Reaction mixture of hydrogenated tallow alkyl amines with sebacic acid and lithium hydroxide

Endpoint summary

Description of key information

There were no signs of systemic toxicity in selected male or female animals at 100, 200 or 450 mg/kg bw/day by oral gavage.Based on these observations the NOAEL was determined as follows:

NOAEL for systemic toxicity of male/female rats: 450 mg/kg bw/day

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

October 3, 2018 - February 4, 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same 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)

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Han:WIST rat

Details on species / strain selection:

The rat is regarded as suitable species for toxicity and reproduction toxicity studies and the test guidelines were designed to use the rat. The Wistar rat was selected due to a wide range of experience with this strain of rat in toxicity.

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: 97-104 days (males and females)
- Weight at study initiation:
Male animals: 375 – 441 g
Female animals: 212 – 264 g
- Housing: Type III polypropylene/polycarbonate
Before mating: 2 animals of the same sex/cage
Mating hours: 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: 34 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 – 70
- Air changes (per hr): above 10 air-exchanges / hour by central air-condition system
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

methylcellulose

Remarks:

1 %

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of formulations was performed by ICP-OES method. Five samples were taken from different places from each concentration (Groups 2, 3 and 4) and were measured on 2 occasions. 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 within the range of 96 and 106 % of the nominal values at both analytical occasions.

Duration of treatment / exposure:

Dams were dosed for 14 days pre-mating, during mating period, through gestation and up to lactation days 13-16 (altogether for 51-71 days depending on day of mating). The day of delivery (when parturition was complete) was defined as day 0 post-partum. Male animals were dosed for 42 days (14 days pre-mating period, mating period an optional extended post-mating period) and then subjected to necropsy one day after the last treatment on Day 42.

Frequency of treatment:

7 days/week

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

200 mg/kg bw/day (nominal)

Dose / conc.:

450 mg/kg bw/day (nominal)

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 test item.

Positive control:

no

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- All parental animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each day). General clinical observations were made once a day, after treatment at approximately the same time.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily
Detailed clinical observations were made on all animals outside the home cage in a standard arena once prior to the first exposure and once weekly thereafter.
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.
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.

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. 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. Body weight was measured on day of necropsy for animals subjected to organ weighing (all male animals and females selected for further examinations).

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

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: one day after the last treatment
- Anaesthetic used for blood collection: Yes, Isofluran anesthesia
- Animals fasted: Yes
- 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, Isofluran anesthesia
- 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: Limited to observations described under "Detailed clinical observations".

IMMUNOLOGY: No

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

Sacrifice and pathology:

GROSS PATHOLOGY
After examination of the external appearance the cranial, thoracic and abdominal cavities were opened and 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. The ovaries, uterus with cervix, vagina, testes, epididymides (total and cauda), prostate, and seminal vesicles with coagulating glands, and all organs showing macroscopic lesions of all adult animals were preserved. Testes and epididymides were fixed in modified Davidson solution, all other organs in 4 % buffered formaldehyde solution. All organs showing macroscopic lesions and the following organs were preserved for dead or moribund animals, for five male and five female animals randomly selected from each group.

HISTOPATHOLOGY
Detailed histological examinations were performed on the sexual organs (ovaries, uterus with oviduct, cervix, 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. 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 and in dead and moribund animals. Some organs of dead animal were missing due to cannibalism (abdominal aorta, intestines, mesenteric lymph nodes, pancreas, prostate, seminal vesicle, skin, spleen, urinary bladder). In addition, histological examinations were performed in organs showing macroscopic findings: thymus (1 female at 100 mg/kg bw/day; 2 males at 200 mg/kg bw/day), kidneys (1 male at 100 mg/kg bw/day; 2 at 450 mg/kg bw/day), skin (1 female at 100 mg/kg bw/day); sexual organs (1 female at 200 mg/kg bw/day), sexual organs, kidneys, liver and pancreas in one non-pregnant female at 200 mg/kg bw/day.

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, fasted body weight, adrenal glands, brain, heart, kidneys, liver, spleen and thymus were weighed. Absolute organ weight was reported. Relative organ weight (to body and brain weight) were calculated and reported. Paired organs were weighed together.

Other examinations:

Furthermore endpoints for toxicity to reproduction were evaluated (described in corresponding IUCLID section).

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.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Adverse signs of systemic toxicity related to the test item were not detected at any dose level at the daily clinical observations (100, 200 and 450 mg/kg bw/day, male or female).
Wet bedding material – indicative of diuresis – was observed in each cage of animals administered with 450 mg/kg bw/day: in male animals during the entire treatment period, in dams during the pre-mating period and in non-pregnant female animal up to Day 28. This was considered treatment related but not adverse.
Dead and moribund animals: Paralysis of hind limbs was noted for one control dam on Day 26. There were no preceding clinical signs in this animal. Piloerection, decreased activity, narrow eye aperture or decreased body tone were observed in one of the high dose treated male animal from Day 8 and this animal was euthanized on Day 17. One animal, which was found dead on Day 23, showed piloerection, decreased activity, narrow eye aperture, decreased body tone or hunched back from Day 9 onwards.
Surviving animals:
The surviving animals were normal in control, 100, 200 and 450 mg/kg bw/day groups (male and female) except for some female animals, which showed some dermal signs – scars, alopecia –as follows:
- small scars on the right side of the head and body at 100 mg/kg bw/day (1/12) during the gestation and lactation periods;
- alopecia on the fore limbs (1/11) and on the left side of the back (1/11) at 200 mg/kg bw/day during the gestation and lactation periods; with small scars on the back during the lactation period (1/11);
- alopecia on the forelimbs (1/11) at 200 mg/kg bw/day during the lactation period;
Alopecia and scar on the skin are species specific findings, which are also observed in untreated experimental rats of this strain with similar age. These were individual findings with low incidence in animals of control or lower dose groups and were not related to the treatment.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There was no test item related mortality in 100, 200 or 450 mg/kg bw/day groups (male or female) during the course of study. One control dam was euthanized for animal-welfare reasons due to paralysis of hindlimbs on gestation day 11 (Day 26). Two male animals at 450 mg/kg bw/day were also subjected to early necropsy because of animal-welfare reasons – moribund condition – and death on Days 17 and 23, respectively. The cause of death was individual lesion for all these animals and considered not test item related.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The body weight development was not influenced by the test item in male or female animals administered with 100, 200 or 450 mg/kg bw/day.
Dead and moribund animals: Significant body weight loss was observed in moribund and dead male animals at 450 mg/kg bw/day from day 7 up to their death.
Surviving animals: The mean body weight was comparable to their control in surviving male and female animals at 100, 200 and 450 mg/kg bw/day during the entire observation period (pre-mating, mating and post-mating periods in male animals, pre-mating, gestation and lactation period in female animals). Statistical significances were detected at the higher mean body weight gain in male animals at 100 mg/kg bw/day between Days 34 and 41 and if summarized (between Day 0 and 41) and at 200 mg/kg bw/day between Days 0 and 7, between Days 34 and 41 and between Day 0 and 41 when compared to the control. In the male animals administered with 450 mg/kg bw/day, the mean body weight gain was lower than in the control during weeks 1-3 – reaching statistical significance on week 1 – and it was higher than in the control group during weeks 4-6 and for the study overall – with statistical significance between Days 20-27, 34-41 and 0-41. In the female animals, statistically significant difference with respect to the control was observed at the lower mean body weight gain at 100 and 200 mg/kg bw/day between Days 7-13 and at 450 mg/kg bw/day between Days 0-7 and 0-13 during the pre-mating period.
The mean body weight gain was comparable in the control and test item treated groups (100, 200 and 450 mg/kg bw/day) during the gestation and lactation periods. Although the differences between the control and test item treated groups reached statistical significances at the lower or higher mean body weight gain these changes did not result in significant changes in the mean body weight values. Therefore, these findings had no biological significances during this study.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

The food consumption was not adversely affected in male or female animals at 100, 200 and 450 mg/kg bw/day. The mean daily food consumption of male animals was slightly higher than in the control group at 100 mg/kg bw/day between Days 20-27 and at 200 mg/kg bw/day during the post-mating period (between Days 20-27, 27-34 and 34-41) and at 450 mg/kg bw/day between Days 27-34 and 34-41. In the female animals, statistical significances were observed at the slightly lower mean daily food consumption at 450 mg/kg bw/day during the pre-mating period (between Day 0-7, 7-13) and between lactation days 4 and 13. These slight differences with respect to the control were in accordance with the bodyweight gain of male and female animals and were of low degree. Therefore, it was not considered to be toxicologically relevant.

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, 200 or 450 mg/kg bw/day.
In the male animals, statistical significances were detected at the higher mean percentage of eosinophil granulocytes (EOS) at 200 and 450 mg/kg bw/day when compared to the control. The examined hematological parameters were comparable in female animals in the control and 100 mg/kg bw/day groups. Statistical significance was detected at the slightly lower mean corpuscular hemoglobin concentration (MCHC) in female animals at 200 and 450 mg/kg bw/day when compared to the control. The individual values of EOS and MCHC were well within the historical control range in male animals and female animals, respectively. There were no related changes in other hematological parameters therefore, the differences in these parameters were considered to have little or no toxicological relevance.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

The examined clinical chemistry parameters were not adversely affected in male and female animals at 100, 200 or 450 mg/kg bw/day. Slightly elevated concentration of albumin in the male animals at 450 mg/kg bw/day and elevated concentration of creatinine (CREA) in female animals at 450 mg/kg bw/day with respect to their controls were judged to have little or no toxicological relevance due to minor degree and in the lack of related findings.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Functional observations did not demonstrate any test item related adverse changes. The behavior, physical condition and reactions to different type of stimuli of animals selected for examination were considered to be normal in all groups (control, 100, 200 and 450 mg/kg bw/day; n=5 animals/sex/group).

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

The weights of examined organs (absolute, relative to body and brain weights) were not affected by the test item at 100, 200 or 450 mg/kg bw/day. In the male animals, statistical significance was detected at the slightly higher mean heart weights – absolute and relative to body and brain weight – as follows:
- absolute heart weight at 100, 200 and 450 mg/kg bw/day;
- heart weight relative to body weight at 450 mg/kg bw/day;
- heart weight relative to brain weight at 100 and 450 mg/kg bw/day.
The weights of all other organs and the fasted mean body weight were similar in the control and test item treated groups. In the female animals, the fasted mean body weight and the weights of the examined organs (absolute, relative to body and brain weights) were comparable with their controls at 100, 200 and 450 mg/kg bw/day The slight changes in the heart weights of male animals were not considered to be toxicologically relevant in the lack of associated histopathological alterations.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Macroscopic alterations related to the effect of the test item were not detected in male or female animals at 100, 200 or 450 mg/kg bw/day at the necropsy.
Dead and moribund animals: In control female animal euthanized early for animal-welfare reasons, enlarged and urine filled urinary bladder was observed. In male animals subjected to early necropsy, the stomach was filled with food and bedding material and the wall of stomach was thin (2/2, each).
The visceral organs were congestive in moribund animal (1/2) and were slightly autolyzed or cannibalized in dead animal (1/2).
Surviving animals:
In the surviving male animals, Hernia diaphragmatica (including a part of liver; 1/12 control), hemorrhages in the thymus (2/12 at 200 mg/kg bw/day) and pyelectasia (2/12 control; 1/12 at 100 mg/kg bw/day and 5/10 at 450 mg/kg bw/day) were observed at the necropsy.
In the surviving dams, the following macroscopic findings were observed:
- thymic hemorrhage (1/12) and small scars on the skin of whole body (1/12) at 100 mg/kg bw/day;
- alopecia on the forelimbs or on the back (3/11) and ovarian cyst (1/11) at 200 mg/kg bw/day;
- pyelectasia (1/11) at 450 mg/kg bw/day;
In one non-pregnant female animal at 200 mg/kg bw/day (1/1), yellow colored pancreas, smaller than normal liver and ovary, both sides pyelectasia and green-yellow fluid filled uterus were observed at the necropsy. The organs and tissues were judged to be normal in all dams in the control group (10/10) and in non-pregnant female animals in control (1/1) and 450 mg/kg bw/day (1/1) groups. Hernia diaphragmatica, pyelectasia, alopecia scar on the skin are common macroscopic findings in experimental rats of this strain with similar age. Pyelectasia is frequently observed in this strain of experimental rats. It was observed with higher incidence in high dose male animals than in the control group. However, histological examination did not reveal degeneration, inflammation or fibrosis. Therefore, this finding was considered as slight individual lesion without toxicological significance. Thymic hemorrhages are indicative of circulatory disturbance developing during the exsanguination and were only present in lower dose animals. Individual findings in one control dam (paralysis of hind limbs, distended urinary bladder) and in one other dam (yellow colored pancreas, smaller than normal liver and ovary, both sides pyelectasia and green-yellow fluid filled uterus) were supported by histological examinations and had no toxicological significance in this study as these findings were with low incidence and were not present in animals of the high dose group.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Histopathological investigations did not reveal any toxic or other test item related lesions detectable by microscopic examination of the investigated reproductive and other organs of male and female animals at 450 mg/kg bw/day.
Dead and moribund animals:
In one control dam, which was subjected to early necropsy due to paralysis of hind limbs, purulent-necrotic metritis was observed. This finding could explain the lameness of posterior limbs and distended urinary bladder with large amount of urine and it was probably due to local infection.
In dead and moribund male animals at 450 mg/kg bw/day, histological examination revealed moderate congestion (2/2) and mild alveolar emphysema (2/2) in the lungs, in connection with a probably suffocation in both animals. No degenerative or other, possible test item related lesion was observed in the liver, kidney or other investigated organs.
Surviving animals:
In the male animals at 450 mg/kg bw/day and control groups, the investigated organs of reproductive system (testes, epididymides, prostate seminal vesicles, coagulating glands) were histological normal and characteristic on the sexually mature organism in all cases. 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, prostate, seminal vesicles, and coagulating glands was normal in all cases as well (12/12 control, 1/1 at 100 mg/kg bw/day; 1/1 at 200 mg/kg bw/day; 10/10 at 450 mg/kg bw/day).
In the female animals at 450 mg/kg bw/day – 11/11 dams and 1/1 non-pregnant female – and control group – 10/10 dam and 1/1 non-pregnant female – the ovaries, uterus, cervix, vagina had a normal structure characteristic of the species, age and phase of the active sexual cycle. The cortical region of ovaries contained primary, secondary and tertiary follicles and corpora lutea, indicating the active maturation of oocytes, and ovulation. The epithelial capsule and ovarian stroma were normal in all cases as well.
Individual histological findings were observed in two female animals at 200 mg/kg bw/day as follows:
- In one non-pregnant female animal at 200 mg/kg bw/day (1/1), subacute serous metritis, dilatation of uterine horn, decreased number of corpora lutea and increase in the number of follicular atresia were detected in the ovaries (ovary atrophy) in accordance with macroscopic findings (serious dilatation of uterus, filled with yellowish fluid, the atrophy of ovaries). The metritis of this animal could be considered as individual disease probably due to local infection.
- The one side cyst in the ovary in one female animal at 200 mg/kg bw/day is a common individual finding without toxicological significance.
In selected animals, minimal alveolar emphysema in the lungs (1/5 control male; 1/5 male at 450 mg/kg bw/day) and mild acute hemorrhage in the thymus occurred sporadically in mid and low dose (2/2 male at 200 mg/kg bw/day; 1/1 female at 100 mg/kg bw/day) and is considered as consequence of hypoxia, dyspnea and circulatory disturbance developed during exsanguinations.
Minimal or mild hyperplasia of bronchus associated lymphoid tissue (BALT) in some animals (1/5 male and 1/5 female control; 1/5 male at 450 mg/kg bw/day) is considered as an immunomorphological phenomenon, without toxicological significance.
One or both sides pyelectasia in some male and female animals (1/1 male at 100 mg/kg bw/day; 1/1 female at 200 mg/kg bw/day; 5/7 male and 1/5 female at 450 mg/kg bw/day) was without degenerative, inflammatory or other histopathological lesions and is a common finding in Wistar rats without toxicological significance.
Multifocal dermatitis in the skin (1/1 female at 100 mg/kg bw/day) was probably in connection with mechanical effects and is considered as individual disease.
Diaphragmatic hernia in one control male animal (1/1) is a developmental disorder.
In one non-pregnant female animal at 200 mg/kg bw/day (1/1), no histological changes were observed in the liver, kidneys or pancreas in-spite of the macroscopic findings at necropsy (smaller than normal liver and kidneys, yellowish color of the pancreas).
No morphological evidence of test item related acute or subacute injury (degeneration, inflammation, necrosis etc.) of the stomach, the small and large intestines, the liver, the pancreas, the cardiovascular system, the respiratory system, the urinary system, the immune system, the hematopoietic system, the skeleton, the muscular system, the central, or peripheral nervous system, the eyes, the integumentary system, the reproductive system was observed.
The cytomorphology of endocrine glands were the same in the control and treated animals.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

The thyroid hormone (FT4 and TSH) levels were not affected in the parental male animals at any dose levels.
There were no statistically significant differences with respect to their control in the FT4 and TSH concentrations in the parental male animals at any dose levels.

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

450 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No test substance related adverse effects were observed.

Key result

Critical effects observed:

no

Conclusions:

Under the conditions of the study, there were no signs of systemic toxicity in selected male or female animals at 100, 200 or 450 mg/kg bw/day. Based on these observations the NOAEL was determined as follows:
NOAEL for systemic toxicity of male/female rats: 450 mg/kg bw/day

Executive summary:

A study according OECD TG 422 was performed to obtain information on the toxic potential of the test item when repeatedly administered orally (by gavage) to rats at doses of 100 mg/kg bw/day, 200 mg/kg bw/day and 450 mg/kg bw/day compared to control animals.

Four groups of Han:WIST rats (n=12/sex/group) were administered with the test item orally (by gavage) once a day at 0 (vehicle), 100, 200 and 450 mg/kg bw/day doses corresponding to concentrations of 0, 20, 40 and 90 mg/mL. The application volume was 5 mL/kg bw. Control animals received the vehicle, 1 % methylcellulose.

The suitability of the vehicle at the intended concentrations of the test item was analytically verified up front (concentration and homogeneity). The concentration of the test item in the dosing formulations used for animal’s treatment was checked two times during the study. Test item in the dosing formulations varied in the acceptable range between 96 % and 106 % of the nominal values and formulations were homogenous thereby confirming the proper dosing.

All animals of the parent (P) generation were dosed prior to mating (14 days) and throughout mating. In addition, males received the test item or vehicle after mating up to the day before the necropsy (altogether for 42 days; except for dead or moribund animals). Dams were additionally exposed through the gestation period and up to lactation days 13-16 (altogether for 51-71 days, except for moribund dam). Observations included mortality, clinical signs, body weight and food consumption. 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. Furthermore endpoints for toxicity to reproduction were evaluated (described in corresponding IUCLID section).

The dams were allowed to litter and were euthanized on postnatal day 13 or shortly thereafter. Blood samples were collected for possible determination of serum levels of thyroid hormones (FT4, TSH) from all dams at termination on post-partum 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. In addition, for five males and females randomly selected from each group, adrenal glands, brain, heart, kidneys, liver, spleen and thymus were weighed.

Histopathology examination was performed on reproductive organs (testes, epididymides, prostate, and seminal vesicles with coagulating glands, uterus with oviduct, cervix, vagina and ovaries) in the control and high dose groups and in non-mated male animal and in non-pregnant female animals and the male this female cohabited with in the low or mid dose groups. 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. In addition, organs showing macroscopic changes (thymus, kidneys, skin, liver, pancreas, ovaries, uterus, vagina) were also processed and examined histologically in animals in low or mid dose groups. The results were interpreted comparing treatment groups with respect to controls, which were treated concurrently with vehicle (1 % methylcellulose) only. Historical control data were also considered.

There was no test item related mortality in 100, 200 or 450 mg/kg bw/day groups (male or female) during the course of study.

One male animal at 450 mg/kg bw/day was found dead, one control dam and one male animal at 450 mg/kg bw/day were euthanized for humane reasons due to moribund state. The cause of death was individual lesion for all these animals: purulent-necrotic metritis in female animal and suffocation in both male animals.

Adverse signs of systemic toxicity related to the test item were not detected at any dose level (100, 200 and 450 mg/kg bw/day, male or female) at the daily or detailed weekly clinical observations or at the terminal functional observations.

Wet bedding material – indicative of diuresis – was observed in each cage of animals administered with 450 mg/kg bw/day from Day 8: in male animals during the entire treatment period, in dams during the pre-mating period and in non-pregnant female animals up to Day 28.

The mean body weight gain and mean body weight were not influenced by the test item in male or female animals administered with 100, 200 or 450 mg/kg bw/day.

The food consumption was not adversely affected in male or female animals at 100, 200 and 450 mg/kg bw/day.

There were no test item related adverse changes in the examined hematological parameters in male or female animals at 100, 200 or 450 mg/kg bw/day.

The examined clinical chemistry parameters were not affected in male and female animals at 100, 200 or 450 mg/kg bw/day.

There were no test item related changes in the serum thyroid hormone (FT4, TSH) levels at any dose.

Macroscopic alterations related to the effect of the test item were not detected in male or female animals at 100, 200 or 450 mg/kg bw/day at the terminal necropsy.

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

The weights of examined organs (absolute, relative to body and brain weights) were not affected by the test item in selected animals at 100, 200 or 450 mg/kg bw/day.

Histopathological examinations of the investigated sexual organs and accessory sex organs (ovaries, uterus, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland) did not reveal any test item related changes at 450 mg/kg bw/day or in not mated male animal or in not fertile animals in the low or mid dose groups. Histopathological investigations did not reveal any toxic or other test item related lesions detectable by microscopic examination of the investigated organs of selected male and female animals at 450 mg/kg bw/day.

Under the conditions of the study, there were no signs of systemic toxicity in selected male or female animals at 100, 200 or 450 mg/kg bw/day. Based on these observations the NOAEL was determined as follows:

NOAEL for systemic toxicity of male/female rats: 450 mg/kg bw/day

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

450 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

OECD TG 422

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Repeated dose toxicity: oral

Key

A study according OECD TG 422 was performed to obtain information on the toxic potential of the test item when repeatedly administered orally (by gavage) to rats at doses of 100 mg/kg bw/day, 200 mg/kg bw/day and 450 mg/kg bw/day compared to control animals.

Four groups of Han:WIST rats (n=12/sex/group) were administered with the test item orally (by gavage) once a day at 0 (vehicle), 100, 200 and 450 mg/kg bw/day doses corresponding to concentrations of 0, 20, 40 and 90 mg/mL. The application volume was 5 mL/kg bw. Control animals received the vehicle, 1 % methylcellulose.

The suitability of the vehicle at the intended concentrations of the test item was analytically verified up front (concentration and homogeneity). The concentration of the test item in the dosing formulations used for animal’s treatment was checked two times during the study. Test item in the dosing formulations varied in the acceptable range between 96 % and 106 % of the nominal values and formulations were homogenous thereby confirming the proper dosing.

All animals of the parent (P) generation were dosed prior to mating (14 days) and throughout mating. In addition, males received the test item or vehicle after mating up to the day before the necropsy (altogether for 42 days; except for dead or moribund animals). Dams were additionally exposed through the gestation period and up to lactation days 13-16 (altogether for 51-71 days, except for moribund dam). Observations included mortality, clinical signs, body weight and food consumption. 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. Furthermore endpoints for toxicity to reproduction were evaluated (described in corresponding IUCLID section).

The dams were allowed to litter and were euthanized on postnatal day 13 or shortly thereafter. Blood samples were collected for possible determination of serum levels of thyroid hormones (FT4, TSH) from all dams at termination on post-partum 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. In addition, for five males and females randomly selected from each group, adrenal glands, brain, heart, kidneys, liver, spleen and thymus were weighed.

Histopathology examination was performed on reproductive organs (testes, epididymides, prostate, and seminal vesicles with coagulating glands, uterus with oviduct, cervix, vagina and ovaries) in the control and high dose groups and in non-mated male animal and in non-pregnant female animals and the male this female cohabited with in the low or mid dose groups. 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. In addition, organs showing macroscopic changes (thymus, kidneys, skin, liver, pancreas, ovaries, uterus, vagina) were also processed and examined histologically in animals in low or mid dose groups. The results were interpreted comparing treatment groups with respect to controls, which were treated concurrently with vehicle (1 % methylcellulose) only. Historical control data were also considered.

There was no test item related mortality in 100, 200 or 450 mg/kg bw/day groups (male or female) during the course of study.

One male animal at 450 mg/kg bw/day was found dead, one control dam and one male animal at 450 mg/kg bw/day were euthanized for humane reasons due to moribund state. The cause of death was individual lesion for all these animals: purulent-necrotic metritis in female animal and suffocation in both male animals.

Adverse signs of systemic toxicity related to the test item were not detected at any dose level (100, 200 and 450 mg/kg bw/day, male or female) at the daily or detailed weekly clinical observations or at the terminal functional observations.

Wet bedding material – indicative of diuresis – was observed in each cage of animals administered with 450 mg/kg bw/day from Day 8: in male animals during the entire treatment period, in dams during the pre-mating period and in non-pregnant female animals up to Day 28.

The mean body weight gain and mean body weight were not influenced by the test item in male or female animals administered with 100, 200 or 450 mg/kg bw/day.

The food consumption was not adversely affected in male or female animals at 100, 200 and 450 mg/kg bw/day.

There were no test item related adverse changes in the examined hematological parameters in male or female animals at 100, 200 or 450 mg/kg bw/day.

The examined clinical chemistry parameters were not affected in male and female animals at 100, 200 or 450 mg/kg bw/day.

There were no test item related changes in the serum thyroid hormone (FT4, TSH) levels at any dose.

Macroscopic alterations related to the effect of the test item were not detected in male or female animals at 100, 200 or 450 mg/kg bw/day at the terminal necropsy.

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

The weights of examined organs (absolute, relative to body and brain weights) were not affected by the test item in selected animals at 100, 200 or 450 mg/kg bw/day.

Histopathological examinations of the investigated sexual organs and accessory sex organs (ovaries, uterus, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland) did not reveal any test item related changes at 450 mg/kg bw/day or in not mated male animal or in not fertile animals in the low or mid dose groups. Histopathological investigations did not reveal any toxic or other test item related lesions detectable by microscopic examination of the investigated organs of selected male and female animals at 450 mg/kg bw/day.

Under the conditions of the study, there were no signs of systemic toxicity in selected male or female animals at 100, 200 or 450 mg/kg bw/day. Based on these observations the NOAEL was determined as follows:

NOAEL for systemic toxicity of male/female rats: 450 mg/kg bw/day

 

Supporting

A 14-Day toxicity study was performed to obtain initial information on the toxic potential of the substance after subacute repeated application in rats at three dose levels and to allow dose-setting for a subsequent Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test (main study according to OECD 422). The test item was administered orally (by gavage) to Han:WIST rats (n=5 animals/sex/group) once a day at 0 (vehicle control), 100, 300 and 1000/750 mg/kg bw/day corresponding to concentrations of 20, 60 and 150/200 mg/mL for 14 days. The application volume was 5 mL/kg bw. Animals of the high dose groups were not administered on Day 8 to ensure one day washout of 1000 mg/kg bw/day. Suitability of the vehicle was analytically verified. Due to the solubility feature of the test item, stability determination was not possible. Formulations were prepared daily and administered within a short period thereafter thus the stability of the formulations was considered to be not relevantly affected. Analysis of formulations was performed once during the scope of this study. The concentrations of the test item in the dosing formulations varied between the range of 104 and 108 % of the nominal concentrations. Detailed clinical observations were made on all animals once a day, after treatment at approximately the same time. Body weight was measured twice a week and food consumption was determined weekly. Clinical pathology and gross pathology examinations were conducted at the end of the treatment period. Selected organs were weighed.

Five male and three female animals were found dead and two moribund female animals were subjected to early necropsy at 1000/750 mg/kg bw/day between Day 8 and 11. Clinical signs and significant body weight loss were noted for all animals before the death. The test item caused clinical signs in male and female animals at 1000/750 mg/kg bw/day. Slight, moderate or marked activity decrease, piloerection, prone position and tremor were observed from Day 4 onwards. Animals were found dead between Days 8 and 11 and two female animals were considered to be in moribund state on Days 8 and 11.

The mean body weight of male and female rats at 1000/750 mg/kg bw/day was significantly lowered during the treatment period. Compared to their control group, the mean body weight gain was reduced in male and female rats at 1000/750 mg/kg bw/day between Days 0 and 3 and the individual body weight decreased in each dead and moribund animal (male and female) from Day 3 onwards.

The mean daily food consumption was reduced with respect to their control at 1000/750 mg/kg bw/day (male and female) in full compliance with the body weight changes.

There were no test item related adverse effects on the investigated hematological parameters at 100 or 300 mg/kg bw/day.

Pathologic changes were not detected in the examined clinical chemistry parameters at 100 and 300 mg/kg bw/day.

Enlargement of the adrenal glands, hemorrhages and sanguineous content in the stomach and reduced size of some organs (thymus, spleen, prostate, seminal vesicles) were observed in animals that died prematurely at 1000/750 mg/kg bw/day.

The weight of the examined organs (absolute and relative to body and brain weights) were comparable in the control and 100 or 300 mg/kg bw/day dose treated male and female animals.

Under the conditions of the present study, the test item caused death, clinical signs, reduced body weight and food consumption and changes in organ pathology in male and female animals following 14-day oral administration at 1000/750 mg/kg bw/day to Han: WIST rats. There were no test item related findings at 100 or 300 mg/kg bw/day.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data is reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on repeated dose toxicity, the test item is not classified according to Regulation (EC) No 1272/2008 (CLP), as amended for the twelfth time in Regulation (EU) No 2019/521.