Disodium 4-[2-[(1-oxoundec-10-enyl)amino]ethyl] 2-sulphonatosuccinate

Repeated dose toxicity: oral

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

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 for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Butanedioic acid, 2(or 3)-sulfo-, 4-[2-[(1-oxo(C12-18(even numbered) and C18 unsaturated)alkyl)amino]ethyl]esters, disodium salts

IUPAC Name:

Butanedioic acid, 2(or 3)-sulfo-, 4-[2-[(1-oxo(C12-18(even numbered) and C18 unsaturated)alkyl)amino]ethyl]esters, disodium salts

Test material form:

other: liquid

Test animals

Species:

rat

Strain:

other: CD® / Crl:CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Research, Models and Services Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age at start of dosing: Males 55 days; Females: 48 days
- Weight range at start of dosing: Males: 281.0 to 308.0 g; Females: 68.9 to 195.7 g
- Fasting period before study: Ad libitum with exception of the night before the day of blood withdrawal for laboratory examination.
- Housing: With exception of the mating period, the animals were kept singly in MAKROLON cages (type III plus) with a basal surface of approx. 39 cm x 23 cm and a height of approx. 18 cm. Granulated textured wood (Granulat A2, J. Brandenburg, 49424 Goldenstedt/ Arkeburg, Germany) was used as bedding material in these cages. The cages were cleaned and changed once a week.
- Diet (e.g. ad libitum): ssniff® R/Z V1324 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany, ad libitum with exception of the night before the day of blood withdrawal for laboratory examination.
- Water (e.g. ad libitum): Tap water was offered daily ad libitum.
- Acclimation period: 5 Days

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

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

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: tap water

Details on oral exposure:

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

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

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

The measured actual concentrations of the test item in the test item vehicle mixtures were between 99.99% and 102.96% of the nominal concentrations (table 26).

Duration of treatment / exposure:

Males: 2 weeks prior to mating, during the mating period and approx. 2 weeks post mating and continuing up to test day 36 (one day before sacrifice).
Females:2 weeks prior to mating and continuing up to, and including, day 3 post-partum or the day before sacrifice.

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

Doses / concentrations 4

Dose / conc.:

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

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose levels have been selected in agreement with the Sponsor based on the results of a 14-day dose-range-finding study in rats dosed at 100, 300 and 1000 mg act. ingr./kg bw by oral gavage (LPT Study No. 28344). None of the animals died prematurely. None of the male and female rats treated orally with 100 or 300 mg act. ingr./kg bw/day revealed any changes in behaviour, external appearance or faeces. Salivation was noted for 2 of 5 male animals treated at 1000 mg/kg bw/day on 1 or 3 test days starting on test day 9 and increased faeces was noted for 3 of 5 male and 2 of 5 female high dosed animals on 6 or 9 test days starting on test day 5. No test item-related changes on body weight and body weight gain were noted for the male and female rats up to 1000 mg act. ingr./kg bw/day. No test item-related changes on food consumption were noted for the male and female rats treated orally with 100 or 300 mg act. ingr./kg bw/day. The food consumption of the male and female animals treated with 1000 mg act. ingr./kg bw/day was slightly increased by 9% for the males and by 10% for the females in test week 2 (statistically significant at p ≤ 0.01 for both sexes). No test item-related influence was noted for the drinking water consumption at any of the tested dose levels. None of the male and female rats treated orally with 100, 300 or 1000 mg act. ingr ./kg bw/day revealed changes at macroscopic inspection at necropsy or organ weights.

Examinations

Observations and examinations performed and frequency:

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

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

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

FOOD CONSUMPTION: Yes
The quantity of food left by individual animals was recorded on a weekly basis throughout the experimental period with the execution of the mating period. Food intake per rat (g/rat/week) was calculated using the total amount of food given to and left by each rat in each group on completion of a treatment week.

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

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

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of the premating period
- Animals fasted: Yes, overnight
- How many animals: 5 male and 5 female animals randomly selected from each group.
- Parameters:
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

OTHER/ REPRODUCTIVE PARAMETERS (See Section 7.8.1 & 7.8.2)

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: shortly before sacrifice (males); during lactation also shortly before sacrifice (females)
- Dose groups that were examined: five males and five females randomly selected from each group
- Battery of functions tested:
1. Observational screening
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
2. Functional tests
Grip strenght
Locomotor activity

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- 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.
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 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), Heart, 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, 8, 9 12, 13, 14, 18, 20
Group 2: 21, 22, 23, 27, 28 33, 35, 36, 39, 40
Group 3: 44, 45, 46, 47, 49 52, 53, 56, 58,60
Group 4: 61, 62, 63, 67, 69 72, 73, 74, 77, 79

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
-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.

Other examinations:

Fertility and reproduction: See section 7.8.1 and 7.8.2
Organ weights: tables 14-1to 14-4 (relative) and 15-1 to 15-4 (absolute)

Statistics:

Toxicology and Pathology data were captured, whenever possible, using the departmental computerized systems (Provantis®8 Integrated preclinical software, Instem LSS Ltd.). Raw data not fully compatible with the computerized systems were maintained on paper according to appropriate SOPs.
The test item-treated groups (2 - 4) were compared with the control group (1 ).
The following statistical methods were used:
STUDENT' s t-test All numerical functional tests (p ≤0.01)
Multiple t-test based on Body weight I Food consumption I
DUNNETT, C. W. Haematology I Clinical chemistry I
New tables for multiple Absolute and relative organ weights
Comparisons with a control (p ≤ 0.05 and ≤0.01)
Biometrics, 482-491 (Sept 1 964)
For all numerical values (e.g. body weight, food consumption and organ weight data) homogeneity of variances was tested by using the BARTLETT chi2-test. lf the variances are homogeneous, the DUNNETT test (p ≤ 0.01) was used to compare the experimental groups with the control group.
In case of heterogeneity of variances, the STUDENT's t-test was carried out; limit of significance is p ≤ 0.01.
Exact test of R. A. FISHER Histopathology, if applicable (p≤0.05)
For the comparison of classification measurements (for example the fertility index) the FISHER's exact test, n < 100 or chi2-test with Yates' correction for continuity, n ≥ 100 (p ≤0.05 and p ≤0.01) were employed.
These statistical procedures were used for all data. Significantly different data were indicated in the tables of the report.
The mean values and standard deviations were calculated to the highest possible degree of accuracy and then rounded to the reported number of decimal places. Hence, deviations to the last decimal place of up to ± 1 may occur caused by rounding.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

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

Mortality:

no mortality observed

Description (incidence):

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

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

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

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

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

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

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

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

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

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

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

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related influence on relative and absolute organ weights was noted for the rats treated with 100, 300 or 1000 mg/kg bw/day in comparison to the control group.
Statistically significant differences in the relative organ weights compared to the control, which are not considered to be test item-related were:
- increased relative weight of left gonads in low and high dose males
- increased relative weight of right kidney in high dose females
- increased relative weight of right adrenal in high dose males
Statistically significant differences in the absolute organ weights compared to the control, which are not considered to be test item-related were:
-decreased absolute brain weight in high dose males
-decreased absolute heart weight in high dose females.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Macroscopic inspection at autopsy for the males was performed on test day 37.
In the male high dose group (1000 mg/kg bw/day) 2 animals with macroscopic changes in the stomach were noted: Animal no. 68 showed a whitish thickening in the cardia part of the stomach and in the stomach of animal no. 69 a yellowish content was noted.
These findings were considered to be test item-related, because they were associated with microscopic changes in the forestomach from the animals of the high dose group.
Macroscopic inspection at autopsy for the females was performed between test days 43 and 54.
No test-item related macroscopic changes were noted in any treatment group (100, 300 and 1000 mg/kg bw/day) in females.

Neuropathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

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

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

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

Histopathological findings: neoplastic:

not examined

Details on results:

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

BODY WEIGHT AND WEIGHT GAIN
A slight reduction in body weight was noted for the male and female rats of the high dose group (1000 mg/kg bw/day). For the male rats the reduction in body weight was noted from test day 8 (4.4%) until test day 36 (5.5%) and for the female rats from gestation day 0 (7.6%) until lactation day 4 (9.5%). The body weight at autopsy was reduced accordingly.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
A slightly statistically significant (p ≤ 0.01) increase in relative food consumption by 10.3% was noted in the high dose males during the 2nd test week. This was caused by the reduced body weight of the rats of the high dose group.
No influence on food consumption was noted in any treatment group in the females.

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

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

NEUROBEHAVIOUR
The functional neurological screenings were performed on test day 36 for the male rats and between test days 40-50 for the female rats, 2 hours after dosing.
No test item-related influence was noted for the fore- and hindlimb grip strength in any male/female treatment group (100, 300 or 1000 mg/kg bw/day).
Hinglimb grip strength in low dose males was statistically significant decreased on test day 36 (p≤0.05) but lacking dose dependence and was considered to be not test item related.
No test item-related influence was noted on the spontaneous motility of the rats in any of the treatment groups.

ORGAN WEIGHTS
No test item-related influence on relative and absolute organ weights was noted for the rats treated with 100, 300 or 1000 mg/kg bw/day in comparison to the control group.
Statistically significant differences in the relative organ weights compared to the control, which are not considered to be test item-related were:
- increased relative weight of left gonads in low and high dose males (p≤0.05)
- increased relative weight of right kidney in high dose females (p≤0.05)
- increased relative weight of right adrenal in high dose males (p≤0.05)
Statistically significant differences in the absolute organ weights compared to the control, which are not considered to be test item-related were:
-decreased absolute brain weight in high dose males (p≤0.05)
-decreased absolute heart weight by 16.6% in high dose females (p≤0.05)

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

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

HISTOPATHOLOGY: NON-NEOPLASTIC (restricted to dose groups 1 and 4)
Histopathological examination performed on one testicle and one epididymis with special emphasis on the qualitative stages of spermatogenesis (proliferative, meiotic and spermiogenic phases) and histopathology of the interstitial testicular structure of all adult male animals of the highest dose group
and the control group following H & E and PAS staining, did not reveal any test item-related effects.
No test item-related microscopic changes were seen in the reproductive organs for both males and fe males.
A pulmonary congestion was noted in the lungs from 4/5 male (control 0/5) and 5/5 female rats (control 4/5) of the high dose group (1000 mg/kg bw/day). As pulmonary congestion is occasionally seen in rats as a background finding, this change was not considered to be test item-related.
A test item-related squamous cell hyperplasia was noted in the fore-stomachs from 5/5 male and 5/5 female animals of the high dose group (1000 mg/kg bw/day). These test item-related stomach changes were localized in the zone adjoining the glandular stomach mucosa (forestomach or non-glandular mucosa) and attained statistical significance for both sexes (p≤0.01). Occasionally the squamous cell hyperplasia with subsequent hyperkeratinization was associated with acute inflammation of the submucosa in the non-glandular stomach (for 2/5 males and 1/5 females).
Examination of the stomachs from the animals (5 per group) of the low- and intermediate dose groups
(100 and 300 mg/kg bw/day) did not reveal any test item-related changes. As humans lack a fore-stomach, the relevance of these changes for humans is questionable.
All other microscopic changes observed were either coincidental, or lie within the normal background alterations which may be seen in untreated rats of this age and strain.

Evaluation of reproduction parameters: see section 7.8.1 & 7.8.2

Effect levels

Dose descriptor:

NOAEL

Remarks:

Parental generation F0

Effect level:

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

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table 1. Mean body weight males

Body Weight (g)
Sex: Male	Day(s) Relative to Start Date
	1	8v	15	22v	29v	36v
Group 1: control	293.02	334.59	350.28	386.70	410.83	440.86
Group 2: 100 mg/kg	293.46	328.96	347.75	380.93	405.63	436.65
Group 3: 300 mg/kg	294.16	336.32	353.72	392.53	416.39	446.90
Group 4: 1000 mg/kg	293.14	319.93*	338.13	367.75	389.07	416.42

Statistical Test Dunnett’s Test (Anova)

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

*-Statistical Test: Dunnett 2 Sided p<0.05

 

Table 2. Mean body weight females

Body Weight (g)
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	14v	20	1vv	4v
Group 1: control	181.25	200.61	207.74	231.36	272.16	301.56	365.20	283.99	301.20
Group 2: 100 mg/kg	180.93	197.80	208.12	226.64	269.64	303.46	376.18	290.81	302.18
Group 3: 300 mg/kg	180.90	194.82	204.24	224.81	254.78	289.41	356.96	271.43	284.22
Group 4: 1000 mg/kg	181.33	193.29	200.78	213.67	245.74*	277.59*	341.34	258.20*	272.58*

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<0.05

 

Table 3. Mean Biochemical Parameters Males and Females (Albumin and ALAT)

Biochemical Parameters
Sex: Male	Albumin (g/L)	ALAT (U/L)v	Sex: Female	Albumin (g/L)	ALAT (U/L)vv
Group 1: control	31.98	38.0	Group 1: control	33.14	38.4
Group 2: 100 mg/kg	31.64	38.6	Group 2: 100 mg/kg	32.80	36.4
Group 3: 300 mg/kg	31.18	43.4	Group 3: 300 mg/kg	33.74	34.6
Group 4: 1000 mg/kg	30.60**	63.2**	Group 4: 1000 mg/kg	32.18	56.2**

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<0.05

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

Table 4.Histopathology Males and Females

Sex	Male				Female
Group	Gr.1	Gr.2	Gr.3	Gr.4	Gr.1	Gr.2	Gr.3	Gr.4
Number of Animals	5			5	5			5
Number of Completed Animals	5			5	5			5
Lungs
congestion	0			4*	2			1
Stomach
No abnormalities detected	5			0	5			0
Non-glandular; submucosa; acute inflammation -slight -moderate	0 0			1 1	0 0			0 1
Non-glandular; squamous cell hyperplasia -slight -moderate -marked	0 0 0 0			5** 1 3 1	0 0 0 0			5** 2 2 1
Non-glandular; keratopurulent debris	0			2	0	0

Fisher’s Two-Tailed Exact Test Performed:

*= 5% Significance

**= 1% Significance

Applicant's summary and conclusion

Conclusions:

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

Executive summary:

The aim of the study was to obtain information on possible effects of the test item on general toxicity, reproduction and/or development according to OECD guideline 422. The test item was administered orally by gavage to rats at dose levels of 100, 300 and 1000 mg active ingredient/kg bw/day. The application started two weeks before mating on test day one and ended on the day or one day before sacrifice. Day of sacrifice was on test day 37 for the male rats and between lactation day 4 and 7 for the female rats. No test item-related premature death was noted in any treatment group (100, 300 and 1000 mg/kg bw/day).

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

The laboratory examinations revealed an increased ALAT activity for the male and female rats of the high dose group (1000 mg/kg bw/day) and a decrease in the albumin concentration for the male rats of the high dose group.

No test item-related changes were noted during the macroscopic inspection at autopsy with the exception of changes in the stomach from 2 male animals from the high dose group (1000 mg/kg bw/day). Microscopic examination revealed the occurrence of squamous cell hyperplasia in the non-glandular mucosa and acute inflammation of the forestomach from the male and female rats of the high dose group (1000 mg test item/kg bw/day). Further microscopic findings occurred in form of pulmonary congestion in the male rats from the high dose group. NOAEL (no-observed-adverse-effect level) for repeated dose toxicity: 300 mg/kg bw/day, p.o.

Effects on reproduction parameters and organs (see section 7.8.1).

Effects on the development of the F1offsprings (pups) (see section 7.8.2).