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Toxicological information

Repeated dose toxicity: oral

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Administrative data

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.
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Details on test material:
- Name of test material (as cited in study report): Butanedioic acid, sulfo-, 1-C12-18-alkyl esters, disodium salts; Disodium Lauryl Sulfosuccinate; Disodium C12-18 alkyl sulfosuccinate
- Physical state: White powder (freeze-dried)
- Analytical purity: At least 93% (correction factor: 1.07)


Test 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: Oktober 25, 2012 To: Males: December 20, 2012; Females: January 3, 2013
10m and 10 f per group

Administration / exposure

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
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 60, 120 and 300 mg/kg bw/day
Basis:
actual ingested
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.

Examinations

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.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
1male and 1 female of the high dose group died on day 33 and day 26 respectively; slight signs of systemic toxicity were noted predominantly in form of pilo-erection and increased salivation in males and females dosed at 120 & 300 mg/kg bw
Mortality:
mortality observed, treatment-related
Description (incidence):
1male and 1 female of the high dose group died on day 33 and day 26 respectively; slight signs of systemic toxicity were noted predominantly in form of pilo-erection and increased salivation in males and females dosed at 120 & 300 mg/kg bw
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
decreased at 120 and 300 mg/kg bw in male rats and at 300 mg/kg bw in female rats prmating, mating, during gestationa and during lactation
Food consumption and compound intake (if feeding study):
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 prmating, mating, during gestationa and during lactation
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
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 famels 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.



Effect levels

open allclose all
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

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.