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REACH

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

EC number: 433-100-1 | CAS number: -

Life Cycle description
Uses advised against

Toxicological information

Endpoint summary

Administrative data

Key value for chemical safety assessment

Toxic effect type:: dose-dependent

Effects on fertility

Description of key information

The substance did not adversely influence the reproductive performance (gonad function, mating behavior, conception, parturition) in parental male and female Han:WIST rats at 100, 200 or 450 mg/kg bw/day by oral gavage.There were no signs of systemic toxicity in selected male or female animals at 100, 200 or 450 mg/kg bw/day. The development of the F1 offspring was not impaired from birth to post-natal day 13 after repeated oral administration of dams at 100, 200 or 450 mg/kg bw/day. Based on these observations the NOAELs were determined as follows:

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

NOAEL for reproductive performance of male/female rats: 450 mg/kg bw/day

NOAEL for F1 Offspring: 450 mg/kg bw/day

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

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

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:

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:

GLP compliance:

yes (incl. QA statement)

Limit test:

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 and reproduction toxicity studies and wellknown 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: 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:

other: 1 % methylcellulose

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in the vehicle in concentrations of 20, 40 and 90 mg/mL. 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. A constant treatment volume of 5 mL dose preparation/kg body weight was administered in all groups.

VEHICLE
- Justification for use and choice of vehicle: The test item is not soluble in water therefore 1 % methylcellulose was used for preparing formulations appropriate for oral administration. 1 % methylcellulose is a suitable vehicle to facilitate formulation analysis for the test item.

Details on mating procedure:

- M/F ratio per cage: 1:1 mating
- Length of cohabitation: Females were cohabited with the same male until copulation occurred or two weeks elapsed.
- Proof of pregnancy: Vaginal smears were examined for the presence of vaginal plug or sperm. Presence of vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (day 0 of pregnancy as defined by OECD 422).
- After successful mating each pregnant female was caged: individually
- Any other deviations from standard protocol: no

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:

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:

Parental animals: Observations and examinations:

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. Placental sign is determined in order to control pregnancy.

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

Other observations:
CLINICAL PATHOLOGY
Clinical pathology examinations including hematology 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 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,TSH) as follows:
- from all dams and at least two pups per litter on day 13 if feasible
- from all parent male animals at termination

Oestrous cyclicity (parental animals):

Estrous cycle was monitored by examining vaginal smears before the treatment started 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 was also prepared on the day of the necropsy.

Sperm parameters (parental animals):

Parameters examined in all male parental generations:
- testis weight, epididymis weight, prostate weight and seminal vesicles with coagulating glands as a whole, spermatogenic cells (the spermatogonia, the spermatocytes, the spermatids and spermatozoa)

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight, anogenital distance (AGD), presence of nipples/areolae in male pups
Blood samples were collected from the surplus pups (pooled and used for determination of serum FT4 and TSH levels on post-natal day 4 (2-6 pups per litter) or on post-natal day 13 (1-7 pups per litter). The number of nipples/areolae in male pups was counted on post-natal day 13.

GROSS EXAMINATION OF DEAD PUPS:
Dead pups found were subjected to necropsy by a macroscopic examination. Any observed abnormalities were recorded.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No

Postmortem examinations (parental animals):

SACRIFICE
One day after the last treatment, surviving animals were euthanized by exsanguination after verification of Isofluran-narcosis.
- Parental male animals: after the optionally extended post-mating period on Day 42
- Non-pregnant female animals: on Days 42, 47 or 54
- Dams not selected for toxicology examinations: on post-partum day 14 or shortly thereafter (Days 51, 54, 55, 57 or 71)
- Dams selected for toxicology examinations: shortly after post-partum day 13 (Day 54; lactation days 14-17)
Moribund animals were sacrified as appropriate. These animals and dead animals were subjected to early necropsy.

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.

Postmortem examinations (offspring):

Dead pups and pups euthanized on post-natal day 13 or shortly thereafter, were carefully examined for gross abnormalities at least externally. Particular attention was paid to the external reproductive genitals.

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:

see table 1

Offspring viability indices:

see table 2

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.

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

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 dams 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.

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

The examined parameters of the estrous cycle were comparable in the control and test item administered animals (100, 200 and 450 mg/kg bw/day).
Statistically significant difference was noted for the slightly shorter estrous cycle of female animals at 450 mg/kg bw/day during the pre-mating period. This minor difference with respect to the control was biologically not relevant as values met well the historical control. There were no statistically significant differences between the control and test item treated groups in the number or percentage of animals with regular cycles, in the mean number of cycles, mean number of days in pro-estrus, estrous or diestrus during the pre-experimental or pre-mating periods or in the mean length of cycles during the pre-experimental period.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

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).

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

The examined parameters of reproductive performance were not affected by the treatment with the test item in male or female animals at 100, 200 or 450 mg/kg bw/day. Statistical significance was observed at the lower copulatory index and at the higher fertility index of male animals at 100 mg/kg bw/day. The percentage of pregnant females was higher than in the control group at 100 mg/kg bw/day. The examined parameters of reproductive performance were comparable in the control and 200 or 450 mg/kg bw/day groups (male and female). These minor changes at 100 mg/kg bw/day were considered to be indicative of biological variation and not related to the test item. Changes in the copulatory index were not detected at the higher dose groups.

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

Dose descriptor:

NOAEL

Remarks:

reproductive performance

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:

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no adverse clinical signs in the offspring from post-natal day 0 to 13. The percentage of offspring, which were cold or did not suckle, were slightly higher at 200 or 450 mg/kg bw/day than in the control group during the first week of the observations. These findings were transient, were only detected in single pups of some litters and met well the historical control. Some other sporadic clinical signs were also observed: alopecia in three litters and cyanotic skin in single pup at 200 mg/kg bw/day. The percentage of missing pups was comparable in all groups. Dead pups were found in low and high dose groups (1-1, each). One female pup was smaller than normal in one litter of the high dose group. Therefore, these signs were considered to be toxicologically not relevant.

Mortality / viability:

no mortality observed

Description (incidence and severity):

There was no test item related effect on offspring’s extra uterine mortality.
The extrauterine mortality was low and comparable in the control, 100, 200 and 450 mg/kg bw/day from birth to post-natal day 13.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The body weight development of the offspring was undisturbed at 100, 200 and 450 mg/kg bw/day. The mean litter weight and litter weight gain was similar in the control and at 100, 200 or 450 mg/kg bw/day groups on postnatal days 0, 4 and 13.
Similarly, there were no statistical significances between the control and test item treated groups (100, 200 and 450 mg/kg bw/day) in the mean pup weight and mean pup weight gain per litter on post-natal 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 at the slightly higher mean weight of male pup at 200 mg/kg bw/day and at the slightly higher mean weight of female pup at 100, 200 and 450 mg/kg bw/day on post-natal day 4. These differences with respect to the control in the body weight of offspring by genders were considered to be toxicologically not relevant due to the minor degree and lack of dose relevance.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

effects observed, non-treatment-related

Description (incidence and severity):

The anogenital distances were not affected by the test item at 100, 200 and 450 mg/kg bw/day in male or female offspring. In the male pups, the mean anogenital distances (absolute and normalized) were comparable in control and all dose groups.
In the female pups, statistical significances were observed at the slightly longer mean absolute anogenital distances at 200 and 450 mg/kg bw/day, while the normalized anogenital distances were similar to that in the control group at 100, 200 and 450 mg/kg bw/day. These differences were with minor degree or without dose relevance and therefore were considered to have no toxicological relevance.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

Nipples/areoles were not visible in any of the examined male offspring in the control or 100, 200 or 450 mg/kg bw/day groups on post-natal day 13.

Organ weight findings including organ / body weight ratios:

not examined

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 (1/1 in the control and 1/1 at 100 mg/kg bw/day) subjected to necropsy on post-natal day 0. Sign of cannibalism (missing head and back) and empty stomach were noted for the single dead pup at 100 mg/kg bw/day (1/1) on post-natal day 0. Autolyzed visceral organs and empty stomach was observed in the other dead pup at 450 mg/kg bw/day (1/1) on post-natal day 4.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

The thyroid hormone (FT4 and TSH) levels were not affected in PN13 offspring at any dose levels.
There were no statistically significant differences with respect to their control in the FT4 and TSH concentrations in PN13 offspring at any dose levels.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

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:

Key result

Reproductive effects observed:

Conclusions:

Under the conditions of the study, the test item did not adversely influence the reproductive performance (gonad function, mating behavior, conception, parturition) in parental male and female Han:WIST rats at 100, 200 or 450 mg/kg bw/day by oral gavage.
There were no signs of systemic toxicity in selected male or female animals at 100, 200 or 450 mg/kg bw/day. The development of the F1 offspring was not impaired from birth to post-natal day 13 after repeated oral administration of dams at 100, 200 or 450 mg/kg bw/day. Based on these observations the NOAELs were determined as follows:
NOAEL for systemic toxicity of male/female rats: 450 mg/kg bw/day
NOAEL for reproductive performance of male/female rats: 450 mg/kg bw/day
NOAEL for F1 Offspring: 450 mg/kg bw/day

Executive summary:

A study according OECD TG 422 was performed to 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, 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. In the dosing formulations the test item 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, 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 before the treatment for two weeks 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 and on the day of the necropsy.

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

Blood samples were collected for possible determination of serum levels of thyroid hormones (FT4, TSH) from 2-6 pups per litter (where it was feasible) on post-natal day 4, from all dams and from 1-7 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.

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.

A test item influence on the estrous cycle was not detected at any dose level (100, 200 and 450 mg/kg bw/day).

There were no test item related differences between the control and test item treated male or female animals in the reproductive performance.

Delivery data of dams was not adversely affected at 100, 200 or 450 mg/kg bw/day dose level. The mean number of implantation sites was slightly lower at 450 mg/kg bw/day than in the control group however this minor difference was considered to be of no toxicological relevance as value remained well within the historical control ranges.

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 (parental male animals or PN13 offspring).

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.

The offspring’s development was not adversely influenced at any dose level. No effects on the mortality, clinical signs, body weight development, anogenital distance (male and female) or nipple retention (male) were detected.

There were no signs of systemic toxicity in selected male or female animals at 100, 200 or 450 mg/kg bw/day. The development of the F1 offspring was not impaired from birth to post-natal day 13 after repeated oral administration of dams at 100, 200 or 450 mg/kg bw/day. Based on these observations the NOAELs were determined as follows:

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

NOAEL for reproductive performance of male/female rats: 450 mg/kg bw/day

NOAEL for F1 Offspring: 450 mg/kg bw/day

Effect on fertility: via oral route

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

Effect on fertility: via inhalation route

Endpoint conclusion:: no study available

Effect on fertility: via dermal route

Endpoint conclusion:: no study available

Additional information

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