Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to GLP and valid methods and is considered relevant and reliable for classification.
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)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
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

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: tap water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: administration volume was 10 mL/kg bw/day.
The test item was dissolved in the vehicle tap water to concentrations of 6, 12 and 30 mg test item/mL tap water. The test item formulations were freshly prepared and adjusted to the animal's current body weight on each administration day.

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



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

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes , cage side observations included skin/fur, eyes, mucous membranes, respiratory and circulatory systems, somatomotor activity and behaviour patterns. The onset, intensity and duration of any signs observed were recorded.
- Time schedule: Throughout the test period, each animal were observed for clinical signs at least once daily. Individual animals were observed before and after dosing at each time of dosing for any signs of behavioural changes, reaction to treatment or illness. Mortality was recorded twice daily. In addition, animals were checked regularly throughout the working day from 7:00 a.m. to 3:45 p.m. On Saturdays and Sundays animals were checked regularly from 7:00 a.m. to 11:00 a.m. with a final check performed at approximately 3:30 p.m.
- Cage side observations checked in table [No.2-1 to 2-14] were included.

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

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

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

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

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

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

HAEMATOLOGY: see Section 7.5.1

CLINICAL CHEMISTRY: See Section 7.5.1

NEUROLOGICAL OBSERVATIONS: see Section 7.5.1

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


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

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

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

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

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

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

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

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

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

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

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

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

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

These statistical procedures were used for all data. Significantly different data were indicated in the tables of the report.
The mean values and standard deviations were calculated to the highest possible degree of accuracy and then rounded to the reported number of decimal places. Hence, deviations to the last decimal place of up to ± 1 may occur caused by rounding.
Reproductive indices:
Gestation Index
Fertility Index
Birth Index
Live Birth Index
Viability Index
Pre-implantation loss [%]
Post-implantation loss [%]
Offspring viability indices:
Gestation length calculated from day 0 of pregnancy
Corpora lutea
lmplantation sites
Number of pups absolute
Number of pups per dam
Number of male and female pups
Number of stillbirths
Number of pups with malformations

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
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):
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
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
hepatocellular hypertrophy and macrovesicular vacuolation in liver and squamous cell hyperplasia in the non-glandular stomach in males and females at 300 mg/kg bw; changes in the mammary glands, the uterus and vagina in females at 300 mg/kg bw
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Test substance intake: actual ingestion

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
reduced gestation index, No. implantation sites and No.(live) born pups per dam and birth index; increased implantation loss index at 300 mg/kg bw

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
One of 10 male animals (no. 64) of the high dose group (300 mg test item/kg bw/day) died on test day 33, showing piloerection and reduced motility before death.
One of 10 female animals (no. 71) of the high dose group (300 mg test item/kg b.w./day) died on gestation day 9, showing piloerection and salivation on a few days during the premating, mating and gestation period.
Male animals
Slight salivation was noted in 2 animals of the intermediate dose group (120 mg test item/kg 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 FOOD CONSUMPTION (PARENTAL ANIMALS)
Male animals
A reduction in body weight was noted in the intermediate dose group (120 mg test item/kg bw/day) from test day 8 by 6.7% until the end of the study by 6.2%, statistically significant (p≤0.05) on test days 8, 22, 29 and 42.
In the high dose group (300 mg test item/kg bw/day) the reduction in body weight was more pronounced, with 9.0% on test day 8 and 13.3% at the end of the study, statistically significant (p≤0.01) from test day 8 to the end of the study.
Accordingly, statistically significant (p≤0.05 or p≤0.01) reductions in body weight gain were noted in the intermediate and the high dose group (120 and 300 mg test item/kg bw/day).
Female animals
A decrease in body weight (300 mg test item/kg bw/day) was noted in the high dose group, starting at the end of the pre-mating period by 5.4%. Statistically significant (p≤0.01) reductions were noted during the gestation period from gestation day 7 (by 9.6%) to 20 (by 23.9%) and on lactation day 1 (by 22.0%) and 4 (by 23.8%).
Statistically significant reductions in body weight gain were noted on gestation day 14 (p≤0.01) and 20 (p≤0.05) in the intermediate dose group (120 mg test item/kg bw/day).
In the high dose group (300 mg test item/kg bw/day) statistically significant reductions in body weight gain were noted on test day 8 (p≤0.01) and during the gestation period from gestation day 7 (p≤0.05) to gestation day 20 (p≤0.01).
Male animals
A statistically significant (p≤0.01) reduction in food consumption by 14.1% was noted in the intermediate dose group (120 mg test item/kg bw/day) during the first test week.
In the high dose group (300 mg test item/kg bw/day) the food consumption was statistically significantly (p≤0.01) reduced by 20.7% during the first and by 15.3% during the second test week.
Female animals
At the intermediate dose group (120 mg test item/kg bw/day) a statistically significant reduction in food consumption was noted during the first week of gestation by 7.3% (p≤0.05) and during the second week of gestation by 13.8% (p≤0.01).
In the high dose group (300 mg test item/kg bw/day) a statistically significant (p≤0.01) reduction in food consumption was noted from the first test week (by 26.3%) until the end of the gestation period (by 6.8%).

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS): gavage

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

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

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

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

No microscopic changes were noted for the reroductive 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 (P0)

Dose descriptor:
NOAEL
Effect level:
120 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: reproductive effects

Results: F1 generation

General toxicity (F1)

Clinical signs:
not specified
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
reduced survival rate of the pups (75.9% versus control) in the high dose group of 300 mg test item/kg
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
reduced at 300 mg/kg bw on lactation day 1 by 17.1% (p≤0.05) and on lactation day 4 by 29.3% (p≤0.01)
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

VIABILITY (OFFSPRING)
The survival rate of the pups was statistically significantly reduced to 75.9% in the high dose group (300 mg test item/kg)

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

GROSS PATHOLOGY (OFFSPRING)
No visible gross abnormalities were noted in the control and the treatment groups.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
120 mg/kg bw/day
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
viability
other: reduced pup weight and number of pups

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Table 1. Fertility and Reproductive parameters Parental generation

Parameter

Group 1

Control

Group 2

60 mg/kg

Group 3

120 mg/kg

Group 4

300 mg/kg

No. females evaluated

10

10

10

10

Mean precoital time (days)

3.8

3.4

2.9

3.8

Number of pregnant dams

10

10

10

8 +1#

Fertility index (%)

100

100

100

90

No. dams with pups (live + dead)

10

10

10

4

Gestation length (days)

22.7

22.9

22.9

23.3

No. dams with live pups

10

10

10

3

Gestation Index (%)

100

100

100

37.5**

No.Corpora lutea(total)

160

142

151

104

No.Corpora lutea(mean)

16.0

14.2

15.1

13.0

No. Implantation sites (total)

143

119

149

86

No. Implantation sites (mean)

14.3

11.9

14.9

10.8v

Number of pups at birth (total)

136

109

134

34vv

Number of pups at birth (mean)

13.6

10.9

13.4

4.3vv

Birth Index (mean %)

95.1

90.3

90.2

36.0

Birth Index (total %)

95.1

91.6

89.9

39.52

Number of stillbirths

3

0

4

5

No. of dams with stillborn pups

2

0

1

2

Number of live born pups (total)

133

109

130

29vv

Number of live born pups (mean)

13.3

10.9

13.0

3.6vv

Live birth index (mean %)

97.7

100.0

96.7

67.3

Live birth index (total %)

97.8

100.0

97.0

85.3b

Pre-implantation loss (mean %)

8.8

16.1

1.3

14.7

Pre-implantation loss (total %)

10.6

16.2

1.3b

17.3

Post-implantation loss (mean %)

7.9

9.7

12.6

68.5

Post-implantation loss (total %)

7.0

8.4

12.8

66.3b

1 p≤0.05 Chi2-test

2 p≤0.01 Chi2-test

*  p≤0.05 Fisher test

**p≤0.01 Fisher test

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

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

ap< 0.05 Chi2-test

bp<0.01 Chi2-test

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

Applicant's summary and conclusion

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

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

Effects on the parental generation (general toxicity) :

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

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

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

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

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

Reproductive toxicity

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

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

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

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

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