Although slight decrease in haematocrit, hemoglobin and white blood cells in females dosed at 4%.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Although indication of increased alkaline phosphatase and SGOT in males and females dosed at 1 and 4%.
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
0.25%: incr. relative kidney weight (F): decr. relative heart weight (M&F) & adrenal weight (F); decr. relative kidney (F) & incr. liver weight (M&F): decr. relative testes (M) & kidney weight (F), incr. liver (M&F); decr. relative ovary weight (F).
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
not examined
Description (incidence and severity):
Except for gross lesions (e.g. respiratory disease observed in control and high dose groups; renal disease observed in high dose groups).
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
All rats except one survived the duration of the experiment. A female rat from the high dose (4.00%) group, rat #466, died at the end of the fifth week of the experiment. Histopathological examination of this rat revealed the presence of mild, chronic renal disease.

BODY WEIGHT AND WEIGHT GAIN
The mean body weight, weight range and weight gain of the rats during the course of the experiment are summarized in Table 1.
The male control group gained an average of 393 g during the course of the experiment, the 0.25% group gained an average of 394 g, the 1.00% group gained an average of 363 g and the 4.00% group gained an average of 213 g during the experiment.
The female control group gained an average of 218 g during the course of the experiment, the 0.25% group gained an average of 205 g, the 1.00% group gained an average of 193 g and the 4.00% group gained an average of 115 g during the experiment.
It is obvious that the intermediate and high level groups, both male and female, did not gain as much as the control and low dose groups did.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
The feed consumption data are summarized in Table 2. The mean feed consumption in g/rat/week for the male control rats was 171, for the 0.25% group 171, for the 1.00% group 166 and for the 4.00% group 172g. The corresponding feed consumption for the female control group was 137, for the 0.25% group 139, for the 1.00% group 136 and for the 4.00% group 156g.
The feed consumption for the high dose (4.00%) female group was higher than the feed consumption of the other female groups.

FOOD EFFICIENCY
The feed efficiency data are summarized in Table 3. The mean feed efficiency in g of feed/week/ g of body weight for the male control group was 0.52, for the 0.25% group 0.51, for the 1.00% group 0.54 and for the 4.00% group 0.69. The corresponding feed efficiency for the female control group was 0.62, for the 0.25% group 0.62, for the 1.00% group 0.63 and for the 4.00% group 0.93.
The feed efficiency for the high dose (4.00%) group of rats both male and female, was less than the feed efficiency of the other groups.

HAEMATOLOGY
A. Hematocrit
The normal hematocrit concentration in the albino rat in our experience has been 33% to 55%. Several rats from all groups at six weeks had hematocrit values lower than normal. By 13 weeks, however, all rats except three exhibited normal hematocrit values; rat #425 (1.00%group), as well as rats #468 and #480 (4.00% group). In addition rat #387 (0.25% group) as well as rats #445 and #474 (4.00% group) also had low hematocrit values, with high dose female mean remaining below normal (28.7%). Histologically the bone marrow of these rats exhibited normal cellularity and composition.
B. Hemoglobin
The normal hemoglobin concentration in the albino rat in our experience has been 12 to 18g%. Female rats #438 (1.00% group) and #476 (4.00% group) had low hemoglobin values at six weeks, however, by 13 weeks these were normal. Two female rats (4.00% group), rat #468 and #474, had low hemoglobin values at six as well as 13 weeks. In addition, the following rats had low hemoglobin values at 13 weeks: #387 (0.25% group), #409 and #425 (1.00% group) and #445 and #480 (4.00% group), with the high dose female mean remaining below normal (10.3 g%). Histologically, however, the bone marrow of these rats exhibited normal cellularity and composition.
C. White Blood Cell Count
The normal white blood cell count in the albino rat in our experience has been 5000 to 25000 cells/mL. All rats except one had normal WBC counts at both time intervals. Rat #459 (4.00% group) had a WBC count of 25800 cells/mL at 13 weeks. Its bone marrow, however, was histologically normal.
D. Differential White Blood Cell Count
The albino rat in our experience displays lymphocytosis normally throughout its lifetime. All rats examined at six and thirteen weeks except control rat #346 at six weeks as well as control rat #357 and 0.25% rat #387 at 13 weeks displayed lymphocytosis. The bone marrow of these rats, however, was not remarkable histologically.

CLINICAL CHEMISTRY
A. Blood Glucose
The normal serum glucose for the adult albino rat in our experience has been 50-250 mg%. All rats except four (4.00% group) had normal glucose values; rats #445, #453, #468 and #479 had glucose values of 40 mg%, 22 mg%, 39 mg% and 40 mg%, respectively. Histologically the liver and pancreas of these rats were normal.
B. Blood Urea Nitrogen
The albino rat in our experience has a normal range of blood urea nitrogen of 5-35 mg%. All rats examined had normal BUN values.
C. Alkaline Phosphatase
In our experience the adult albino rat has an alkaline phosphatase range of 10-80 units per liter. Most rats, including most of the controls, had abnormally high serum alkaline phosphatase values.
D. Serum Glutamic Oxaloacetic Transaminase
The normal adult albino rat has an SGOT range of 10-100units per liter according to our experience. Almost all rats, including the control, had abnormally high SGOT values. Because of the high levels of serum alkaline phosphatase and SGOT, a diligent histopathological search was made of the H&E sections of the high dose rats to find out any underlying pathology. Particular emphasis was placed on the parathyroid-skeletal renal system and on the hepato-biliary system. Since some histopathological lesions were found –see below under Histology- which could account for three values, the following additional study was performed to clarify this matter.

Five one year old male control rats as well as five one year old male rats which had been receiving 4.00% of A-268 for one year (terminal sacrifice from a reproduction study) had the same assays performed with the results tabulated in Table 9A. As can be seen from this table, all rats had normal glucose, BUN and alkaline phosphatase values. The SGOT values, however, continue to be abnormally high, both in the control as well as in the high dose rats. Since our control sera on the day of the assays were in phase, we consider this SGOT “elevation” to be spurious findings not related to the administration of A-268.

URINALYSIS
The six-week urinalysis results were basically negative for both male and female rats. At 13 weeks, however, several rats in all groups exhibited proteinuria to a certain degree. Certain of these rats at autopsy exhibited mild chronic renal disease, an entity not uncommon in this strain of rats ( see below under Histology).

ORGAN WEIGHTS
The mean body weight for the male control rats was 502 g, for the 0.25% group 495 g, for the 1.00% group 464g and for the 4.00% group 346g. The corresponding mean body weight for the female control rats was 301 g, for the 0.25% group 296 g, for the 1.00% group 293 g and for the 4.00% group 215g.
It is, again, obvious that the intermediate and high dose group gained less than the other two groups.
The mean thyroid weight for the male control group was 0.04 g, for the 0.25% group 0.03 g, for the 1.00% group 0.03 g and for the 4.00% group 0.02 g.
The mean heart weight for the male control group was 1.39 g, for the 0.25% group 1.28g, for the 1.00% group 1.15g and for the 4.00% group 0.90 g. The corresponding mean heart weight for the female control group was 0.96 g, for the 0.025% group 0.94 g, for the 1.00% group 0.83 g and for the 4.00% group 0.69 g.
The mean adrenal weight for the male control group was 0.08 g, for the 0.25% group 0.08 g, for the 1.00% group 0.07 g and for the 4.00% group 0.07 g. The corresponding mean adrenal weight for the female control group was 0.10 g, for the 0.25% group 0.10 g, for the 1.00% group 0.08 g and for the 4.00% group 0.06 g.
The mean kidney weight for the male control group was 3.14 g, for the 0.25% group 3.06 g, for the 1.00% group 3.29 g and for the 4.00% group 2.39 g. The corresponding mean kidney weight for the female control group was 1.87 g, for the 0.25% group 2.05 g, for the 1.00% group 2.09 g and for the 4.00 % group 1.67 g.
The mean liver weight for the male control group was 17.56 g, for the 0.25% group 17.76 g, for the 1.00% group 19.18 g and for the 4.00% group 15.88 g. The corresponding mean liver weight for the female control group was 9.74 g, for the 0.25% group 10.91g, for the 1.00% group 12.98 g and for the 4.00% group 12.21 g.
The mean testicular weight for the male control group was 3.46 g, for the 0.25% group 3.38 g, for the 1.00% group 3.31 g and for the 4.00% group 3.15 g. The corresponding mean ovarian weight for the female control group was 0.21 g, for the 0.25% group 0.23 g, for the 1.00% group 0.18 g and for the 4.00% group 0.13 g.
The organ weights of the high dose groups were in general, smaller than those of the control group. Histologically, however, these organs were not remarkable.

GROSS PATHOLOGY
The high dose animal which died on the 35th day of dosing (rat #466) had mild chronic renal disease. The rest of the findings consisted of six cases of respiratory disease and one case of fatty infiltration of the liver in the control group as well as five cases of respiratory disease, seven cases of renal disease and one case of cloudy swelling of the liver in the high dose group. None of these findings was compound related, since these conditions are endemic in this strain of rat.
Dose descriptor:
NOEL
Effect level:
188 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: 0.25% in the diet
Dose descriptor:
NOAEL
Effect level:
750 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: 1% in the diet; increased relative liver weights in both sexes.
Critical effects observed:
not specified

The findings in the 90-day rat study with CAS No. 37294-19-8 up to 1% in the diet are not really adverse. The organ weight changes at 1% were mainly seen in females, showing marginal weight gain loss but no gross pathological changes. Findings were more pronounced at 4%, and the chronic renal disease in males and females may be considered – possibly partly – test article related.

Conclusions:
When the read-across test item (purity ca. 50%) is fed to rats at concentrations as high as 4.00%, it causes a significant difference in body weight gain only at the high dose (4.00% ) level. This finding is probably related to an effect by the compound during absorption from the gastro-intestinal tract.
The high dose was considered to be toxic, whereas the changes of the medium dose may be considered adaptive. Therefore 0.25% (188 mg/kg) is considered NOEL, whereas 1% (750 mg/kg) may be considered as NOAEL.
Executive summary:

The read-across test item containing 50% active ingredient, a colorless, clear viscous liquid with a nut-like odor, was fed to three groups of 40 young random bred albino rats (20 male and 20 female) at 0.25%, 1.00% and 4.00% with a control group also consisting of 40 rats (corresponding to mean test article intake of 188, 750 and 3000 mg act.ingr./kg bw/day). The rats were housed individually in an environmentally controlled room. The experiment lasted for 90 days during which time feed consumption and body weights were recorded weekly. At six and 13 weeks five hematological parameters and complete urinalysis (five parameters) were performed while at 13 weeks an additional four biochemical parameters were examined. The rats were subsequently autopsied and 20 rats (10 of each sex) from the control and the high dose groups had careful histological examination performed on 30 separate tissues from each. Organ weights were taken from 20 rats (10 of each sex) per level. The following parameters were calculated: feed consumption, feed efficiency and organ weight to body ratios.
From these results we conclude that the substance caused toxicity by means of a significant difference in body weight gain only at the high dose (4.00% ) level. This finding is probably related to an effect by the compound during absorption from the gastro-intestinal tract. There was one mortality in the high dosed females. Haematology and serum analysis also indicated toxicity at the high dose, whereas at the medium dose, only some organ-to-body weight changes were observed (e.g. liver weight increases in both sexes). Other organ-to-body weight changes were also observed at the high dose group (e.g. increased gonad weights in males; decreased gonad weight in females); increased relative kidney weights were observed in all female dose groups. Chronic renal disease (mild) was observed in 1/20 males and 6/20 females of the high dose groups.

The high dose was considered to be toxic, whereas the changes of the medium dose may be considered adaptive. Therefore 0.25% (188 mg/kg) is considered NOEL, whereas 1% (750 mg/kg) may be considered as NOAEL for this read-across substance CAS 37294 -49 -8.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
60 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Klimisch 1
System:
other: digestive system and urogenital system
Organ:
kidney
liver
stomach

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Subacute toxicity

- A key study for subacute toxicity was performed by means of an oral combined repeated dose and reproduction/development screening study according to OECD guideline 422 (Hansen, 2013a). The test item was a solid formulation (containing >93% active ingredient) which was administered as a watery solution orally by gavage to rats at dose levels of 60, 120 and 300 mg act. ingr./kg bw/day for at least 28 days in male rats up to 54 days in female rats. One of 10 male and one of 10 female animals at 300 mg/kg bw/day died prematurely on test day 33 or on gestation day 9. Slight to moderate salivation was noted in a few male and female animals at 120 mg/kg bw/day; at 300 mg/kg bw/day, piloerection and a slight to extreme salivation was noted for several to all male and female animals on several days. Reduced body weight was noted for the male animals at 120 mg/kg bw/day and for both sexes at 300 mg/kg bw/day. Increases in ALAT, aP and ASAT and decreases in globulin, cholesterol, chloride and potassium concentrations were noted for the male and/or female animals of the intermediate and/or the high dose group (120 and/or 300 mg/kg bw/day). Several organ weights were seen in males and females dosed at 120 and 300 mg/kg bw/day, most remarkably for the thymus and liver weights of the animals of the high dose group. Macroscopic inspection revealed changes in the stomach of male animals dosed at 300 mg/kg bw/day and female animals dosed at 120 and 300 mg/kg bw/day. Histopathological examination revealed changes in the liver (hepatocellular hypertrophy and macrovesicular vacuolation) and the non-glandular stomach (squamous cell hyperplasia) of male and female animals dosed at 300 mg/kg bw/day. These latter changes are considered to be related to a local activity of the test item. As humans lack a forestomach, the relevance of these changes for humans is questionable.

NOAEL-level were as follows: 60 mg/kg bw for paternal/maternal toxicity and 120 mg/kg bw for reproductive and developmental toxicity (see Section 7.8.1 and 7.8.2).

- A supporting 14-day dose-range-finding study (Hansen, 2013b) was conducted with test item containing >95 % active ingredient to select the dose levels for a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test of the test item in rats. The animals were treated once daily with100, 300 and 1000 mg act. ingr./kg bw/day by oral administration. Four of 5 males and all (5) females treated orally with 1000 mg/kg bw/day died prematurely. Oral treatment with 1000 mg/kg bw/day caused signs of systemic toxicity in form of pilo-erection, reduced motility, pultaeous faeces/diarrhea, salivation, increased drinking water consumption, ataxia or decreased body temperature in the male and/or female rats. A decrease in body weight, body weight at autopsy and food consumption was noted for the male and female rats treated with the intermediate and the high dose. At necropsy, whitish deposits on the stomach mucosa were observed in the male rats treated orally with 300 mg/kg bw/day. The high dose of 1000 mg/kg bw/day led to further changes in the gastro-intestinal tract in both sexes such as inflation or discolourations. The examination of organ weights revealed a dose-related increase of liver weights. The dose of 300 mg/kg bw was considered as NOAEL, and dose levels selected for the repeated dose and reproduction/developmental toxicity screening test were 60, 120 and 360 mg/kg bw/day.

 

In conclusion, NOAEL-levels of 60 mg/kg bw for paternal/maternal toxicity, 60 mg/kg bw for reproductive toxicity and 120 mg/kg bw for developmental toxicity were obtained in a combined repeated dose and reproductive/developmental screening study which was performed with registered substance according to OECD guideline 422. Therefore, no classification is needed.

 

Subchronic toxicity

A 90 -day study was not available for the registered substance, however read across data were available from a category member, CAS No. 37294 -49 -8 (Butanedioic acid, sulfo-, 1,4-diisodecyl ester, sodium salt).

- For this read across substance, a 14 -day dose rang finding study was also conducted as supporting study (Hansen, 2013c). Rats were treated once daily with a test item (47% purity) at100, 300 and 1000 mg act. ingr./kg bw/day by oral gavage administration. None of the animals died prematurely. The body weight of the male rats treated orally at 1000 mg/kg bw/day was slightly decreased on test days 8 and 15 compared to the control group. Body weight gain and body weight at autopsy changed accordingly. None of the male and female rats treated orally at 100, 300 or 1000 mg/kg bw/day revealed any test item-related changes in behaviour, external appearance or faeces. No changes in the relative food consumption were noted at any of the tested dose levels. At macroscopic inspection at necropsy, test item-related changes in the stomach (detachment of mucosa, haemorrhagic foci, mucosa thickened/swollen, ulcer and cardia thickened) were noted in the animals treated with 1000 mg /kg bw/day. Further, the absolute kidney weights of the high dosed animals were decreased. In conclusion, 300 mg/kg body weight can be considered as NOAEL. This was comparable to the registered substance, therefore read across was considered valid.

- A 90-day toxicity study was performed in rats with this read across test item containing ca. 50% active ingredient (Tegeris and Underwood, 1975a) at 0.25%, 1.00% and 4.00% in the diet (corresponding to mean calculated test article intake of 188, 750 and 3000 mg act.ingr./kg bw/day). At the highest dose levels, it caused a significant difference in body weight gain, most probably related to an effect by the compound during absorption from the gastro-intestinal tract. There was one mortality in the high dosed females. Haematology and serum analysis also indicated toxicity at the high dose, whereas at the medium dose, only some organ-to-body weight changes were observed (e.g. liver weight increases in both sexes). Other organ-to-body weight changes were also observed at the high dose group (e.g. slightly decreased gonad weights in males; decreased gonad weight in females); increased relative kidney weights were observed in all female dose groups. Chronic renal disease (mild) was observed in 1/20 males and 6/20 females of the high dose groups. The high dose was considered to be toxic, whereas the changes of the medium dose may be considered adaptive. Therefore 1% (750 mg/kg bw/day) may be considered as NOAEL.

- A 90 -day toxicity study in dogs was performed with the same read-across test item containing ca. 50% active ingredient (Tegeris and Underwood, 1975b) at 0.12%, 0.50% and 2.00% in the diet (corresponding to mean calculated test article intake of 30, 125 and 500 mg act. ingr. /kg bw/day). There was a decreased feed consumption, feed efficiency and corresponding weight loss in the high dose (2.00%) group of dogs. Also testicular atrophy and hepatic fatty infiltration were observed in the same group without functional impairment of the liver. These observations were not considered to be direct toxic manifestations of the test item, as disturbed fat absorption is known as a physiological action of the sulfosuccinate class of compounds. Effects at the highest dose were considered to be secondary manifestations to disturbed fat absorption at these high multiples of human intake. The dose of 500 mg/kg bw (2% in the diet) was considered NOAEL.

- The dog species was considered to be less appropriate due to the disturbed fat absorption, by which systemic effects could not be correctly interpreted. The NOAEL of 750 mg/kg bw/day in rats was selected as main endpoint; this was also consistent with the selection of the rat as main species and NOAELs from the Di-ester substances.

In conclusion for the subchronic toxicity a NOAEL of 1% in the diet, corresponding with 750 mg/kg bw, was obtained in a 90 -day study with read across substance CAS No. 37294 -49 -8.

 

Conclusion

- For risk characterisation, the paternal/maternal NOAEL of 60 mg/kg bw in the OECD 422 study with registered substance was selected as most conservative value.

- Further information supporting the safety of the test substance is provided in the read across justification for the Mono-ester subgroup, (justification with data matrix separately attached in Section 13).

 

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint: Key study for the 28-day study. Note however that there is also a key study for the 90-day oral toxicity with a read across substance. 

Justification for classification or non-classification

As there were no relevant findings below classification threshold of 100 mg/kg bw, classification for repeated dose toxicity is not warranted according to CLP (No. 1272/2008 of 16 December 2008).