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Description of key information

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

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Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012/2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
adopted 1996
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3650, adopted July 2000
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:Charles River Laboratories, Research Models and Services, Germany GmbH, Sulzfeld, Germany
- Age at study initiation: 10-11 wks;
- Weight at study initiation: Males: 294g; Females: 197g, on average
- Fasting period before study: no
- Housing: individually (except during overnight mating and lactation) in Makrolon type M III cages
- Diet (e.g. ad libitum): Kliba maintenance diet mouse-rat “GLP” meal, ad lib.
- Water (e.g. ad libitum): tap water ad lib.
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 30-70%
- Air changes (per hr):15
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:The test substance was applied as suspension, which was prepared at least once a week. An appropriate amount of the test substance was weighed, and corn oil was added up to the desired volume. The suspension was kept homogenous during administration by stirring with a magnetic stirrer.

The administration volume was 4ml/kg b.w.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The test substance is poorly soluble in water.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability of the test substance in corn oil for a period of 7 days at room temperature was proven before the start of the study (BASF Project No. 11L00386).
For homogeneity and concentration control analyses, each 6 samples of all concentrations was drawn at the start of the study and towards the end of the administration period. Homogeneity was confirmed, all concentrations measured were within 90-110% of the target concentration.
Duration of treatment / exposure:
males: 36 days
females: 51 days
Frequency of treatment:
daily, except to animals being in labor
Remarks:
Doses / Concentrations:
100, 300, 1000mg/kg (high dose was reduced to 600mg/kg on day 19)
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on results of a range-finding study - no severe signs of toxicity were observed in animals treated with 1000mg/kg of the test substance for 14 days.
Positive control:
no
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily on workdays, daily on weekends and public holidays
- Cage side observations: check for moribund animals, pertinent behavioral changes, signs of overt toxicity, parturation, littering and lactation behavior of the dams

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to the first administration, weekly thereafter
- The following parameters were examined: abnormal behavior during “handling”, fur, skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, impairment of gait, lacrimation, palpebral closure, exophthalmus, feces (appearance/consistency), urine, pupil size

BODY WEIGHT: Yes
- Time schedule for examinations: day 0, weekly thereafter with the following exceptions for females: During the mating period the parental females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20. Females with litter were weighed on the day of parturition (PND 0) and on PND 4.

FOOD CONSUMPTION:
- Food consumption for each animal determined: Yes, except during mating

WATER CONSUMPTION: Monitored by daily visual inspection

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the day of necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (16-20h)
- How many animals: 5 per sex and group
- The following parameters were examined: Leukocyte count, Erythrocyte count, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet count, differential blood count, reticulocytes, prothrombin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on the day of necropsy
- Animals fasted: Yes (16-20h)
- How many animals: 5 per sex and group
- The following parameters were examined: ALT, AST, ALP, GGT, sodium, potassium, chlorid, inorganic phosphate, calcium, urea, creatinine, glucose, total bilirubin, total protein, albumin, globulins, triglycerides, cholesterol

URINALYSIS: Yes
- Time schedule for collection of urine: day 30 (males) and day 50 (females)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: yes, during collection
- How many animals: 5 per sex and group
- The following parameters were examined: pH, protein, glucose, ketones, urobilinogen, bilirubin, blood, specific gravity, sediment, color, turbity, volume

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: 3 days prior to necropsy (day 32 males, day 49 females)
- Dose groups that were examined: 5 animals per sex and group (only females with litter)
- Battery of functions tested: home cage and open field observation, motor activity, sensory motor test / reflexes (including: Reaction to an object being moved towards the face, touch sensitivity, vision (Visual placing response), pupillary reflex, pinna reflex, auditory startle response, righting response, behavior during handling, vocalization, pain perception (Tail pinch), grip strength of forelimbs and hindlimbs, landing foot-splay test)
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
The following tissues were weighed
- in all animals: epididymides, testes
- in 5 males and females of each group: adrenal glands, brain, heart, kidneys, liver, spleen, thymus
The following tissues were examined histotechnically in at least 5 animals per sex of the control and high dose group unless noted otherwise
adrenal glands, gross lesions (all affected animals in all dosage groups), bone marrow (femur), brain, cecum, cervix, coagulation glands, colon, duodenum, epididymides, heart, ileum, jejunum, kidneys, liver, lungs, lymph nodes (auxillary and mesenteric), ovaries, oviducts, prostate gland, peyer's patches, rectum, sciatic nerve, seminal vesicles, spinal cord, spleen, forestomach (all animals from all dose groups), glandular stomach, testes, thymus, thyroid glands, trachea, urinary bladder, uterus, vagina
Other examinations:
Reproductive performance: see entry in chapter 7.8.1
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
1000 (study days 0 to 18) and 600 mg/kg bw/d (from study day 19 onwards):
- One female animal was found dead on study day 16 (mating day 3) and another one was sacrificed moribund on study day 18 (gestation day 2).
- One male animal was found dead on study day 35 (post-mating day 7).
- Piloerection was observed in 3 of 10 female animals during DCO, in 1 female animal during premating and post mating as well as in 2 females in mating and gestation.
- Smeared fur was observed in 1 of 10 female animals during gestation.
- Respiratory sounds were observed in 1 of 10 females during mating, in 2 of 10 females during gestation and in 1 of 10 females during lactation, gasping was observed in 1 of 10 female animals during gestation and lactation.
- Poor general condition was observed in 2 of 10 female animals during gestation.
- Hunched posture was observed in 1 of 10 female animals during gestation.
- Semiclosed eyelids were observed in 1 of 10 female animals during gestation.
Mortality:
mortality observed, treatment-related
Description (incidence):
due to local effects: ersoion/ulcer in the forstomach,
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
In test group 3 (1000 and 600 mg/kg bw/d) female animal No. 80 was found dead on study day 17 (Mating Day 3), female animal No. 74 was sacrificed moribund on study day 19 (GD 2), and male animal No. 32 was found dead on study day 35. These premature deaths were assessed as being related to the test substance. One female animal of test group 2 (300 mg/kg bw/d) was also found dead on study day 44. The animal died because of a gavage error. Therefore, a relation to treatment was excluded.

Almost all animals of both sexes of the mid and high dose group showed mostly slight, sometimes moderate salivation within 2 hours after the test substance administration on several days of the study. The same was true for individual low dose animals. From the temporary, short appearance immediately after dosing it could be assumed that salivation was induced by a bad taste of the test substance or local affection of the upper digestive tract. The effect was related to the test substance but assessed as being non-adverse.

Animal No. 74 of test group 3 (1000 and 600 mg/kg bw/d) showed poor general condition, piloerection and respiration sounds on GD 1 and GD 2. Therefore, the animal was sacrificed in a moribund condition on GD 19. Animal No. 75 of the same group showed semiclosed eyelids, poor general condition, hunched posture, smeared fur, piloerection (up to GD 7) and gasping on GD 2 and respiration sounds on GD 3. The same animal showed respiration gasping and sounds on LD 1. These findings were assessed as being related to treatment.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
No treatment-related changes in body weight data were observed in animals of all test groups.

FOOD CONSUMPTION
No treatment-related changes in food consumption were observed.

HAEMATOLOGY
No treatment-related changes among hematological parameters were observed.

CLINICAL CHEMISTRY
No treatment-related changes were measured.

URINALYSIS
No test substance-related effects were observed.

NEUROBEHAVIOUR
No test substance-related effects were observed.

ORGAN WEIGHTS
Absolute thymus weights were significantly decreased in high dose male animals when compared to control group animals. This finding was not considered to be treatment related, since no relative organ weights were significantly altered.

GROSS PATHOLOGY
Erosions, ulcers, and in one case a thickened forstomach were observed in all high dose male and females (with the exception of one female) and 5 mid dose females and 4 mid dose males.

HISTOPATHOLOGY:
Five male and 6 female animals of test group 3 (1000 and 600 mg/kg bw/d) as well as each 2 males and females of test group 2 (300 mg/kg bw/d) revealed multifocal erosions/ulcers in the forestomach. Accompanying these erosions/ulcers a mild to extreme diffuse or focal squamous hyperplasia with hyperkeratosis and a mild to severe submucosal edema was present. When the hyperplasia of the squamous epithelium was focal, it was located at the cardia region (stomach entrance). Within the edema a higher number of inflammatory cells were observed. Not all animals with a macroscopically diagnosed erosion/ulcer showed this finding. But the consistency of the accompanying findings (hyperplasia with hyperkeratosis and edema) which were observed in animals with and without erosion/ulcer led to the conclusion that this all belongs to the same complex. As the erosions/ulcer that could be seen microscopically were very small it is possible that they were not on the level of section. But taken all these findings together they point towards an irritating effect of the substance on the squamous epithelium of the forestomach. Especially the cardia region (entrance of the stomach) was affected.
The three animals of test group 3 (1000 and 600 mg/kg bw/d) which were found dead or were sacrificed in a moribund (animal Nos. 32, 74 and 80) did not show any other findings that could explain the death beside the ones in the stomach. Two of them revealed a moderate to severe thymus atrophy which was regarded to be a consequence to the stress caused by the findings in the forestomach. But it is not clear whether these observed findings were the cause of the death although death was regarded to be a consequence to the treatment with the test substance.
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
>= 300 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: deaths and poor general state in high dose animals
Dose descriptor:
NOAEL
Remarks:
local
Effect level:
>= 100 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: irritation in the stomach
Critical effects observed:
not specified
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06 November 2018 -
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
25 June 2018
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
30 May 2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
- Batch No.of test material: 180006PO40
- Expiration date of the batch: Stable until 04 July 2019
- Storage condition of test material: Avoid temperatures >45 °C; ambient (room temperature)
- Stability under test conditions: The stability of the test substance under storage conditions over the test period was guaranteed.
Species:
rat
Strain:
Wistar
Remarks:
Crl:WI(Han)
Details on species / strain selection:
The rat is a frequently used laboratory animal, and there is comprehensive experience with this animal species and the Wistar strain. Moreover, the rat has been proposed as a suitable animal species by the OECD and the EPA for this type of study.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services GmbH, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 42 ± 1 days
- Weight at study initiation: male: 150.8 - 171.8 g; female: 116.4 - 144.6 g
- Fasting period before study: no
- Housing: group housing (5 animals/cage), polysulfonate cages (floor area about 2065 cm^2), dust-free wooden bedding, wooden gnawing blocks and large play tunnels
- Diet: ad libitum, mouse and rat maintenance diet “GLP” (Granovit AG, Kaiseraugst, Switzerland)
- Water: ad libitum, tap water in water bottles
- Acclimation period: 8 days

DETAILS OF FOOD AND WATER QUALITY:
The supplier assayed the food used in the study for chemical and microbiological contaminants. On the basis of duration of use and the analytical findings with respect to chemical and microbiological contaminants, the diet was found to be suitable.
The drinking water is regularly assayed for chemical contaminants by the municipal authorities of Frankenthal and by the Environmental Analytics Water/Steam Monitoring Department of BASF SE as well as for the presence of microorganisms by a contract laboratory. On the basis of the analytical findings, the drinking water was found to be suitable. German “Trinkwasserverordnung” (Drinking Water Regulation) served as a guideline for maximum tolerable contaminants.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 45 - 65
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Details on route of administration:
The oral route was selected since this was proven to be suitable for the detection of a toxicological hazard.
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance was applied as a suspension. To prepare this suspension, the appropriate amount of test substance was weighed out depending on the desired concentration. Then, corn oil was filled up to the desired volume and homogenized with a magnetic stirrer.
- Rate of preparation: once per week

VEHICLE
- Concentration in vehicle: 60 mg/kg bw: 1.5 g/100 mL; 180 mg/kg bw: 4.5 g/100 mL; 540 mg/kg bw: 13.5 g/100 mL
- Amount of vehicle: 4 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Homogeneous distribution of the test substance in preparations was performed in the highest and lowest concentration. Additionally, concentration control was performed in all concentrations at the beginning and towards the end of the administration period.
The test substance was determined to be homogeneously distributed in the vehicle and the determined concentrations corresponded to 99 - 103 % of the nominal concentrations.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
once daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
60 mg/kg bw/day (nominal)
Dose / conc.:
180 mg/kg bw/day (nominal)
Dose / conc.:
540 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The selection of dose levels based mainly on the results of a test study in female Wistar rats conducted at dose levels of 0, 600 and 1000 mg/kg bw/d. In this study, a NOAEL was not established due to erosions and ulcerations in the stomach of different animals at both dose levels of 600 and 1000 mg/kg bw/d as well as clinical findings like poor general condition, piloerection and body weight loss after 1 week of treatment.
In a subsequently performed OECD 422 study male and female parental animals were treated at dose levels of 50, 150 and 500 mg/kg bw/d. In this study, neither signs of general systemic toxicity nor of local irritation in the stomach were observed.
Considering dose levels of both previous studies the dose levels of this study were selected.

- Fasting period before blood sampling for clinical biochemistry: at least 16 hours
Positive control:
No positive control was used.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily, before administration and within 2 and 5 hours after administration

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to the administration period and thereafter at weekly intervals
- Parameters checked in table No.1 were examined.

BODY WEIGHT: Yes
- Time schedule for examinations: prior to the administration period, on day 0 and afterwards weekly

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption was determined weekly and calculated as mean food consumption in grams per animal and day.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: Yes
- Drinking water consumption was observed by daily visual inspection of the water bottles for any overt changes in volume.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to the start of the administration period on day -1 / 0 and on study day 91
- Dose groups that were examined: day -1/0: all animals; day 91: control and high-dose animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: before sacrifice
- Anaesthetic used for blood collection: Yes, isoflurane
- Animals fasted: Yes, at least 16 hours
- How many animals: all
- Parameters checked in table No.5 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: before sacrifice
- Animals fasted: Yes, at least 16 hours
- How many animals: all
- Parameters checked in table No.6 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine:
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes, during urine collection (overnight)
- Parameters checked in table No.7 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: at the end of the administration period starting
- Dose groups that were examined: all (also all animals)
- Battery of functions tested: Home cage and open field observations, sensory activity (incl. grip strength) and motor activity
- Parameters checked in table No.2-4 were examined.
- Details on motor activity assessement: The examinations were performed using the TSE Labmaster System (TSE Systems GmbH, Bad Homburg, Germany). The number of beam interrupts was counted over 12 intervals for 5 minutes per interval. The sequence in which the rats were placed in the cages was selected at random. The measurement period began when the 1st beam was interrupted and finished exactly 1 hour later. No food or water was offered to the rats during these measurements and the measurement room was darkened.

IMMUNOLOGY: No

OTHER:
- Thyroid hormones (total triiodothyronine (T3), total thyroxine (T4), thyroid stimulating hormone (TSH)) were determined using the blood samples collected.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
The animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology. Organ weights were determined (table No. 8). All paired organs were weighed together (left and right).

HISTOPATHOLOGY: Yes
Organs or tissues that were fixed in 4 % neutral buffered formaldehyde solution or in modified Davidson’s solution are listed in table No. 9. The assessement was done for all organs and animals for control and high dose groups. For low and median dose groups assessment of gross lesions was done in animals affected.
Statistics:
Please refer to table No. 10 for details on statistical methods.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
No test substance-related, adverse effects were obtained in any test group (60, 180 and 540 mg/kg bw/d).

Salivation shortly after treatment was observed in all male and female animals treated with 180 mg/kg bw/d and 540 mg/kg bw/d as well as in 9 male and 7 female animals of test group 1 (60 mg/kg bw/d) on several days of the application period.
From the temporary, short appearance immediately after dosing it was concluded that the finding was induced by a bad taste of the test substance or local affection of the upper digestive tract. The effect was related to the test substance but assessed as being non-adverse as no lesions in the upper digestive tract were observed in male and female animals during pathological examinations.
One female animal of the control group showed respiration sounds on study day 51. Since the animal belonged to the control group, a relation to the test substance was excluded.
Mortality:
no mortality observed
Description (incidence):
No animal died prematurely.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No test substance-related, adverse effects on body weight development were obtained in any test group (60, 180 and 540 mg/kg bw/d).
Mean body weights and body weight change values of male and female animals did not show any significant deviations to the control.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No test substance-related, adverse changes with regard to food consumption were observed.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
No test substance-related, adverse changes with regard to water consumption were observed.
Ophthalmological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related findings were observed.
All other apparent findings were also assessed as being incidental in nature since they occurred in control as well as in treated animals and did not show a dose-response relationship.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes among hematological parameters were observed.
At the end of the administration period, platelet counts were significantly decreased in males of test group 3 (540 mg/kg bw/d; 650 giga/L, -12 % compared to control group, p ≤ 0.01), but the mean was within the historical control range (males, platelets 592-768 giga/L).
In males of test groups 1 and 3 (60 and 540 mg/kg bw/d), relative basophil counts were significantly increased (60 mg/kg bw/d 78.6 % (mean 0.2 %) and 540 mg/kg bw/d 171.4 % (mean 0.4 %) compared to control group, p ≤ 0.05), and in females of test group 3 relative neutrophil counts were significantly decreased (-22.5 % (mean 15.4 %), p ≤ 0.01). However, all values were within historical control ranges (males, relative basophils 0.1-0.4 %; females, relative neutrophils 14.6-24.9 %).
Therefore, all mentioned alterations were regarded as incidental and not treatment-related.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes among clinical chemistry parameters were observed.
At the end of the administration period, glucose levels were significantly decreased in males of test groups 2 and 3 (180 mg/kg bw/d: -13.44 % (mean 5.62 mmol/L) p ≤ 0.01, 540 mg/kg bw/d: -11.82 % (mean: 5.72 mmol/L) p ≤ 0.05), but the means were within the historical control range (males, glucose 5.46-6.98 mmol/L).
In males of test group 1 (60 mg/kg bw/d), inorganic phosphate levels were significantly decreased, but the change was not dose-dependent.
Therefore, all mentioned alterations were regarded as incidental and not treatment-related.
Urinalysis findings:
no effects observed
Description (incidence and severity):
No treatment-related changes among urinalysis parameters were observed.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Deviations from "zero values" were obtained in several rats. However, as most findings were equally distributed between test-substance treated groups and controls, were without a dose response relationship or occurred in single rats only, these observations were considered to have been incidental.
Home cage observations: No test substance-related effects were observed.
Open field observations: No test substance-related effects were observed.
Sensorimotor tests/reflexes: No test substance-related effects were observed.
Quantitative parameters: No test substance-related effects were observed.

Regarding the overall motor activity as well as single intervals, no test substance-related deviations to the control animals were noted for male and female animals of test groups 1-3 (60, 180 and 540 mg/kg bw/d).
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Absolute weights
When compared with control group 0 (2.18 g, set to 100 %), the mean absolute brain weights were significantly reduced in all test groups (60 mg/kg bw/d: -4.2 % (2.09 g), 180 mg/kg bw/d: -3.5 % (2.11 g), 540 mg/kg bw/d: -4.7 % (2.08 g); all p ≤ 0.05). All other mean absolute weight parameters did not show significant differences when compared to the control group 0.

Relative organ weights
All mean relative weight parameters did not show significant differences when compared to the control group 0.

The significant, but only minimally decreased mean absolute brain weights in male animals of test groups 1 to 3 (60, 180 and 540 mg/kg bw/d) did not show a dose-response relationship. In addition, no significance was observable for the relative weights and all absolute values were within the historical control data (2.015 - 2.199 g). Thus, the changes were regarded not to be related to treatment.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
All findings occurred individually. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
All findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Thyroid hormones:
In males and females of test groups 1, 2 and 3 (60, 180 and 540 mg/kg bw/d) no treatment related alterations of T3, T4 and TSH levels were observed.
Key result
Dose descriptor:
NOAEL
Effect level:
540 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
no
Organ:
other: no adverse effect

Discussion

The test substance was administered orally by gavage to groups of 10 male and 10 female Wistar rats at dose levels of 0 (test group 0), 60 (test group 1), 180 (test group 2) and 540 mg/kg bw/d (test group 3) over a period of 3 months.

Regarding clinical examinations, signs of general systemic toxicity were not observed in male or female parental animals of test groups 1-3 (60, 180 and 540 mg/kg bw/d) during the entire study period.

Salivation after treatment was seen in all animals of test groups 2 and 3 (180 and 540 mg/kg bw/d) and sporadically in a few animals of test group 1 (60 mg/kg bw/d). From the temporary, short appearance immediately after dosing it is likely, that this finding was induced by a bad taste of the test substance or local affection of the upper digestive tract. This finding was not considered to be an adverse and toxicologically relevant effect.

Concerning clinical pathology, no treatment-related, adverse effects were observed up to a dose level of the compound of 540 mg/kg bw/d.

Regarding pathology, all findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

 

CONCLUSION

Under the conditions of this study the oral administration of the test substance by gavage to male and female Wistar rats over a period of 3 months did not reveal adverse signs of toxicity even at a dose level of 540 mg/kg bw/d.

Thus, the no observed adverse effect level (NOAEL) for was set to 540 mg/kg bw/d for male and female Wistar rats.

Additional information

OECD 422 study

Propylidynetrimethanol, ethoxylated, esters with acrylic acid, reaction products with 1-Butanamine, N-butyl- was administered daily in corn oil via gavage to groups of 10 male and 10 female Wistar rats at dose levels of 100, 300, and 1000mg/kg. Because of the premature death of 2 female animals, the high dose was reduced from 1000 to 600 mg/kg bw/d from study day 19 onwards. The duration of treatment covered 36 days in males and 51 days in females, including premating, mating, gestation and 4 days of lactation.

A detailed clinical observation was performed in all animals before initial test substance administration and at weekly intervals thereafter. Food consumption and body weight of the animals were determined in app. weekly intervals. Towards the end of the administration period a functional observational battery was performed and motor activity was measured in 5 animals per sex and test group. Clinicochemical and hematological examinations as well as urinalyses were also performed towards the end of the administration period in 5 animals per sex and test group. All animals were sacrificed by decapitation, under isoflurane anesthesia, and were assessed by gross pathology. Weights of selected organs were recorded and a histopathological examination was performed.

Of the high dose animals, one male and one female were found dead, and one female was sacrificed in moribund condition. Piloerection, smeared fur, respiratory sounds, poor general state, hunched posture, and semiclosed eyelids were observed in single animals of this group at different time points. Pathology revealed erosion/ulcer in the forestomach of all male and 9/10 female high dose animals, mild to severe diffuse or focal hyperplasia with hyperkeratosis in the forestomach of all animals, and mild to severe submucosal edema in the forestomach of 8 male and 3 female animals. Animals receiving 300mg/kg b.w. showed comparable signs of local irritation in the forestomach, though the incidence was clearly reduced. No clinical signs were noted in this group and in low dose animals, which also showed no local irritation in the stomach. Reproductive performance was unaffected in all groups. Though the clinical signs and deaths of high dose animals are most likely secondary to the severe irritation in the stomach, systemic toxicity cannot be completely excluded. Following the precautionary principle, the NOAEL was thus set to 300mg/kg b.w. for systemic effects, and to 100mg/kg for local irritation.

90 day oral repeated dose study

In a 90 -day repeated dose toxicity study according to OECD TG 408 and GLP Propylidynetrimethanol, ethoxylated, propoxylated, esters with acrylic acid (>1<6.5 E0) was administered orally by gavage to groups of 10 male and 10 female Wistar rats at dose levels of 0 (test group 0), 60 (test group 1), 180 (test group 2) and 540 mg/kg bw/d (test group 3) over a period of 3 months. Corn oil served as vehicle, control animals were dosed daily with the vehicle only.

Food consumption and body weight were determined weekly. The animals were examined for signs of toxicity or mortality at least once a day. Detailed clinical examinations in an open field were conducted prior to the start of the administration period and weekly thereafter. Ophthalmological examinations were performed before the beginning of the first administration and at the end of the administration period. A functional observational battery (FOB) as well as measurement of motor activity (MA) were carried out at the end of the administration period.

Clinico-chemical and hematological examinations as well as urinalyses were performed towards the end of the administration period. After the administration period, all animals were sacrificed and assessed by gross pathology. Organ weights were determined followed by histopathological examinations.

Analytical determination confirmed the stability of the test substance preparation in corn oil for a period of 7 days at room temperature and the correctness of the prepared concentrations (99 - 103 % of the nominal concentrations).

Regarding clinical examinations, signs of general systemic toxicity were not observed in male or female parental animals of test groups 1-3 (60, 180 and 540 mg/kg bw/d) during the entire study period.

Salivation after treatment was seen in all animals of test groups 2 and 3 (180 and 540 mg/kg bw/d) and sporadically in a few animals of test group 1 (60 mg/kg bw/d). From the temporary, short appearance immediately after dosing it is likely, that this finding was induced by a bad taste of the test substance or local affection of the upper digestive tract. This finding was not considered to be an adverse and toxicologically relevant effect.

Concerning clinical pathology, no treatment-related, adverse effects were observed up to a dose level of the compound of 540 mg/kg bw/d.

Regarding pathology, all findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Under the conditions of this study the oral administration of the test substance by gavage to male and female Wistar rats over a period of 3 months did not reveal adverse signs of toxicity even at a dose level of 540 mg/kg bw/d.

Thus, the no observed adverse effect level (NOAEL) for was set to 540 mg/kg bw/d for male and female Wistar rats.

Justification for classification or non-classification

No specific target organ toxicity was oberseved; only local irritation occured in the repeated dose study. Thus Propylidynetrimethanol, ethoxylated, esters with acrylic acid, reaction products with 1-Butanamine, N-butyl- does not have to be classified according to 67/548/EEC or CLP/EU-GHS.