Reaction mass of lauric acid, compound with morpholine (1:1) and 2-ethylhexyl dihydrogen phosphate, compound with morpholine (1:2) and bis(2-ethylhexyl) hydrogen phosphate, compound with morpholine (1:1)

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:

25 October 2917 - March 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

October 25, 2017 - January 9, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA Health Effects Test Guidelines: OPPTS 870.3650 Combined Repeated Dose Toxicity with the Reproduction/Developmental Toxicity Screening Test

Version / remarks:

July 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Toxi-Coop Zrt. Berlini utca 47-49. 1045 Budapest Hungary, Arácsi út 97. H-8230 Balatonfüred, Hungary

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Toxi-Coop Zrt. Cserkesz u. 90. H-1103 Budapest, Hungary
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 81-84 days old males and females
- Weight at study initiation: (P) Males: 309-390 g; Females: 198-250 g
- Fasting period before study: no
- Housing: Type III polypropylene/polycarbonate
Before mating: 2 animals of the same sex/cage
Mating: 1 male and 1 female/cage
Pregnant females were housed individually.
Males after mating: 2 animals/cage
- Diet: ad libitum, ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest, Germany
- Water: ad libitum, tap water from municipal supply
- Acclimation period: 20 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): above 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

distilled

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in the vehicle in concentrations of 150/200, 60 and 20 mg/mL. High dose was reduced on Day 21, thus concentration of the high dose formulation was also reduced from 200 to 150 mg/mL on Day 21. A constant treatment volume of 5 mL dose preparation/kg body weight was administered in all groups. The individual volume of the treatment based on the most recent individual body weight measurement of the animals. In the first week of the pre-mating period, animals received volumes based on the actual body weight on Day 0.
VEHICLE
The suitability of the chosen vehicle for the test item at the intended concentrations was analytically verified up front (Toxi-Coop study no: 685-100-1247). A sufficient stability and homogeneity in the chosen vehicle were verified over the range of relevant concentrations at the appropriate frequency of preparation. (Recovery from distilled water: 96% and 99 % of nominal concentrations at ca. 1 mg/mL and ca. 400 mg/mL; the substance preparation was stable at the intended concentrations for at least one day at room temperature and for three days in refrigerator.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: Females remained with the same male until copulation occurs or 14 days have elapsed
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- Further matings after two unsuccessful attempts: yes
Four female animals at the high dose failed to mate during the 14-days period, therefore mating period was prolonged and their partners were exchanged with each other. Two females failed to mate during the additional seven days therefore their partners were exchanged with each other for three or five additional days.
- After successful mating each pregnant female was caged: individually

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of formulations was performed in the Analytical Laboratory of Test Facility. Five samples were taken from different places from each concentration (Groups 2, 3 and 4) and were measured on 2 occasions, during the first and last treatment weeks. Similarly, five samples were taken from the control solution (Group 1) from different places and analyzed. Concentration of the test item in the dosing formulations varied in the range of 97 and 103 % of the nominal values.

Duration of treatment / exposure:

Males received the test item or vehicle after mating up to the day before the necropsy (altogether for 42 days). Females were additionally exposed through the gestation period and up to lactation days 13-20, up to the day before necropsy (altogether for 54-75 days). Animals (male and female) were not administered one day before the dose reduction (Day 21).

Frequency of treatment:

daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

750 mg/kg bw/day (nominal)

Remarks:

The high dose is reduced from 1000 mg/kg bw/day to 750 mg/kg bw/day on Day 21 because 1000 mg/kg bw/day was lethal for two male and two female animals.

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose setting is based on findings obtained in a 14-day dose range finding study performed with the substance (study no. 685-400-1237, non-GLP).

Positive control:

none

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once a day, after treatment
Observations were performed on the skin, fur, eyes and mucous membranes, 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. Special attention was directed towards the observation of tremors, convulsions, salivation, diarrhea, lethargy, sleep and coma.
The mating period was different for individual animals, therefore clinical signs were evaluated by phases as follows:
-Male animals: pre-mating, mating and post-mating period: from Day 0 up to the day before the necropsy;
-Dams: pre-mating (From Day 0 up to day of positive vaginal smear), gestation and lactation periods;
-non-pregnant and not delivered females: pre-mating and post-mating periods

BODY WEIGHT: Yes
- Time schedule for examinations: Parental males were weighed on the first day of dosing (Day 0), weekly thereafter and on the day of necropsy. Male animals in the high dose were weighed daily for a period (from Day 2 or 3 to Day 22) due to their poor condition and ensuring more appropriate dosing. Parental females were weighed on the first day of dosing (Day 0) then weekly, on gestation days 0, 7, 14 and 21 and on post-partum day 0 (within 24 hours after parturition), 4 and 13. Additionally, female animals were weighed on gestational day 10 in order to give accurate treatment volumes, but these data were not evaluated statistically. Some female animals in the high dose was also weight daily due to their poor condition and ensuring more appropriate dosing.

FOOD CONSUMPTION AND COMPOUND INTAKE :
The food consumption was determined weekly by reweighing the non-consumed diet with an accuracy of 1 g during the treatment period except mating phase: premating Days 0, 7 and 13 and by weekly interval during post-mating period for male animals; premating Days 0, 7 and 13, gestation days 0, 7, 14 and 21, lactation days 0, 4 and 13 for female animals

OTHER:
Sensory reactivity to different type of stimuli (e.g. auditory, visual and proprioceptive), assessment of grip strength and motor activity were conducted on five male and five female animals randomly selected from each group during the last exposure week but before the blood sampling. General physical condition and behavior of animals were tested. A modified Irwin test was performed. (Irwin, 1968).

Oestrous cyclicity (parental animals):

Estrous cycle was monitored by examining vaginal smears before the treatment starts from each animal being considered for study for two weeks. Vaginal smears were also prepared and estrous cycle was monitored daily from the beginning of the treatment period (two weeks pre-mating period) and during the mating period until evidence of copulation.Vaginal smear were also prepared on the day of the necropsy.

Sperm parameters (parental animals):

Parameters examined in male parental generation [P]:
- testis weight, epididymis weight, prostate weight and seminal vesicles with coagulating glands as a whole

Litter observations:

STANDARDISATION OF LITTERS
On post-partal day 4, the size of each litter was adjusted to four pups per sex per litter.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, runts (pups that are significantly smaller than normal pups), and litters were weighed (with an accuracy of 0.1 g) within 24 hours of parturition (on the day when parturition was complete, post-natal day 0) and on postnatal day 4. The anogenital distance of each pup was determined on postnatal day 4. The anogenital distance was normalized to the cube root of the body weight. Therefore, individual body weight of pups was also determined with an accuracy of 0.01 g on postnatal day 4 (litter weight was calculated for postnatal day 4). Blood samples were collected from the surplus pups (at least two pups per litter), pooled and used for determination of serum T4 levels. The number of nipples/areolae in male pups was counted on postnatal day 13.

GROSS EXAMINATION OF DEAD PUPS: Yes

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No

Postmortem examinations (parental animals):

CLINICAL PATHOLOGY
Clinical pathology examinations including hematology, blood coagulation and clinical chemistry were conducted in five male and five female animals randomly selected from each group one day after the last treatment (i.e. on the day of necropsy). Animals were food deprived for approximately 16 hours (overnight) prior to blood collection. Blood samples were harvested from the retro-orbital venous plexus under Isofluran CP® anesthesia. Three samples were taken from each animal: one for hematology, one for determination of blood clotting times and the third one to obtain serum samples for clinical chemistry. In addition, blood samples were collected for determination of serum levels of thyroid hormones.
HEMATOLOGY
WBC (White Blood Cell leukocyte count), RBC (Red Blood Cell erythrocyte count), HGB (Hemoglobin concentration), HCT (Hematocrit, relative volume of erythrocytes), MCV (Mean Corpuscular erythrocyte Volume), MCH (Mean Corpuscular erythrocyte Hemoglobin), MCHC (Mean Corpuscular erythrocyte Hemoglobin Concentration), PLT (Platelet thrombocyte count), RET (Reticulocytes),Differential white blood cell count, APTT (Activated partial Thromboplastin Time), PT (Prothrombin Time)
CLINICAL CHEMISTRY
ALT (Alanine Aminotransferase activity), AST (Aspartate Aminotransferase activity), TBIL (Total Bilirubin concentration), CREA (Creatinine concentration), UREA (Urea concentration), GLUC (Glucose concentration),CHOL (Cholesterol concentration), Na+(Sodium concentration),K+ (Potassium concentration), ALB (Albumin concentration), TPROT (Total Protein concentration)
DETERMINATION OF SERUM LEVELS OF THYROID HORMONES
Blood samples were collected for determination of serum levels of thyroid hormones (T4) as follows:
-from all parent male animals at termination
SACRIFICE
Parental male animals: after the optionally extended post-mating period on Day 43.
Non-pregnant or not-delivered (but mated) female animals: on Day 43.
Dams not selected for toxicology examinations: on post-partum day 13 or shortly thereafter (Days 55, 59, 70 or 76).
Dams selected for toxicology examinations: shortly after post-partum day 13 (Day 57).
Offspring: on postnatal day 13 or shortly thereafter.
GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera. The appearance of the tissues and organs were observed, and any abnormality was recorded including details of the location, color, shape and size. Special attention was paid to the organs of the reproductive system.
HISTOPATHOLOGY
Detailed histological examinations were performed on the ovaries, uterus, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland in the control and high dose groups with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure; on the ovaries covering the follicular, luteal, and interstitial compartments of the ovary as well as the epithelial capsule and ovarian stroma.
In addition, these organs were processed and examined histologically in non-pregnant or not delivered females and males these females cohabited with in the low and middle dose groups. Full histopathology examinations were performed on the preserved organs and tissues of the randomly selected animals (n=5 animals/sex/group) in the control and high dose groups.
In addition, stomach was also processed and examined histologically in four males and one female from the 300 mg/kg bw/day group due to necropsy findings (thickened or erythematous mucosa). Seminal vesicle with coagulating gland was also processed and examined for one male at 100 mg/kg bw/day due to the necropsy observation (smaller than normal, one side). Uterus of three females in the mid dose group was processed and examined as well due to macroscopic finding (hydrometra). The following organs were preserved: Adrenal glands, Aorta, Bone with marrow and joint (femur), Brain (representative regions: cerebrum, cerebellum and pons and medulla oblongata), Eyes (lachrymal gland with Harderian glands), Female mammary gland, Gonads (testes with epididymides, ovaries, uterus with vagina), Gross lesions, Heart, Kidneys, Large intestines (caecum, colon, rectum, including Peyer’s patches), Liver, Lungs (with main stem bronchi; inflation with fixative and then immersion), Lymph nodes (submandibular, mesenteric), Muscle (quadriceps), Esophagus, Pancreas, Pituitary, Prostate, Salivary glands (submandibular), Sciatic nerve, Seminal vesicle with coagulating gland, Skin, Small intestines(representative regions: duodenum, ileum, jejunum), Spinal cord (at three levels: cervical, mid-thoracic and lumbar), Spleen, Sternum, Stomach, Thymus, Thyroid +parathyroid, Trachea, Urinary bladder.
ORGAN WEIGHT
At the time of termination, fasted body weight (all male animals) and weight of the testes, epididymides as well as 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, adrenal glands, brain, heart, kidneys, liver, spleen and thymus were weighed.

Postmortem examinations (offspring):

On the day of birth, pups found dead were subjected to a lung flotation test to differentiate pups died intrauterine (stillborn; negative lung flotation test) from pups died after the birth (dead pups; positive lung flotation test). Dead pups found were subjected to necropsy by a macroscopic examination. Any observed abnormalities were recorded.

Statistics:

The statistical evaluation of appropriate data (marked with † above) were 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) was 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.

Reproductive indices:

Please refer to "Any other information on materials and methods incl. tables"

Offspring viability indices:

Please refer to "Any other information on materials and methods incl. tables"

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

In dead animals, nuzzling up the bedding material (2/2 male, 2/2 female), salivation (2/2 male, 2/2 female), decreased activity (2/2 male, 1/2 female), piloerection (2/2 male, 1/2 female) and dyspnea (2/2 male, 1/2 female), signs of diarrhea (faeces around the anus, 1/2 male) and polyuria (wet, yellowish bedding material 2/2 male) were observed during the days before the death.
Test item related clinical signs were observed at 300 and 750/1000 mg/kg bw/day (male and female). Nuzzling up the bedding material and salivation were findings with the highest incidence in male and female animals of these groups. Most of signs – piloerection, hunched back, prone position, narrow eye aperture, dyspnea, decreased activity, noisy breathing, wet, yellowish bedding material, faeces around the anus – ceased in most of animals within 10 days after the dose reduction. Male animals proved to be more sensitive than females on the basis of the number and incidence of signs at 750/1000 mg/kg bw/day.
Some clinical signs were observed in four male and two female animals at 750/1000 mg/kg bw/day as well as in one dam at 300 mg/kg bw/day at the detailed weekly observation indicating that these animals did not fully recover 24 hours after the daily administration (piloerection or noisy breathing in male animals and piloerection or decreased activity or noisy breathing in female animals). Additionally, noisy breathing was noted for one female animal at 300 mg/kg bw/day on Gestation day 0.
There were no clinical signs during the course of the entire study in animals of control (12/12 male and 12/12 female) and 100 mg/kg bw/day group (12/12 male and 12/12 female), i.e. these parental animals exhibited normal behavior and physical condition with no abnormalities during the treatment period.

Mortality:

mortality observed, treatment-related

Description (incidence):

Two male and two female animals at 1000 mg/kg bw/day were found dead on Day 3, on Day 6, on Day 17 and on Day 20. There was no mortality in control, 100, 300 or 750 mg/kg bw/day groups (later after Day 21; male or female) during the course of study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The body weight gain and body weight were reduced in male animals at 750/1000 mg/kg bw/day. The mean body weight gain was slightly lower with respect to their control in male animals at 300 and at 100 mg/kg bw/day reaching statistical significance between Days 34 and 41, and in male animals at 300 mg/kg bw/day for the study overall (between Day 0 and 42). These changes however did not result in significant differences to their control in the mean body weight at 100 and 300 mg/kg bw/day therefore were considered to be toxicologically relevant. There were no statistically significant differences between the control and test item treated groups in the mean body weight of female animals at 100, 300 during the pre-mating period. The mean body weight and body weight gain was similar in the control and test item treated female animals at 750/1000 mg/kg bw/day during the pre-mating period. The mean body weight gain of female animals at 750/1000 mg/kg bw/day was slightly lower with respect to the control at between gestation days 14-21 and 0-21, which however did not result in significant changes in the mean body weight value of these animals. The body weight development was similar in the control and test item administered dams during the lactation period.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The food consumption was lower than in the control group in male and female animals at 750/1000 mg/kg bw/day. There were no toxicologically relevant differences in the mean daily food consumption of male and female animals in the control and of 100 and 300 mg/kg bw/day groups during the entire study. Although, statistical significance was noted for the slightly lower mean daily food consumption n female animals at 300 mg/kg bw/day between gestation days 7-14, this slight change was transient and judged to be toxicologically not significant.

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):

There were no test item related adverse changes in the examined hematological parameters in male or female animals at 100, 300 or 750/1000 mg/kg bw/day. Slight elevation of mean percentage of neutrophil granulocytes in male and female animals at 750/1000 mg/kg bw/day was probably an accompanying phenomenon of changes in gastric mucosa. Statistical significance was noted for the lower mean percentage of lymphocytes (LYM) and for the higher mean percentage of neutrophils (NEU) in male animals at 750/1000 mg/kg bw/day. In female animals at 750/1000 mg/kg bw/day, the mean percentage of neutrophils was higher and the mean corpuscular (erythrocyte) hemoglobin concentration (MCHC) and mean activated partial thromboplastin time were lower than in the control group. The individual values of these parameters (NEU, LYM, RET, MCHC and APTT) were within the historical control range and were judged to be toxicologically not relevant as there was no dose dependence (RET, APTT) and findings were with minor degree.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Evaluation of the clinical chemistry parameters did not reveal pathologic changes in the examined biochemical parameters.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Functional observations did not demonstrate any test item related changes at 100 and 300 mg/kg bw/day in male and female animals and at 750/1000 mg/kg bw/day in male animals. The behavior, physical condition and reactions to different type of stimuli of animals selected for examination were considered to be normal in these groups.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histopathological examinations revealed test item related alterations in the stomach of male and female animals at 300 and 750/1000 mg/kg/bw/day (inflammation of the non-glandular area).

Dead animals at 1000 mg/kg bw: Histological examination revealed inflammation of the stomach (non-glandular area) and ulceration of mucous membrane in all cases. Inflammation was accompanied with squamous cell hyperplasia, infiltration of inflammatory cells and fibroblast proliferation in the lamina propria.
In addition, congestion and alveolar emphysema (grade 3) was seen in the lungs, in all four cases, in connection with agony.
In one of the dead male animals (no.405) lymphocyte depletion occurred in the spleen which is in good correlation with the macroscopic observations (smaller than normal spleen). In this animal decreased amount of secretum in the seminal vesicles was also observed.

Terminal sacrifice: In male animals, the investigated organs of reproductive system (testes, epididymides, prostate and seminal vesicles with coagulating gland) were histologically normal and characteristic on the sexually mature organism except one animal (no.406) in which decreased amount of spermatozoa in the epididymides and decreased intensity of spermatogenesis in the testes were observed. 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 histological picture of epididymides, seminal vesicles, and coagulating glands was normal in all animals as well. In the female animals the investigated organs of reproductive system (ovaries, uterus with cervix, vagina) had a normal structure characteristic of the species, age and phase of the active sexual cycle in all cases. 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 was normal in all cases as well.
Test item related inflammation of stomach (non-glandular area) accompanied with squamous cell hyperplasia and infiltration of inflammatory cells and fibroblast proliferation in the lamina propria were observed in all male and female animals belonging to the 750/1000 mg/kg bw/day treated group (10/10 males and 10/10 females) and in some animals from the 300 mg/kg bw/day treated group (4 males and 1 female).
No ulceration or dysplasia and down growths of the hyperplastic basal cells (suspected neoplasm formation) were detectable in the altered areas. No inflammatory or hyperplastic lesions were seen in the glandular stomach.
There was no morphological evidence of acute or subacute injury (degeneration, inflammation, necrosis, etc.) of the small and large intestines, the liver, the pancreas, the cardiovascular system, the urinary system, the immune system, the hematopoietic system, the skeleton, the muscular system, the male and female reproductive system or the central or peripheral nervous system in the animals.
The cytomorphology of the endocrine glands were the same in the control and the treated animals.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Serum Levels of Thyroid Hormones (T4):
There were no statistically significant differences with respect to the control in the thyroid hormone (free T4) level in parental male animals or in offspring sampled on postnatal day 13 at any dose levels.

Reproductive function: oestrous cycle:

effects observed, treatment-related

Description (incidence and severity):

A test item influence on the estrous cycle was detected at 1000 mg/kg bw/day as the number and percentage of females with regular cycle were significantly lower with respect to their control. The mean number of cycles, mean number of days in pro-estrous and estrous and mean length of cycles, mean number of days in diestrous, the number and percentage of females in prolonged diestrous were affected at the high dose. (The dose was reduced after estrous cycle examination during the pre-meting period therefore 1000 mg/kg bw/day was considered at the cycle evaluation.) An influence on the estrous cycle was detected at 1000 mg/kg bw/day prior to dose reduction in the form of a significant lower percentage of animals with regular estrus cycle, primarily due to a higher mean number of animals with prolonged diestrous stage and subsequent impairment of the other estrus stages. Noteworthy to mention that these effects occurred only at 1000 mg/kg bw/day which was associated with mortalities. Furthermore, histopathology of the female reproductive system (ovaries, uterus with cervix, vagina) showed normal structure characteristics of the species, age and phase of the active sexual cycle in all cases. 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 was normal in all cases as well.
During the pre-experimental period, statistical significance was observed at the slightly higher mean number of cycles and at the lower mean number of days in diestrous at 300 mg/kg bw/day as an indication of the biological variation (see table 1)

Reproductive function: sperm measures:

effects observed, non-treatment-related

Description (incidence and severity):

see table 2

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

The examined parameters of reproductive performance were comparable in the control and test item treated (100, 300 or 750/1000 mg/kg bw/day) male or female animals.
Statistically significant differences were detected between the control and female animals at 750/1000 mg/kg bw/day at the higher fertility index because of the two pairs of control group that mated without pregnancy resulting in lower fertility indices. This finding was not considered as biologically relevant. Statistical significance was detected at the slightly lower gestation index in female animals at 750/1000 mg/kg bw/day comparing to their control. In this group 1 out of 10 females did not deliver. Considering the minor degree of this change and the historical control data, this difference with respect to their control were not considered to be toxicologically relevant. The percentage of pregnant females, non-pregnant females, and dams delivered, pregnants with liveborn(s) were comparable in female animals of control and test item treated animals. There were no significant differences in the copulatory indices of males and females and in the fertility index of males between the control and test item treated groups.

Delivery data of dams:
Higher percentage and higher mean of post-implantation loss and stillborns, lower mean number of liveborns and lower mean number of viable pups per litter on post-natal day 0 were observed at 750/1000 mg/kg bw/day treated animals (see table 4)

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

body weight and weight gain

food consumption and compound intake

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive performance

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive function (oestrous cycle)

Key result

Dose descriptor:

NOAEL

Remarks:

local

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

300 mg/kg bw/day (nominal)

System:

gastrointestinal tract

Organ:

stomach

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Test item related clinical signs were not detected in the offspring between postnatal days 0 and 13.
The percentage of offspring with signs (cold, not suckled) at 750/1000 mg/kg bw/day was lower than in the control group. The number of dead pups (including missing pups) was higher in the 750/1000 mg/kg bw/day group than in the control. Occasionally, other clinical signs were also observed: cyanotic skin (1 % at control), alopecia (10 % at 750/1000 mg/kg bw/day; one whole litter), which however were not considered to have toxicological relevance.

Mortality / viability:

no mortality observed

Description (incidence and severity):

There was no test item related effect on offspring’s extra uterine mortality.
The mean number of dead offspring per litter was comparable in the control and test item treated groups on postnatal day 0 and between postnatal day 0 and 13.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

A test item related effect on the body weight development of the offspring was not found.
The mean litter weight gain was lower between postnatal days 0-4, 4-13 and 0-13 reaching statistical significance between postnatal days 0-4 and 4-13. Similarly, the mean litter weight was also lower than in the control group in offspring at 750/1000 mg/kg bw/day on postnatal days 0, 4 and 13 reaching statistical significance on postnatal days 0 and 4. This is in good correlation with numbers of pups per dose groups, therefore has no toxicological relevance.
The mean body weight and body weight gain of pups were similar in the control and in all test item treated groups (100, 300 and 750/1000 mg/kg bw/day) on postnatal days 0, 4 and 13. Considering the offspring’s body weight in males and females, separately, statistically significant difference with respect to the control was detected for the higher male and female pup weights at 750/1000 mg/kg bw/day on postnatal day 4.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Test item related macroscopic alterations were not found in offspring subjected to gross pathological examination.
There were no macroscopic changes in the organs or tissues of stillborn offspring (5/5 at 750/1000 mg/kg bw/day) subjected to necropsy on postnatal day 0.
In some of the pups found dead, there were no macroscopic changes in the organs or tissues (4/4 at 750/1000 mg/kg bw/day on postnatal day 0) . In other dead pups the visceral organs were autolyzed and there was no milk in their stomach (2/2 at control on post-natal day 1, and 1/1 at 750/1000 mg/kg bw/day on postnatal day 1).

Anogenital Distance and Nipple Retention:
The anogenital distances (in male or female offspring) or nipple retention (male) were not affected by the test item at 100, 300 and 750/1000 mg/kg bw/day.
The absolute anogenital distances were slightly shorter with respect to their control in female pups at 100 and 300 mg/kg bw/day. The normalized anogenital distances were similar in male and female pups in the control and test item treated groups. These slight changes were not considered to be toxicologically relevant.
Nipples/areoles were not visible in any of the examined male offspring in the control or 100, 300 or 750/1000 mg/kg bw/day groups on postnatal day 13.
Sex Distribution and Survival of Offspring:
There were no test item related differences between the control and test item treated groups in the ratio or in the litter means of genders on postnatal days 0, 4 or 13.
There were no statistically significant differences between the control and test item treated (100, 300 or 750/1000 mg/kg bw/day) groups in the survival indices, however the survival indices of the 750/1000 mg/kg bw/day on postnatal day 0 and 4 were slightly lower than the control (post-natal day 0: control: 100%, high dose: 94%; post-natal day 4: control: 98%, high dose: 93%), but 4 out of the total death in the high dose were in the same litter. Thus, it was considered to have no toxicological relevance. The mean number of liveborns was statistically significantly lower than in the control group at 750/1000 mg/kg bw/day on postnatal day 0. Similarly, the mean number of viable pups per litter was statistically significantly lower than in the control group at 750/1000 mg/kg bw/day on postnatal days 0 and 4.

Key result

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Generation:

F1

Effect level:

>= 750 - <= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: highest dose tested

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Table 1: SUMMARY DATA OF REPRODUCTIVE ABILITY (FEMALE)

Values		Control	Group (mg/kg bw/day) 100                     300 750/1000
No. of females paired		12	12	12	10
Unmated females	N	0	0	0	0
	%	0	0	0	0
Sperm positive females	N	12	12	12	10
	%	100	100	100	100
Non-pregnant females	N	2	1	2	0
	%	17	8	17	0
Pregnant females	N	10	11	10	10
	%	83	92	83	100	NS
Dams delivered	N	10	11	10	9	NS
	%	100	100	100	90
Pregnants	N	10	11	10	9
with liveborn(s)	%	100	100	100	100
Pregnants	N	0	0	0	0
with stilborns only	%	0	0	0	0
Pregnants	N	0	0	0	1
not delivered	%	0	0	0	10
Precoital interval (days)	Mean	1.3	1.9	1.1	2.6
	SD	1.4	2.5	1.2	2.1
	N	12	12	12	10	NS
Conceiving days	Mean	2.5	2.8	2.3	3.6
	SD	1.4	2.6	1.2	2.1
	N	10	11	10	10	NS
Copulatory index	%	100	100	100	100	NS
Fertility index	%	83	92	83	100 **
Gestation index	%	100	100	100	90

Remarks: High dose was reduced from 1000 mg/kg bw/day to 750 mg/kg bw/day on Day 21.
* = p< 0.05 CH2

**= p < 0.01 CH2

U = Mann-Whitney U-Test Versus Control
DN = Duncan's Multiple Range Test

Table 2: SUMMARY DATA OF REPRODUCTIVE ABILITY (MALE)

Values		Control	Group (mg/kg bw/day) 100                     300		750/1000
No. of males paired		12	12	12	10
Not mated males	N	0	0	0	0
	%	0	0	0	0
Mated males	N	12	12	12	10
	%	100	100	100	100
Fertile males	N	10	11	10	10	NS
	%	83	92	83	100
Infertile males	N	2	1	2	0	NS
	%	17	8	17	0
Copulatory index	%	100	100	100	100	NS
Fertility index	%	83	92	83	100	**

Remarks: High dose was reduced from 1000 mg/kg bw/day to 750 mg/kg bw day on Day 21.

** = p< 0.01 CH2

 

Table 3: Tabular summary

	VALUES PER GROUP
OBSERVATIONS	Control	100	300	750/1000
		mg/kg bw/day	mg/kg bw/day	mg/kg bw/day
Pairs started (n)	12	12	12	10
Estrous cycle — mean length (days) *	4.0	3.9	4.0	6.8
- frequency of irregular cycle **	1/12	2/12	1/12	8/10
Females showing evidence of copulation (n)	12	12	12	10
Females achieving pregnancy (n)	10	11	10	10
Conceiving days 0 - 5 (n)	10	10	10	8
Conceiving days 6 - 7 (n)	0	1	0	2
Pregnancy - 21 days (n)	3	5	1	3
Pregnancy = 22 days (n)	7	5	9	5
Pregnancy 1 23 days (n)	0	1	0	1
Dams with live young born (n)	10	11	10	9
Dams with live young at day 4 pp (n)	10	11	10	9
Implants dam (mean: n)	12.0	10.4	11.1	9.8
Live pups dam at birth (mean: n)	10.9	9.7	10.2	7.8
Live pups/dam at day 4 (mean; n)	10.7	9.4	10.1	7 2
Sex ratio (m f) at birth (mean: n/n)	4.7/6.2	4.5/5.3	4.8/5.4	3.1/4.7
Sex ratio (m f) at day 4 (mean: n/n)	4.7/6.0	4.3/5.1	4.8/5.3	2.8/4.4
Litter weight at birth (mean; g)	67.3	58.2	62.6	47.4
Litter weight at day 4 (mean; g)	113.9	105.8	106.1	83.1
Pup weight at birth (mean; g)	6.3	6.0	6.2	6.5
Pup weight at day 4 (mean; g)	11.0	10.4	10.7	11.5
Pup weight at day 4 (mean; g; m/f)	10.7/10.6	10.5/10.1	10.8/10.3	12.0/11.2
Normalized anogenital distance at day 4 (mean: mm: m/f)	2.9/1.8	2.8/1.7	2.8/1.7	2.8/1.8
Pup weight at day 13 (mean; g)	30.5	29.1	29.3	28.8
Number of nipples - male pups at day 13 (mean;n)	0	0	0	0
ABNORMAL PUPS
Dams with 0	0	0	0	0
Dams with 1	0	0	0	0
Dams with ≥2	0	0	0	0
LOSS OF OFFSPRING
Pre-natal / post-implantations (implantations minus live births)
Females with 0	4	6	3	1
Females with 1	2	3	6	3
Females with 2	3	2	0	2
Females with 3	1	0	1	4
Post-natal (live births minus alive at post-natal day 13)
Females with 0	8	9	9	6
Females with 1	2	0	1	2
Females with 2	0	2	0	0
Females with 3	0	0	0	1

Remark: * = estrous cycle examined during the pre-mating (treatment) period

** = frequency of irregular cycle = number of animals with irregular cycle/number of animals examined
n = number of dams or pups

g  = gram

m = male

f  = female

Two male (on Day 6 and 20) and two female (on Day 3 and 17) animals died in the high dose.
High dose was reduced from 1000 mg/kg bw/day to 750 mg/kg bw/day on Day 21.

Table 4: SUMMARY OF DELIVERY DATA OF DAMS

			Group (mg/kg bw/day)
Values		Control	100	300	750 / 1000
No. of pregnants	N	10	11	10	10
No of dams delivered	N	10	11	10	9
No. of Implantation	Sum	120	114	111	88
	N	10	11	10	9
Post-implantation loss	Sum	11	7	9	18 *
	N	10	11	10	9
	%	9	6	8	20
No. of implantation	Mean	12.0	10.4	11.1	9.8
	SD	2.8	2.7	2.6	1.8
	N	10	11	10	9            NS
Post-implantation loss	Mean	1.1	0.6	0.9	2.0
	SD	1.1	0.8	0.9	1.4
	N	10	11	10	9            NS

Remarks:

* = p < 0.05 CH2

High dose was reduced from 1000 mg/kg bw/day to 750 mg/kg bw/day on Day 21

 

Table 4: SUMMARY OF DELIVERY DATA OF DAMS (continued)

Values		Control	Group (mg/kg bw/dav) 100                    300		750 / 1000
No. of pregnants	N	10	11	10	10
Dams delivered	N	10	11	10	9
	%	100	100	100	90
Dams with Liveborns	N	10	11	10	9
	%	100	100	100	100
Dams with stillborns only	N	0	0	0	0
	%	0	0	0	0
Duration of	Mean	22.20	22.12	22.24	22.41
pregnancy (days)	SD	0.37	0.57	0.31	0.55
	N	10	11	10	9	NS
Dams	N	0	0	0	0
with prolonged pregnancy	%	0	0	0	0
No. of Total Births	Sum	109	107	102	75
	N	10	11	10	9
No. of Liveborns	Sum	109	107	102	70
	N	10	11	10	9
Live birth index	%	100	100	100	93
No. of Stillborns	Sum	0	0	0	5 **
	N	10	11	10	9
	%	0	0	0	7
No. of Viable Pups (Day 0)	Sum	109	107	102	66
	N	10	11	10	9
	%	100	100	100	94
No. of Total Births/Litter	Mean	10.90	9.73	10.20	8.33
	SD	3.07	3.20	2.70	1.87
	N	10	11	10	9	NS
No. of Liveborns /Litter	Mean	10.90	9.73	10.20	7.78
	SD	3.07	3.20	2.70	1.09
	N	10	11	10	9*	U
No. of Stillborns / Litter	Mean	0.00	0.00	0.00	0.56
	SD	0.00	0.00	0.00	1.33
	N	10	11	10	9	NS
No. of Viable Pups/Litter (Day 0)	Mean	10.90	9.73	10.20	7.33
	SD	3.07	3.20	2.70	1.32
	N	10	11	10	9 *	DN

Remarks:

* = p < 0.05 CH2
** = p<0.01 CH2
U = Mann-Whitney U-Test Versus Control
DN = Duncan's Multiple Range Test

High dose was reduced from 1000 mg/kg bw/day to 750 mg/kg bw/day on Day 21

 

Conclusions:

In an OECD TG 422 study the test item was administered by oral gavage in Hsd.Han: Wistar rats and caused mortality (2/12 males and 2/12 females) at 1000 mg/kg bw/day. At 750/1000 mg/kg bw/day, clinical signs (male and female), reduced body weight development (male) and food consumption (male and female), irregular estrous cycle (female), changed delivery data of dams, changes in stomach mucosa (male and female) indicative of localized inflammation of the non-glandular area were observed (male and female). 300 mg/kg bw/day caused clinical signs, which recover shortly after administration and alterations in the stomach mucosa (male and female) indicative of localized inflammation of the non-glandular area. The development of the F1 offspring was not impaired from birth to post-natal day 13 at any dose level after repeated oral administration of dams.
Based on these observations the NOAEL for systemic toxicity of male and female rats was 300 mg/kg bw/day, the NOAEL for local toxicity was 100 mg/kg bw/day, the NOAEL for reproductive performance of male and female rats was 300 mg/kg bw/day and the NOAEL for developmental toxicity 750/1000 mg/kg bw/day.

Executive summary:

A combined repeated dose toxicity study with the reproduction/developmental toxicity screening test was performed obtain initial information on the toxic potential of the test item and its possible effects on male and female reproductive performance such as gonadal function, mating behavior, conception, pregnancy, parturition as well as development of the F1 offspring from conception to day 13 post-partum when repeatedly administered orally (by gavage) to parental animals at doses of 100 mg/kg bw/day, 300 mg/kg bw/day and 750/1000 mg/kg bw/day compared to control animals.

The high dose was reduced from 1000 mg/kg bw/day to 750 mg/kg bw/day on Day 21 due to unexpected toxicity of the test item at the high dose.

Four groups of Hsd.Han:Wistar rats (n=12/sex/group) were administered with the test item orally (by gavage) once a day at 0 (vehicle), 100, 300 and 750/1000 mg/kg bw/day doses corresponding to concentrations of 0, 20, 60 and 150/200 mg/mL. The application volume was 5 mL/kg bw. Control animals received the vehicle, distilled water.

The suitability of the vehicle at the intended concentrations of the test item was analytically verified up front. The concentration of the test item in the dosing formulations was checked two times during the study. Test item concentrations in the dosing formulations varied between 97 % and 103 % of the nominal values confirming the proper dosing.

All animals of the parent (P) generation were dosed prior to mating (14 days) and throughout mating (except Day 20). In addition, males received the test item or vehicle after mating up to the day before the necropsy (altogether for 42 days). Females were additionally exposed through the gestation period and up to lactation days 13-20, up to the day before necropsy (altogether for 54-75 days). Animals (male and female) were not administered one day before the dose reduction (Day 21). Observations included mortality, clinical signs, body weight, food consumption, mating, pregnancy and delivery process, as well as development of offspring. Five dams and the male mating partners were randomly selected from each group to examine further signs of toxicity such as functional observations, hematology, clinical chemistry, gross necropsy, organ weighing and histopathology. Estrous cycle was monitored by examining vaginal smears for two weeks before the treatment and for two weeks from the beginning of the treatment period (two weeks pre-mating period) and during the mating period until evidence of mating.

The dams were allowed to litter and rear their offspring up to day 13 post-partum. Litters were weighed and offspring were observed for possible abnormalities and were euthanized on postnatal day 13 or shortly thereafter.

Blood samples were collected for determination of serum levels of thyroid hormones (T4) from at least two pups per litter (where it was feasible) on post-natal day 4, from all dams and at least two pups per litter at termination on post-partum/post-natal day 13 and from all parent male animals at termination.

All parental animals were subjected to gross pathology one day after the last treatment. The body weight, brain weight, weight of the testes, epididymides and prostate and seminal vesicles with coagulating glands as a whole of all adult male animals were determined.

Histopathology examination was performed on reproductive organs (testes, epididymides, uterus and ovaries) in the control and high dose groups. In addition, these organs were processed and examined histologically in non-pregnant or not delivered females and males these females cohabited with in the low and middle dose groups. Additionally, full histopathology examinations were performed on the organs and tissues of parental animals (male and female) selected for general toxicological examinations in the control and high dose groups including dead animals.

In addition, organs showing macroscopic findings were processed and examined histologically in animals of the low and mid dose groups based on the macroscopic findings at the necropsy.

The results were interpreted comparing treatment groups with respect to controls, which were treated concurrently with vehicle (distilled water) only. Historical control data were also considered.

Test item related mortality was observed (two males and females) at 1000 mg/kg bw/day groups during first 20 days of the study. Histological examinations revealed local inflammation in the non-glandular area of stomach accompanied with ulceration and leading to fatal death of these animals.

Test item related clinical signs were observed at 300 and 750/1000 mg/kg bw/day (male and female). Nuzzling up the bedding material and salivation were findings with the highest incidence in male and female animals of these groups. Most of signs – piloerection, hunched back, prone position, narrow eye aperture, dyspnea, decreased activity, noisy breathing, wet, yellowish bedding material, faeces around the anus – ceased in most of animals within 10 days after the dose reduction. Male animals proved to be more sensitive than females on the basis of the number and incidence of signs at 750/1000 mg/kg bw/day.

Some clinical signs were observed in four male and two female animals at 750/1000 mg/kg bw/day as well as in one dam at 300 mg/kg bw/day at the detailed weekly observation indicating that these animals did not fully recover 24 hours after the daily administration (piloerection or noisy breathing in male animals and piloerection or decreased activity or noisy breathing in female animals). Additionally, noisy breathing was noted for one female animal at 300 mg/kg bw/day on Gestation day 0.

Functional observations revealed test item or treatment related noisy breathing in one female animal at 750/1000 mg/kg bw/day group.

The body weight gain and body weight were reduced in male animals at 750/1000 mg/kg bw/day.

The food consumption was lower than in the control group in male and female animals at 750/1000 mg/kg bw/day.

An influence on the estrous cycle was detected at 1000 mg/kg bw/day prior to dose reduction in the form of a significant lower percentage of animals with regular estrus cycle, primarily due to a higher mean number of animals with prolonged diestrous stage and subsequent impairment of the other estrus stages. Noteworthy to mention that these effects occurred only at 1000 mg/kg bw/day which was associated with mortalities. Furthermore,histopathologyof the female reproductive system (ovaries, uterus with cervix, vagina)showed normal structure characteristics of the species, age and phase of the active sexual cycle in all cases. The cortical region of ovaries contained primary, secondary and tertiary follicles and corpora lutea, indicating the active maturation of oocytes, and ovulation. Theepithelial capsule and ovarian stroma was normal in all cases as well.

The examined parameters of the reproductive performance were comparable in the control and test item treated groups.

Higher percentage and higher mean of post-implantation loss and stillborns, lower mean number of liveborns and lower mean number of viable pups per litter on post-natal day 0 were observed at 750/1000 mg/kg bw/day treated animals.

There were no test item related adverse changes in the examined hematological parameters in male or female animals at 100, 300 or 750/1000 mg/kg bw/day. Elevated mean percentage of neutrophils in male and female animals at 750/1000 mg/kg bw/day was related to gastric changes (local inflammation in the non-glandular area).

Evaluation of the clinical chemistry parameters did not reveal pathologic changes in the examined biochemical parameters.

There were no test item related changes in the serum thyroid hormone levels at any dose (male or offspring).

Macroscopic finding (thickened mucous membrane in the stomach) related to test item was found in male and female animals at 300 and 750/1000 mg/kg bw/day

There were no test item related changes in the weights (absolute and relative to body or brain weights) of brain, testes and epididymides of male animals at any dose level.

Test item related toxic changes were not detected in the weights of examined organs of selected animals at any dose levels.

Histopathological examinations of the investigated reproductive organs (ovaries, uterus, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland) did not reveal any test item related changes at 750/1000 mg/kg bw/day.

In the stomach of male or female animals at 300 and 750/1000 mg/kg/bw/day inflammation of the non-glandular area was observed.

No adverse effect on the mortality, clinical signs body weight development or necropsy findings were detected in the offspring terminated as scheduled. The anogenital distance (male and female) or nipple retention (male) were not affected.

Under the conditions of the present study the test item caused mortality (2/12 males and 2/12 females) at 1000 mg/kg bw/day. At 750/1000 mg/kg bw/day, clinical signs (male and female), reduced body weight development (male) and food consumption (male and female), irregular estrous cycle (female), changed delivery data of dams, changes in stomach mucosa (male and female) indicative of localized inflammation of the non-glandular area were observed (male and female). 300 mg/kg bw/day caused clinical signs, which recover shortly after administration and alterations in the stomach mucosa (male and female) indicative of localized inflammation of the non-glandular area. The development of the F1 offspring was not impaired from birth to post-natal day 13 at any dose level after repeated oral administration of dams.

Based on these observations the NOAEL for systemic toxicity of male and female rats was 300 mg/kg bw/day, the NOAEL for local toxicity was 100 mg/kg bw/day, the NOAEL for reproductive performance of male and female rats was 300 mg/kg bw/day and the NOAEL for developmental toxicity 750/1000 mg/kg bw/day.

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2018

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EPA Health Effects Test Guidelines: OPPTS 870.3650 Combined Repeated Dose Toxicity with the Reproduction/Developmental Toxicity Screening Test

Version / remarks:

July 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Toxi-Coop Zrt. Berlini utca 47-49. 1045 Budapest Hungary, Arácsi út 97. H-8230 Balatonfüred, Hungary

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of lauric acid, compound with morpholine (1:1) and 2-ethylhexyl dihydrogen phosphate, compound with morpholine (1:2) and bis(2-ethylhexyl) hydrogen phosphate, compound with morpholine (1:1)

EC Number:

915-372-8

Molecular formula:

not applicable (multi-const.substance)

IUPAC Name:

Reaction mass of lauric acid, compound with morpholine (1:1) and 2-ethylhexyl dihydrogen phosphate, compound with morpholine (1:2) and bis(2-ethylhexyl) hydrogen phosphate, compound with morpholine (1:1)

Test material form:

liquid

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Hsd.Han

Details on species / strain selection:

The rat is regarded as suitable species for reproduction studies and the test guideline is designed to use the rat. The Wistar rat was selected due to a wide range of experience with this strain of rat in reproduction toxicity studies and well-known fertility parameters.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Toxi-Coop Zrt. Cserkesz u. 90. H-1103 Budapest, Hungary
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 81-84 days old males and females
- Weight at study initiation: (P) Males: 309-390 g; Females: 198-250 g
- Fasting period before study: no
- Housing: Type III polypropylene/polycarbonate
Before mating: 2 animals of the same sex/cage
Mating: 1 male and 1 female/cage
Pregnant females were housed individually.
Males after mating: 2 animals/cage
- Diet: ad libitum, ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest, Germany
- Water: ad libitum, tap water from municipal supply
- Acclimation period: at least 20 days

DETAILS OF FOOD AND WATER QUALITY: The food is considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study. The drinking water is periodically analyzed and is considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): above 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

distilled

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in the vehicle in concentrations of 150/200, 60 and 20 mg/mL. High dose was reduced on Day 21, thus concentration of the high dose formulation was also reduced from 200 to 150 mg/mL. A constant treatment volume of 5 mL dose preparation/kg body weight was administered in all groups. The individual volume of the treatment based on the most recent individual body weight measurement of the animals. In the first week of the pre-mating period, animals received volumes based on the actual body weight on Day 0.
VEHICLE
The suitability of the chosen vehicle for the test item at the intended concentrations was analytically verified up front (Toxi-Coop study no: 685-100-1247). A sufficient stability and homogeneity in the chosen vehicle were verified over the range of relevant concentrations at the appropriate frequency of preparation. (Recovery from distilled water: 96% and 99 % of nominal concentrations at ca. 1 mg/mL and ca. 400 mg/mL; the substance preparation was stable at the intended concentrations for at least one day at room temperature and for three days in refrigerator.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of formulations was performed in the Analytical Laboratory of Test Facility. Five samples were taken from different places from each concentration (Groups 2, 3 and 4) and were measured on 2 occasions, during the first and last treatment weeks. Similarly, five samples were taken from the control solution (Group 1) from different places and analyzed. Concentration of the test item in the dosing formulations varied in the range of 97 and 103 % of the nominal values.

Duration of treatment / exposure:

Males received the test item or vehicle after mating up to the day before the necropsy (altogether for 42 days). Females were additionally exposed through the gestation period and up to lactation days 13-20, up to the day before necropsy (altogether for 54-75 days). Animals (male and female) were not administered one day before the dose reduction (Day 21).

Frequency of treatment:

daily

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.:

750 mg/kg bw/day (nominal)

Remarks:

The high dose is reduced from 1000 mg/kg bw/day to 750 mg/kg bw/day on Day 21 because 1000 mg/kg bw/day was lethal for two male and two female animals.

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose setting is based on findings obtained in a 14-day dose range finding study performed with the substance (study no. 685-400-1237, non-GLP).

Positive control:

none

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once a day, after treatment
Observations were performed on the skin, fur, eyes and mucous membranes, 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. Special attention was directed towards the observation of tremors, convulsions, salivation, diarrhea, lethargy, sleep and coma. The mating period was different for individual animals, therefore clinical signs were evaluated by phases as follows:
-Male animals: pre-mating, mating and post-mating period: from Day 0 up to the day before the necropsy;
-Dams: pre-mating (From Day 0 up to day of positive vaginal smear), gestation and lactation periods;
-non-pregnant and not delivered females: pre-mating and post-mating periods

BODY WEIGHT: Yes
- Time schedule for examinations: Parental males were weighed on the first day of dosing (Day 0), weekly thereafter and on the day of necropsy. Male animals in the high dose were weighed daily for a period (from Day 2 or 3 to Day 22) due to their poor condition and ensuring more appropriate dosing. Parental females were weighed on the first day of dosing (Day 0) then weekly, on gestation days 0, 7,14 and 21 and on post-partum day 0 (within 24 hours after parturition), 4 and 13. Additionally, female animals were weighed on gestational day 10 in order to give accurate treatment volumes, but these data were not evaluated statistically. Some female animals in the high dose was also weight daily due to their poor condition and ensuring more appropriate dosing.

FOOD CONSUMPTION AND COMPOUND INTAKE :
The food consumption was determined weekly by reweighing the non-consumed diet with an accuracy of 1 g during the treatment period except mating phase: premating Days 0, 7 and 13 and by weekly interval during post-mating period for male animals; premating Days 0, 7 and 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
- Anaesthetic used for blood collection: Yes, Isofluran CP anesthesia
- Animals fasted: Yes, Animals were food deprived for approximately 16 hours (overnight) prior to blood collection.
- How many animals: five male and five female animals randomly selected from each group
- Parameters checked: WBC (White Blood Cell leukocyte count), RBC (Red Blood Cell erythrocyte count), HGB (Hemoglobin concentration), HCT (Hematocrit, relative volume of erythrocytes), MCV (Mean Corpuscular erythrocyte Volume), MCH (Mean Corpuscular erythrocyte Hemoglobin), MCHC (Mean Corpuscular erythrocyte Hemoglobin Concentration), PLT (Platelet thrombocyte count), RET (Reticulocytes),Differential white blood cell count, APTT (Activated partial Thromboplastin Time), PT (Prothrombin Time)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: one day after the last treatment
- Animals fasted: Yes, Animals were food deprived for approximately 16 hours (overnight) prior to blood collection.
- How many animals: five male and five female animals randomly selected from each group
- Parameters checked: ALT (Alanine Aminotransferase activity), AST (Aspartate Aminotransferase activity), TBIL (Total Bilirubin concentration), CREA (Creatinine concentration), UREA (Urea concentration), GLUC (Glucose concentration),CHOL (Cholesterol concentration), Na+(Sodium concentration),K+ (Potassium concentration), ALB (Albumin concentration), TPROT (Total Protein concentration)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during the last exposure week but before the blood sampling
- Dose groups that were examined: each group
- Battery of functions tested: Sensory reactivity to different type of stimuli (e.g. auditory, visual and proprioceptive), assessment of grip strength and motor activity

IMMUNOLOGY: No

OTHER:

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera. The appearance of the tissues and organs were observed, and any abnormality was recorded including details of the location, color, shape and size. Special attention was paid to the organs of the reproductive system.

HISTOPATHOLOGY: Yes
Detailed histological examinations were performed on the ovaries, uterus, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland in the control and high dose groups with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure; on the ovaries covering the follicular, luteal, and interstitial compartments of the ovary as well as the epithelial capsule and ovarian stroma. In addition, these organs were processed and examined histologically in non-pregnant or not delivered females and males these females cohabited with in the low and middle dose groups. Full histopathology examinations were performed on the preserved organs and tissues of the randomly selected animals (n=5 animals/sex/group) in the control and high dose groups. In addition, stomach was also processed and examined histologically in four males and one female from the 300 mg/kg bw/day group due to necropsy findings (thickened or erythematous mucosa). Seminal vesicle with coagulating gland was also processed and examined for one male at 100 mg/kg bw/day due to the necropsy observation (smaller than normal, one side). Uterus of three females in the mid dose group was processed and examined as well due to macroscopic finding (hydrometra). The following organs were preserved: Adrenal glands, Aorta, Bone with marrow and joint (femur), Brain (representative regions: cerebrum, cerebellum and pons and medulla oblongata),
Eyes (lachrymal gland with Harderian glands), Female mammary gland, Gonads (testes with epididymides, ovaries, uterus with vagina), Gross lesions, Heart, Kidneys, Large intestines (caecum, colon, rectum, including Peyer’s patches), Liver, Lungs (with main stem bronchi; inflation with fixative and then immersion), Lymph nodes (submandibular, mesenteric), Muscle (quadriceps), Esophagus, Pancreas, Pituitary, Prostate, Salivary glands (submandibular), Sciatic nerve, Seminal vesicle with coagulating gland, Skin, Small intestines(representative regions: duodenum, ileum, jejunum), Spinal cord (at three levels: cervical, mid-thoracic and lumbar), Spleen, Sternum, Stomach, Thymus, Thyroid +parathyroid, Trachea, Urinary bladder.

Other examinations:

DETERMINATION OF SERUM LEVELS OF THYROID HORMONES
Blood samples were collected for determination of serum levels of thyroid hormones (T4) as follows:
-from all parent male animals at termination

Statistics:

The statistical evaluation of appropriate data were 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) was 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.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

In dead animals, nuzzling up the bedding material (2/2 male, 2/2 female), salivation (2/2 male, 2/2 female), decreased activity (2/2 male, 1/2 female), piloerection (2/2 male, 1/2 female) and dyspnea (2/2 male, 1/2 female), signs of diarrhea (faeces around the anus, 1/2 male) and polyuria (wet, yellowish bedding material 2/2 male) were observed during the days before the death.
Test item related clinical signs were observed at 300 and 750/1000 mg/kg bw/day (male and female). Nuzzling up the bedding material and salivation were findings with the highest incidence in male and female animals of these groups. Most of signs – piloerection, hunched back, prone position, narrow eye aperture, dyspnea, decreased activity, noisy breathing, wet, yellowish bedding material, faeces around the anus – ceased in most of animals within 10 days after the dose reduction. Male anim
als proved to be more sensitive than females on the basis of the number and incidence of signs at 750/1000 mg/kg bw/day.Some clinical signs were observed in four male and two female animals at 750/1000 mg/kg bw/day as well as in one dam at 300 mg/kg bw/day at the detailed weekly observation indicating that these animals did not fully recover 24 hours after the daily administration (piloerection or noisy breathing in male animals and piloerection or decreased activity or noisy breathing in female animals). Additionally, noisy breathing was noted for one female animal at 300 mg/kg bw/day on Gestation day 0.There were no clinical signs during the course of the entire study in animals of control (12/12 male and 12/12 female) and 100 mg/kg bw/day group (12/12 male and 12/12 female), i.e. these parental animals exhibited normal behavior and physical condition with no abnormalities during the treatment period.

Mortality:

mortality observed, treatment-related

Description (incidence):

Two male and two female animals at 1000 mg/kg bw/day were found dead on Day 3, on Day 6, on Day 17 and on Day 20. There was no mortality in control, 100, 300 or 750 mg/kg bw/day groups (later after Day 21; male or female) during the course of study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The body weight gain and body weight were reduced in male animals at 750/1000 mg/kg bw/day. The mean body weight gain was slightly lower with respect to their control in male animals at 300 and at 100 mg/kg bw/day reaching statistical significance between Days 34 and 41, and in male animals at 300 mg/kg bw/day for the study overall (between Day 0 and 42). These changes however did not result in significant differences to their control in the mean body weight at 100 and 300 mg/kg bw/day therefore were considered to be toxicologically relevant. There were no statistically significant differences between the control and test item treated groups in the mean body weight of female animals at
100, 300 during the pre-mating period. The mean body weight and body weight gain was similar in the control and test item treated female animals at 750/1000 mg/kg bw/day during the pre-mating period. The mean body weight gain of female animals at 750/1000 mg/kg bw/day was slightly lower with respect to the control at between gestation days 14-21 and 0-21, which however did not result in significant changes in the mean body weight value of these animals. The body weight development was similar in the control and test item administered dams during the lactation period.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The food consumption was lower than in the control group in male and female animals at 750/1000 mg/kg bw/day. There were no toxicologically relevant differences in the mean daily food consumption of male and female animals in the control and of 100 and 300 mg/kg bw/day groups during the entire study. Although, statistical significance was noted for the slightly lower mean daily food consumptio in female animals at 300 mg/kg bw/day between gestation days 7-14, this slight change was transient and judged to be toxicologically not significant.

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):

There were no test item related adverse changes in the examined hematological parameters in male or female animals at 100, 300 or 750/1000 mg/kg bw/day. Slight elevation of mean percentage of neutrophil granulocytes in male and female animals at 750/1000 mg/kg bw/day was probably an accompanying phenomenon of changes in gastric mucosa. Statistical significance was noted for the lower mean percentage of lymphocytes (LYM) and for the higher mean percentage of neutrophils (NEU) in male animals at 750/1000 mg/kg bw/day. In female animals at 750/1000 mg/kg bw/day, the mean percentage of neutrophils was higher and the mean corpuscular (erythrocyte) hemoglobin concentration (MCHC) and mean activated partial thromboplastin time were lower than in the control group. The individual values of these parameters (NEU, LYM, RET, MCHC and APTT) were within the historical control range and were judged to be toxicologically not relevant as there was no dose dependence (RET, APTT) and findings were with minor degree.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Evaluation of the clinical chemistry parameters did not reveal pathologic changes in the examined biochemical parameters.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Functional observations did not demonstrate any test item related changes at 100 and 300 mg/kg bw/day in male and female animals and at 750/1000 mg/kg bw/day in male animals. The behavior, physical condition and reactions to different type of stimuli of animals selected for examination were considered to be normal in these groups.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

The absolute and relative (to body weight; to brain weight) weights of examined organs did not demonstrate any test item related adverse alterations in male or female animals at 100, 300 or 750/1000 mg/kg bw/day. Observed changes were not considered to be toxicologically relevant in the lack of associated histopathological alterations.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Macroscopic findings related to the effect of the test item were detected in male and female animals at 300 and 750/1000 mg/kg bw/day (thickened mucous membrane in the stomach).
Two male and two female animals administered with 1000 mg/kg bw/day were found dead on Days 6 and 20, and on Days 17 and 3, respectively.
Gross pathology examination of these animals revealed the following findings:
Males:
-stomach: erythematous mucosa at the cardia (1/2); grey-black colored at the cardia (1/2), full with gas (2/2)
-intestines: full with gas (2/2); brownish-reddish mucilagionus fluid content (1/2) yellow-red fluid content (1/2)
-seminal vesicle, prostate: smaller than normal (1/2)
-spleen: smaller than normal (1/2)
Females:
-stomach: dark discoloration at the cardia (1/2); slightly erythematous mucosa at the cardia (1/2); full with gas (1/2)
-intestines: yellowish-brownish mucilagionus fluid content (1/2); full with gas (1/2)
-lungs: spongy texture (1/2)
-liver: dark red (1/2)
Findings in the gastrointestinal tract are clearly related to the test item treatment and findings related to the stomach were due to inflammation of the non-glandular area of the stomach. The following findings were observed in the surviving animals of 300 and 750/1000 mg/kg bw/day at the gross pathology examination:
Males:
-stomach: thickened, yellow colored mucosa at the cardia or at the pylorus (10/10 at high dose, 4/12 at mid dose); erythematous mucosa at the fundus (2/10 at high dose);
-testes, epididymides: smaller than normal (1/10 at high dose)
-seminal vesicle: smaller than normal, right side (1/12 in low dose, 1/10 in high dose)
-thymus: hemorrhage (2/12 at control)
Dams:
-stomach: erythematous mucosa (1/10 at mid dose, 2/9 at high dose); thickened, grey-yellow colored mucosa at the cardia (8/9 at high dose);
-lungs: moderate emphysema (1/9 at high dose)
-uterus: hydrometra (2/10 at control, 3/9 at mid dose)
Not delivered female at the high dose:
-stomach: thickened, yellow colored mucosa at the cardia (1/1)
-stomach, intestines: full with gas (1/1)
Findings in the gastrointestinal tract are clearly related to the test item treatment and findings related t
o the stomach were due to inflammation of the non-glandular area of the stomach.
Hemorrhage in the thymus was probably due to circulatory disturbance developed during exsanguination procedure.
Hydrometra, related to the female sexual cycle, is a frequent observation in experimental rats. In the lack of related histopathological alterations (inflammatory or other pathological lesion) these were judged not to be toxicologically relevant.
Smaller than normal seminal vesicle and prostate or epididymides are common findings in experimental rats of this strain with similar age.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histopathological examinations revealed test item related alterations in the stomach of male and female animals at 300 and 750/1000 mg/kg/bw/day (inflammation of the non-glandular area). Dead animals at 1000 mg/kg bw: Histological examination revealed inflammation of the stomach (nonglandular area) and ulceration of mucous membrane in all cases. Inflammation was accompanied with squamous cell hyperplasia, infiltration of inflammatory cells and fibroblast proliferation in the lamina
propria.In addition, congestion and alveolar emphysema (grade 3) was seen in the lungs, in all four cases, in connection with agony.In one of the dead male animals lymphocyte depletion occurred in the spleen which is in good correlation with the macroscopic observations (smaller than normal spleen). In this animal decreased amount of secretum in the seminal vesicles was also observed. Terminal sacrifice: In male animals, the investigated organs of reproductive system (testes, epididymides, prostate and seminal vesicles with coagulating gland) were histologically normal and characteristic on the sexually mature organism except one animal in which decreased amount of
spermatozoa in the epididymides and decreased intensity of spermatogenesis in the testes were observed. 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 histological picture of epididymides, seminal vesicles, and coagulating glands was normal in all animals as well. In the female animals the investigated organs of reproductive system (ovaries, uterus with cervix, vagina) had a normal structure characteristic of the species, age and phase of the active sexual cycle in all cases. 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 was normal in all cases as well. Test item related inflammation of stomach (non-glandular area) accompanied with squamous cell hyperplasia and infiltration of inflammatory cells and fibroblast proliferation in the lamina propria were
observed in all male and female animals belonging to the 750/1000 mg/kg bw/day treated group (10/10 males and 10/10 females) and in some animals from the 300 mg/kg bw/day treated group (4 males and 1 female). No ulceration or dysplasia and down growths of the hyperplastic basal cells (suspected neoplasm formation) were detectable in the altered areas. No inflammatory or hyperplastic lesions were seen in the glandular stomach. There was no morphological evidence of acute or subacute injury (degeneration, inflammation, necrosis, etc.) of the small and large intestines, the liver, the pancreas, the cardiovascular system, the urinary system, the immune system, the hematopoietic system, the skeleton, the muscular system, the male and female reproductive system or the central or peripheral nervous system in the animals. The cytomorphology of the endocrine glands were the same in the control and the treated animals.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Serum Levels of Thyroid Hormones (T4):
There were no statistically significant differences with respect to the control in the thyroid hormone (free T4) level in parental male animals or in offspring sampled on postnatal day 13 at any dose levels.

Details on results:

For results on offspring please refer to IUCLID Section 7.8.1.

Effect levels

1

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

mortality

2

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive performance

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: irregular estrous cycle

3

Key result

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

300 mg/kg bw/day (nominal)

System:

gastrointestinal tract

Organ:

stomach

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Applicant's summary and conclusion

Conclusions:

In an OECD TG 422 study the test item was administered by oral gavage in Hsd.Han: Wistar rats and caused mortality (2/12 males and 2/12 females) at 1000 mg/kg bw/day. At 750/1000 mg/kg bw/day, clinical signs (male and female), reduced body weight development (male) and food consumption (male and female), irregular estrous cycle (female), changed delivery data of dams, changes in stomach mucosa (male and female) indicative of localized inflammation of the non-glandular area were observed (male and female).
300 mg/kg bw/day caused clinical signs, which recover shortly after administration and alterations in the stomach mucosa (male and female) indicative of localized inflammation of the non-glandular area. The development of the F1 offspring was not impaired from birth to post-natal day 13 at any dose level after repeated oral administration of dams.
Based on these observations the NOAEL for systemic toxicity of male and female rats was 300 mg/kg bw/day, the NOAEL for local toxicity was 100 mg/kg bw/day, the NOAEL for reproductive performance of male and female rats was 300 mg/kg bw/day and the NOAEL for developmental toxicity 750/1000 mg/kg bw/day.

Executive summary:

A combined repeated dose toxicity study with the reproduction/developmental toxicity screening test was performed obtain initial information on the toxic potential of the test item and its possible effects on male and female reproductive performance such as gonadal function, mating behavior, conception, pregnancy, parturition as well as development of the F1 offspring from conception to day 13 post-partum when repeatedly administered orally (by gavage) to parental animals at doses of 100 mg/kg bw/day, 300 mg/kg bw/day and 750/1000 mg/kg bw/day compared to control animals.

The high dose was reduced from 1000 mg/kg bw/day to 750 mg/kg bw/day on Day 21 due to unexpected toxicity of the test item at the high dose.

Four groups of Hsd.Han:Wistar rats (n=12/sex/group) were administered with the test item orally (by gavage) once a day at 0 (vehicle), 100, 300 and 750/1000 mg/kg bw/day doses corresponding to concentrations of 0, 20, 60 and 150/200 mg/mL. The application volume was 5 mL/kg bw. Control animals received the vehicle, distilled water.

The suitability of the vehicle at the intended concentrations of the test item was analytically verified up front. The concentration of the test item in the dosing formulations was checked two times during the study. Test item concentrations in the dosing formulations varied between 97 % and 103 % of the nominal values confirming the proper dosing.

All animals of the parent (P) generation were dosed prior to mating (14 days) and throughout mating (except Day 20). In addition, males received the test item or vehicle after mating up to the day before the necropsy (altogether for 42 days). Females were additionally exposed through the gestation period and up to lactation days 13-20, up to the day before necropsy (altogether for 54-75 days). Animals (male and female) were not administered one day before the dose reduction (Day 21). Observations included mortality, clinical signs, body weight, food consumption, mating, pregnancy and delivery process, as well as development of offspring. Five dams and the male mating partners were randomly selected from each group to examine further signs of toxicity such as functional observations, hematology, clinical chemistry, gross necropsy, organ weighing and histopathology. Estrous cycle was monitored by examining vaginal smears for two weeks before the treatment and for two weeks from the beginning of the treatment period (two weeks pre-mating period) and during the mating period until evidence of mating.

The dams were allowed to litter and rear their offspring up to day 13 post-partum. Litters were weighed and offspring were observed for possible abnormalities and were euthanized on postnatal day 13 or shortly thereafter.

Blood samples were collected for determination of serum levels of thyroid hormones (T4) from at least two pups per litter (where it was feasible) on post-natal day 4, from all dams and at least two pups per litter at termination on post-partum/post-natal day 13 and from all parent male animals at termination.

All parental animals were subjected to gross pathology one day after the last treatment. The body weight, brain weight, weight of the testes, epididymides and prostate and seminal vesicles with coagulating glands as a whole of all adult male animals were determined.

Histopathology examination was performed on reproductive organs (testes, epididymides, uterus and ovaries) in the control and high dose groups. In addition, these organs were processed and examined histologically in non-pregnant or not delivered females and males these females cohabited with in the low and middle dose groups. Additionally, full histopathology examinations were performed on the organs and tissues of parental animals (male and female) selected for general toxicological examinations in the control and high dose groups including dead animals.

In addition, organs showing macroscopic findings were processed and examined histologically in animals of the low and mid dose groups based on the macroscopic findings at the necropsy.

The results were interpreted comparing treatment groups with respect to controls, which were treated concurrently with vehicle (distilled water) only. Historical control data were also considered.

Test item related mortality was observed (two males and females) at 1000 mg/kg bw/day groups during first 20 days of the study. Histological examinations revealed local inflammation in the non-glandular area of stomach accompanied with ulceration and leading to fatal death of these animals.

Test item related clinical signs were observed at 300 and 750/1000 mg/kg bw/day (male and female). Nuzzling up the bedding material and salivation were findings with the highest incidence in male and female animals of these groups. Most of signs – piloerection, hunched back, prone position, narrow eye aperture, dyspnea, decreased activity, noisy breathing, wet, yellowish bedding material, faeces around the anus – ceased in most of animals within 10 days after the dose reduction. Male animals proved to be more sensitive than females on the basis of the number and incidence of signs at 750/1000 mg/kg bw/day.

Some clinical signs were observed in four male and two female animals at 750/1000 mg/kg bw/day as well as in one dam at 300 mg/kg bw/day at the detailed weekly observation indicating that these animals did not fully recover 24 hours after the daily administration (piloerection or noisy breathing in male animals and piloerection or decreased activity or noisy breathing in female animals). Additionally, noisy breathing was noted for one female animal at 300 mg/kg bw/day on Gestation day 0.

Functional observations revealed test item or treatment related noisy breathing in one female animal at 750/1000 mg/kg bw/day group.

The body weight gain and body weight were reduced in male animals at 750/1000 mg/kg bw/day.

The food consumption was lower than in the control group in male and female animals at 750/1000 mg/kg bw/day.

An influence on the estrous cycle was detected at 1000 mg/kg bw/day prior to dose reduction in the form of a significant lower percentage of animals with regular estrus cycle, primarily due to a higher mean number of animals with prolonged diestrous stage and subsequent impairment of the other estrus stages. Noteworthy to mention that these effects occurred only at 1000 mg/kg bw/day which was associated with mortalities. Furthermore, histopathology of the female reproductive system (ovaries, uterus with cervix, vagina) showed normal structure characteristics of the species, age and phase of the active sexual cycle in all cases. 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 was normal in all cases as well.

The examined parameters of the reproductive performance were comparable in the control and test item treated groups.

Higher percentage and higher mean of post-implantation loss and stillborns, lower mean number of liveborns and lower mean number of viable pups per litter on post-natal day 0 were observed at 750/1000 mg/kg bw/day treated animals.

There were no test item related adverse changes in the examined hematological parameters in male or female animals at 100, 300 or 750/1000 mg/kg bw/day. Elevated mean percentage of neutrophils in male and female animals at 750/1000 mg/kg bw/day was related to gastric changes (local inflammation in the non-glandular area).

Evaluation of the clinical chemistry parameters did not reveal pathologic changes in the examined biochemical parameters.

There were no test item related changes in the serum thyroid hormone levels at any dose (male or offspring).

Macroscopic finding (thickened mucous membrane in the stomach) related to test item was found in male and female animals at 300 and 750/1000 mg/kg bw/day

There were no test item related changes in the weights (absolute and relative to body or brain weights) of brain, testes and epididymides of male animals at any dose level.

Test item related toxic changes were not detected in the weights of examined organs of selected animals at any dose levels.

Histopathological examinations of the investigated reproductive organs (ovaries, uterus, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland) did not reveal any test item related changes at 750/1000 mg/kg bw/day.

In the stomach of male or female animals at 300 and 750/1000 mg/kg/bw/day inflammation of the non-glandular area was observed.

No adverse effect on the mortality, clinical signs body weight development or necropsy findings were detected in the offspring terminated as scheduled. The anogenital distance (male and female) or nipple retention (male) were not affected.

Under the conditions of the present study the test item caused mortality (2/12 males and 2/12 females) at 1000 mg/kg bw/day. At 750/1000 mg/kg bw/day, clinical signs (male and female), reduced body weight development (male) and food consumption (male and female), irregular estrous cycle (female), changed delivery data of dams, changes in stomach mucosa (male and female) indicative of localized inflammation of the non-glandular area were observed (male and female).

300 mg/kg bw/day caused clinical signs, which recover shortly after administration and alterations in the stomach mucosa (male and female) indicative of localized inflammation of the non-glandular area. The development of the F1 offspring was not impaired from birth to post-natal day 13 at any dose level after repeated oral administration of dams.

Based on these observations the NOAEL for systemic toxicity of male and female rats was 300 mg/kg bw/day, the NOAEL for local toxicity was 100 mg/kg bw/day, the NOAEL for reproductive performance of male and female rats was 300 mg/kg bw/day and the NOAEL for developmental toxicity 750/1000 mg/kg bw/day.