Sulphophthalic acid derivatives reaction products of fatty alcohol C12-C14 and sodium hydroxide

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From January 29 to April 05, 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

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)

Version / remarks:

29 July 2016

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Dilasoft TF

IUPAC Name:

Dilasoft TF

Test material form:

liquid

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Han:WIST

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Toxi-Coop Zrt. 1103 Budapest, Cserkesz u. 90. Hungary
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: male 89 – 92 days (12-13 weeks) old and female animals 89– 92 days (12-13 weeks) old
- Weight at study initiation: 325 – 415 g for male animals and 205 – 239 g for female animals (the weight variation did not exceed  20 per cent of the mean weight)
- Housing:
Before mating 2 animals of the same sex/cage. Mating: 1 male and 1 female / cage. Pregnant females: individually. Males after mating: 2 animals / cage
- Diet (e.g. ad libitum): ssniff® SM R/M-Z+H complete diet for rats and mice ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: 20 days

DETAILS OF FOOD AND WATER QUALITY:
The food was considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study. The supplier provided an analytical certificate of the standard diet for the batch used. Water quality control analysis and microbiological assessment are performed once in every six months by Government Office of Capital Budapest Department of Public Health and Medical Officer Service (Váci út 172-174. Budapest, H-1138 Hungary).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 - 70 %
- Air changes (per hr): Above 10 air-exchanges/ hour by a central air-condition system
- Photoperiod (hrs dark / hrs light): Artificial light, from 6 a.m. to 6 p.m.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

Aqua purificata (Distilled water)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in the vehicle (distilled water) in concentrations of 20, 60 and 200 mg/mL (corrected concentrations respectively to uncorrected concentrations of 36, 108 and 360 mg/mL), and the corresponding doses for 5 ml of solutions are 100, 300 and 1000 mg/kg bw/day. Formulations were prepared in the formulation laboratory of the Test Facility beforehand not longer than for three days and stored at room temperature until use.

VEHICLE
- Concentration in vehicle: 20, 60 and 200 mg/mL
- Amount of vehicle (if gavage): 5 ml
- Lot/batch no. (if required): 201910069

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Five aliquots of 10 mL of each formulation and five aliquots of control substance (vehicle) were taken from different places and analyzed. The samples were stored at room temperature until analysis.
Date of sampling: February 06, 2020 and March 10, 2020
Date of analysis: February 07, 2020 and March 11, 2020
Concentration of the test item in the dosing formulations varied between the range of 100 % and 106 % in comparison to the nominal values. Results and details of analysis are reported in section 8 of this IUCLID file.
The suitability of the chosen vehicle (recovery and stability) for the test item at the intended concentrations was analytically verified up front.
The recovery of the test item from the vehicle was within the acceptance criteria (96 and 97 % relative to nominal concentrations) at ca. 1 mg/mL and at ca. 400 mg/mL.
The test item proved to be stable in distilled water at the intended concentrations at room temperature and at 5 ± 3 °C for three days.

Duration of treatment / exposure:

The experimental period involved 20 days of acclimatization (including 14 days for examination of estrous cycle) and 41-67 days treatment/observation period (depending on the effectiveness of mating) and necropsy days.
The day of first treatment is considered as day 0 of examination.

Frequency of treatment:

The test item was administered in a single dose by oral gavage on a 7 days/week basis, every day at a similar time (±2 hours)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

12 animals/sex in the control and dose groups except

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: doses selected based on results of a range finding test
- Fasting period before blood sampling for clinical biochemistry: 16 hours (overnight) prior to blood collection

Positive control:

None

Examinations

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once a day, after the administration at approximately the same time

BODY WEIGHT: Yes
- Time schedule for examinations: Parental males were weighed on the first day of dosing (Day 0) and weekly thereafter and on the day of the necropsy.
Parental females were weighed on the first day of dosing (Day 0) then weekly, on gestation days 0, 7, 14 and 21 and on days 0 (within 24 hours after parturition), 4 and 13 post-partum.
Body weight of the female animals was additionally weighed on gestational day 10 in order to give accurate treatment volumes, but these data were not evaluated. Body weight was measured on the day of necropsy for female animals subjected to organ weighing.

FOOD CONSUMPTIONS:
- The food consumption was determined weekly by reweighing the given and non-consumed diet with a precision of 1 g during the treatment period except mating phase (pre-mating days 7, 13 and post-mating days 20, 27, 34 and 40 for male animals, pre-mating days 7, 13, gestation days 0, 7, 14 and 21, lactation days 0, 4 and 13 for female animals).

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: one day after the last treatment (i.e. on the day of necropsy).
- Anaesthetic used for blood collection: Yes, Isofluran CP®
- Animals fasted: Yes
- How many animals: five male and five female animals randomly selected from each group
- Parameters checked in the table reported in "Any other information on materials...."

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: ne day after the last treatment (i.e. on the day of necropsy).
- Animals fasted: Yes
- How many animals: five male and five female animals randomly selected from each group
- Parameters checked in the table reported in "Any other information on materials...."

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: on parental animals once a day, after the administration at approximately the same time
- Dose groups that were examined: all groups
- Battery of functions tested: autonomic activity (lachrymation, piloerection, pupil size, respiratory pattern, occurrence of secretions and excretions), circulatory and central nervous system, somatomotor activity and behavior pattern, changes in gait, posture and response to handling

IMMUNOLOGY: No

OTHER:
- DETERMINATION OF SERUM THYROID HORMONE Blood samples were collected from animals as follows:
from all dams and from 3-7 pups per litter on post-partum/ post-natal day 13

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table in "Any othet information on materials....)

HISTOPATHOLOGY: Yes (see table in "Any othet information on materials....)

Statistics:

The statistical evaluation of appropriate data (marked †above) was performed with the statistical program package SPSS PC+4.0.
The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test.
Where no significant heterogeneity was detected a one-way analysis of variance (ANOVA) is carried out. If the obtained result was significant Duncan Multiple Range test was used to access the significance of inter-group differences. Getting significant result at Bartlett’s test, the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test. Chi2 test was performed if feasible.
Frequency of toxic response, pathological and histopathological findings by sex and dose was calculated.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Daily clinical observations
Test item related clinical signs were not detected in any group (100, 300 or 1000 mg/kg bw/day). Parental animals (male and female) exhibited normal behavior and physical condition with no abnormalities at the daily clinical observations except for one dam at 1000 mg/kg bw/day with individual clinical signs. Sporadically, alopecia – as species specific findings – was also noted for some female animal.
There were no clinical signs in male animals in the control, 100, 300 or 1000 mg/kg bw/day groups during the entire observation period (pre-mating, mating and post-mating periods).
In one female animal at 1000 mg/kg bw/day, paralysis-like state on hindlimbs and tail, flattened body position, spreaded hindlimbs were observed from gestation day 20 up to lactation day 0. Marked vaginal bleeding and paleness were noted for this dam on the day of delivery.
Alopecia was detected on the skin of the left side of the body (1/12, control), of the abdomen (1/12, at 300 mg/kg bw/day) and of the neck (1/12, at 1000 mg/kg bw/day) in female animals from lactation days 1 or 3 up to termination of the study.
All other female animals were normal in the control, 100, 300 and 1000 mg/kg bw/day groups during the entire observation period (pre-mating, mating, gestation and lactation periods).

Detailed weekly clinical observations
Detailed weekly clinical observations did not reveal test item related changes in the behavior or physical condition of all animals (control, 100, 300 and 1000 mg/kg bw/day) during the pre-mating, mating and post-mating periods in male animals and during the pre-mating, mating, post-mating, gestation and lactation periods in female animals.
Individual clinical signs of one dam at 1000 mg/kg bw/day – paralysis-like state on hindlimbs and tail, flattened body position, spreaded hindlimbs – were also recorded at the detailed weekly observations on gestation day 21 and on the day of parturition (lactation day 0). Paleness and marked bleeding from vaginal orifice were also noted for this dam on the day of delivery and animal was euthanized because there were no viable pups.
Alopecia – as described above – was detected in some female animal in the control (1/12, on the left side of the body), at 300 mg/kg bw/day (1/12, on the abdomen) and at 1000 mg/kg bw/day (1/11, on the neck) on lactation days 4 and 13.

All other parental animals (male and female) exhibited normal behavior and physical condition in control, 100, 300 and 1000 mg/kg bw/day at the detailed weekly observations during the entire treatment period.

Mortality:

no mortality observed

Description (incidence):

There was no mortality in any groups of control, 100, 300 or 1000 mg/kg bw/day during the course of study (male and female).

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The body weight development was undisturbed in male and female animals at 100, 300 and 1000 mg/kg bw/day during the entire treatment period.
The mean body weight was comparable in the control and at 100, 300 and 1000 mg/kg bw/day groups in male animals during the pre-mating, mating and post-mating periods. Statistical significance was noted for the slightly lower mean body weight gain in male animals at 1000 mg/kg bw/day – when compared to the control – between Days 13 and 20. This minor difference did not result in significant changes in the mean body weight with respect to the control.
The mean body weight and body weight gain were comparable in female animals in the control and 100, 300 and 1000 mg/kg bw/day during the pre-mating, gestation and lactation period. There were no statistically or biologically significant differences in the mean body weight or body weight gain of female animals in the control and test item administered groups.

Food efficiency:

no effects observed

Description (incidence and severity):

There was no test item related adverse effect in the mean daily food consumption of male or female animals at 100, 300 or 1000 mg/kg bw/day.
The mean daily food consumption was comparable in the control and 100, 300 or 1000 mg/kg bw/day groups in male animals during pre-mating and post mating periods and in female animals during the pre-mating, gestation and lactation periods.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Hematological evaluation did not reveal adverse test item related changes in the examined parameters at 100, 300 or 1000 mg/kg bw/day.
Statistically significant difference with respect their control was detected only at some parameters in the low and mid dose group.
The mean white blood cell count (WBC) exceeded the control value in male animals at 300 mg/kg bw/day.
In the female animals, the mean platelet count (PLT) was higher than in the control at 100 and 300 mg/kg bw/day.

All these variations were considered to be toxicologically not relevant due to the minor degree and the lack of dose relevancy and because of the individual values of test item treated animals were well within the historical control ranges.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item related adverse effects on the examined clinical chemistry parameters at 100, 300 or 1000 mg/kg bw/day (male or female).
In the male animals, statistical significance with respect to the control was detected at the slightly higher mean activity of alanine aminotransferase (ALT) and at the lower mean concentration of sodium (Na+) at 1000 mg/kg bw/day.
The examined clinical chemistry parameters were similar to their control in female animals at 100, 300 and 1000 mg/kg bw/day.
The statistically significant differences in ALT and Na+ in male animals were considered to have little or no toxicological importance because values – mean and individual – corresponded well to the historical control values, in addition there was no related histopathological alterations.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Functional observation battery did not demonstrate any alterations in the behavior or reactions to different type of stimuli of selected male or female animals in the control, 100, 300 or 1000 mg/kg bw/day groups at the end of the treatment period (on Day 40 for male animals and on Day 54 for female animal).
There were no changes in the physical condition, behavior or in reactions to different types of stimuli in the male or female animals of control and test item treated groups in the examined parameters during the course of the functional observations.
Alopecia was detected in female animals as mentioned above (1/5 control and 1/5 at 1000 mg/kg bw/day) at the functional observations.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item related effects on the weights of examined organs in male or female animals at any dose level (100, 300 and 1000 mg/kg bw/day).

Minor change in the mean weights of heart relative to brain weight in male animals at 1000 mg/kg bw/day compared to their control were considered to be of no biological relevance in the related findings or histological alterations.

The weights of examined organs (absolute and relative to body and brain weights) were comparable to their control in female animals at 100, 300 and 1000 mg/kg bw/day.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Specific macroscopic alterations were not detected in the organs or tissues at any dose levels (100, 300 or 1000 mg/kg bw/day) at the necropsy.
Necropsy observation revealed findings commonly detected also in untreated experimental rats of this strain with similar age.
In the male animals, one sided pyelectasia (3/12 control; 2/12 100 mg/kg bw/day; 1/12 300 mg/kg bw/day) and thymic hemorrhage (2/12 at 300 mg/kg bw/day) were detected.
In dams in the control group, alopecia on the skin of the left-side of the body (1/12) and renal changes (1/12; enlargement, pyelectasia, 0.4x0.5 mm stone content on the left side and smaller than normal, deformation on the right side) were observed.

At 100 mg/kg bw/day, enlarged (2x4 cm), vesicle -like, liquid filled kidney on the left side (1/12), both sides pyelectasia (1/12) and slight hydrometra (1/12) were revealed in dams.
At 300 mg/kg bw/day, alopecia was observed on the skin of abdomen in one dam (1/12).
In female animal at 1000 mg/kg bw/day, which was subjected to early necropsy on lactation day 0, pale liver, extremely enlarged spleen, dilated and urine filled urinary bladder, one pup inserted in the birth canal (vagina) and enlarged compact placenta were observed. These findings refer to lymphocyte leukemia in accordance with the results of histopathological examinations.
Right side pyelectasia (2/12), alopecia on the skin of the neck (1/12) were also observed in dams at 1000 mg/kg bw/day.
Pyelectasia (without related histopathological lesions) and other renal changes occurred independently from doses and with low incidence. Therefore, these findings were judged to be toxicologically not relevant on the basis of literary data and necropsy observations of several years.
Hydrometra, related to the female sexual cycle and alopecia is a frequent observation in untreated experimental rats of this strain and the frequency noted here fit the ranges seen for control animals. In the lack of related histopathological alterations (inflammatory or another pathological lesion) these were considered to be toxicologically not relevant.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Histopathological examinations did not reveal any toxic or other test item related lesions in the investigated reproductive and other organs of experimental male and female animals at 1000 mg/kg bw/day.
In the male animals of control and 1000 mg/kg bw/day groups, the investigated organs of reproductive system (testes, epididymides, prostate, seminal vesicles, coagulating glands) were histologically normal and characteristic on the sexually mature organism in all cases (12/12, each). The various spermatogenic cells (the spermatogonia, the spermatocytes, the spermatids and spermatozoa); representing different phases in the development and differentiation of the spermatozoons and the interstitial cells were the same in quantity and morphologically in the testes of investigated control and treated animals.

The histology picture of testes, epididymides, prostate, seminal vesicles and coagulating glands was normal in all animals in the control groups (12/12).
In the female animals belonging to the high dose (1000 mg/kg bw/day) treated and control groups, the ovaries, uterus, cervix, vagina had a normal structure characteristic of the species, age and phase of the active sexual cycle (11/12, in control and high dose groups) except for uterus of one dam at 1000 mg/kg bw/day, in which metritis – inflammation of mucous membrane of the uterus; 1/12 – was observed as an individual lesion. The cortical region of ovaries contained primary, secondary and tertiary follicles and corpora lutea, indicating the active maturation of oocytes, and ovulation. The epithelial capsule and ovarian stroma were normal in all cases as well.
The histological structure and the cellularity of pituitary with special attention on the cytomorphology and proportion of acidophilic and basophilic cells in the adenohypophysis were the same in the control and treated male (5/5 in both groups) and female (5/5 in both groups) animals selected for examination.
Acute hemorrhage was detected in the thymus in two male animals (2/2) at 300 mg/kg bw/day examined on the basis of the necropsy findings. The acute hemorrhage in the thymus is considered as consequence of hypoxia, dyspnea and circulatory disturbance developing during exsanguination procedure.
Atrophy of hair follicles – in accordance with alopecia detected at the necropsy – occurred in three animals (1/5 control female and 1/1 female at 300 mg/kg bw/day, 1/6 at 1000 mg/kg bw/day). This finding is common in laboratory rats of this strain. Under conventional conditions dermal infections (Trichophyton spp, Microsporon spp, Demodex infection etc.) may occur. In these cases, dermatitis is seen. In non-infectious cases of alopecia, imbalance of hormonal factors with age, malnutrition or focal ischemia could play role in development of atrophy of hair follicles and alopecia. Corticosteroids (stress) reduce the metabolic activity of epidermal cells.
Pyelectasia was observed in one or both sides kidneys in male and female animals in accordance with necropsy findings as follows:
- 3/5 male in control group (one side), 2/2 male at 100 mg/kg bw/day (one side) and 1/1 male at 300 mg/kg bw/day (one side);
- 1/6 female in control group (one side), 1/2 female at 100 mg/kg bw/day (both sides) and 3/5 female at 1000 mg/kg bw/day (one side, each);
Pyelectasia without degenerative, inflammatory or other histological (fibrotic etc.) lesion is considered as a common finding in laboratory rats without toxicological significance.
Additional individual renal lesions were seen in some animal as follows:
- purulent pyelitis and fibrosis with pyelectasia in one female animal in the control group (1/6);
- hydronephrosis in accordance with the necropsy observations in one female animal at 100 mg/kg bw/day (1/2). The kidney is enlarged, lobulated and may consist of a translucid fluid-filled sac. Affected kidneys often contain multilocular cavities separated by incomplete trabecula in the medulla and cortex. Microscopic features include atrophy of the renal tubules, glomeruli and the pelvic urothelium.
In animals, selected for full histological examinations, no morphological evidence of test item related acute or subacute injury (degeneration, inflammation, necrosis etc.) of the stomach, the small and large intestines, the liver, the cardiovascular system, the respiratory system, the urinary system, the immune system, the hematopoietic system, the skeleton, the muscular system, the central, or peripheral nervous system, the eyes, the integumentary system, the reproductive system was observed.
The cytomorphology of endocrine glands were the same in the control and treated animals.

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Lymphocyte leukemia was detected in one female animal at 1000 mg/kg bw/day (1/6) as an individual disease. Histological examination of rats with large granular lymphocyte leukemia revealed neoplastic cells in vascular lumens of different organs or infiltrating the parenchyma of the tissue of affected organs. Lymphocyte leukemia is the most commonly observed leukemia in laboratory rats and is occasionally observed as an incidental finding in subchronic studies.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Serum thyroid hormones

The FT3, FT4 and TSH levels were not affected by the test item in parental male animals (100, 300 and 1000 mg/kg bw/day) and in offspring sampled on postnatal day 13.
Statistical significance was only detected at the slightly higher mean concentration of FT4 in pups at 1000 mg/kg bw/day sampled on post-natal day 13 compared to their control. The values were well within the historical control ranges; therefore, this finding was considered a random finding and to be toxicologically not relevant.
The TSH values of parental male animals were below the detection limit in the control and test item administered groups. Therefore, it was considered to be within the physiological ranges in animals at 100, 300 and 1000 mg/kg bw/day.

Effect levels

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

NOAEL for systemic toxicity of male/ female rats = 1000 mg/kg bw

Executive summary:

The objective of this study was to obtain initial information on the toxic potential of test item and on the possible effects of the test item on reproduction and developmentwhen repeatedly administered orally (by gavage) to rats at doses of 100, 300 and 1000 mg/kg bw/day (calculated by active ingredient) compared to control animals according to OECD 422.

As a screening test, it was intended to provide initial informationon the possible health hazards likely to arise from repeated exposure over a relatively limited period of time and on the possible effects onmale and female reproductive performance such as gonadal function, mating behavior, conception, pregnancy, parturition as well as on development of the F1 offspring from conception to day 13 post-partum associated with administration of repeated maternal doses

The test item was administered orally (by gavage) once daily at 0 (vehicle only), 100, 300 and 1000 mg/kg body weight (mg/kg bw/day) doses to four groups of Han:WIST rats consisting of 12 animals per sex per groupin concentrations of 20, 60 and 200 mg/mL (calculated by active ingredient) corresponding to a 5 mL/kg bw dosing volume. A group of vehicle (distilled water) treated animals (n= 12/sex) served as a control.

The suitability of the chosen vehicle for the test item at the intended concentrations was analytically verified up front. The test item was stable in the vehicle in concentrations of 1 mg/mL and 400 mg/mL at room temperature and in a refrigerator (at 5 ± 3 °C) for three days.

The concentration of the test item in the dosing formulations administered to the animals was checked two times during the study. Test item concentrations in the dosing formulations varied within the rangeof 100 % and 106 % (in comparison to the nominal values)and confirming the proper preparation of the dosing formulations.

All animals of the parent (P) generation were dosed prior to mating (14 days) and throughout mating. In addition, males received the test item or vehicle after mating up to the day before the necropsy (altogether for 41 days). Dams were additionally exposed through the gestation period and up to lactationdays 13-16,i.e. up to the day before necropsy (altogether for 51, 54 or 67 days). One dam for which no living pups remained was administered for 44 days.

Observations included mortality, clinical signs, body weight, food consumption, mating, pregnancy and delivery process, as well as development of offspring.

Blood samples were collected for possible determination of serum levels of thyroid hormones (FT3, FT4 and TSH) from all dams.

Five dams and their male mating partners were randomly selected from each group to examine further signs of toxicity such as functional observations, hematology and blood coagulation, clinical chemistry, gross necropsy, organ weighing and histopathologic examinations.

All parental animals were subjected to gross pathology one day after the last treatment. The body weight, brain weight and weight of the testes and epididymides and prostate and seminal vesicles with coagulating glands as a whole of adult male animals were determined. In addition, for five males and females randomly selected from each group, adrenals, brain, heart, kidneys, liver, spleen and thymus were weighed.

Thyroid gland was preserved from all adult males and females for the intended subsequent histopathological examination.

Full histopathology examinations were performed on the preserved organs and tissues of the randomly selected animals in the control and high dose groups. Additionally, full histopathological examinations were performed for organs and tissues of one dam with severe clinical signs and macroscopic findings at 1000 mg/kg bw/day.

In addition, organs showing macroscopic findings at the necropsy (kidneys, skin and thymus) were processed and examined histologically in some animals of low and mid dose groups.

 

The results of this study were summarized – indicating only the doses of 100, 300 and 1000 mg/kg bw/day by active ingredient – as follows:

Mortality

There was no mortality in any group of control, 100, 300 or 1000 mg/kg bw/day.

 

Clinical and functional observation

Clinical signs of systemic toxicity related to the test item were not detected at any dose level, neither at the daily nor at the detailed weekly clinical observations or at the functional observations. The behavior and physical condition of the animals was not impaired at any dose level (100, 300 or 1000 mg/kg bw/day) during the entire treatment period.

 

Body weight and body weight gain

The body weight development was not affected by the test item in male or in female animals at 100, 300 or 1000 mg/kg bw/day during the entire treatment period (pre-mating and post-mating period for male animals; pre-mating, gestation and lactation periods for female animals).

Food consumption

The mean daily food consumption was similar in male or female animals in control and at 100, 300 and 1000 mg/kg bw/day during the entire study.

Hematology and blood coagulation

Hematologicaland blood coagulation investigation did not reveal test item related changes in the examined parameters at 100, 300 or 1000 mg/kg bw/day.

 

Clinical chemistry

There were no test item related effects on the examined clinical chemistry parameters at 100, 300 or 1000 mg/kg bw/day (male or female).

 

Serum thyroid hormones

Therewereno test item related changes in the serum thyroid hormone (FT3, FT4 and TSH) levels at any dose (parental male or 13-day offspring).

 

Necropsy

Macroscopic findings related to the effect of the test item were not found in male and female animals at 100, 300 or 1000 mg/kg bw/day.

 

Organ weight

There were no test item related changes in the weights (absolute and relative to body or brain weights) of brain, testes and epididymides,prostate and seminal vesicles with coagulating glands as a wholeof male animals at any dose level.

The weights of organs of selected animals were comparable in the control and test item treated groups (male and female).

 

Histopathology

There were no toxic or other test item related lesions detectable by histological examination in the investigated reproductive organs (ovaries, uterus, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland) in male or female animals administered with 1000 mg/kg bw/day.

Histopathological examinations did not indicate any toxic or other test item related lesions in the investigated organs of selected male and female animals at 1000 mg/kg bw/day.

 

Conclusion

Under the conditions of the present study, no signs of systemic toxicity in selected male or female animals at 100, 300 or 1000 mg/kg bw/day were found.

Based on these observations the No Observed Adverse Effect Levels (NOAEL) by active ingredient was found to be 1000 mg/kg bw/day.