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Administrative data

Description of key information

28-day oral Parental systemic NOAEL: at least 500 mg/kg.

28-day oral Parental local NOAEL: 50 mg/kg, based on adverse histopathological changes in the stomach (glandular and non-glandular) in males starting at 150 mg/kg.

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:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
31-July-2018 to 28-January-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:
2016
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
- Purity: UVCB; solid matter 41.4%; Correct for % solid matter
Species:
rat
Strain:
Wistar
Remarks:
Crl: WI(Han)
Details on species / strain selection:
The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: Yes
- Age at study initiation: 14-15 weeks old; Study males were slightly older than the standard age (14-15 weeks instead of 10-12 weeks) at initiation of dosing. As the test item was dosed undiluted, slightly older males were used to increase the actual dose volume.
- Weight at study initiation: Males: 332 to 393 g; Females: 217 to 256 g
- Housing:
On arrival and following the pretest (females only) and pre-mating period, animals were group housed, up to 5 animals of the same sex and same dosing group together. During the mating phase, males and females were cohabitated on a 1:1 basis. During the post-mating phase, males were housed in their home cage with a maximum of 5 males/cage. Females were individually housed. During the lactation phase, females were housed with pups, except during locomotor activity monitoring of the dams, when the pups were kept warm in their home cage using bottles filled with warm water. In order to avoid hypothermia of pups, pups were not left without their dam or a bottle filled with warm water for longer than 30-40 minutes. During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage without cage enrichment, bedding material, food and water.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 22°C
- Humidity (%): 42 to 73%.
- Air changes (per hr): 10 or more per hour
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Details on route of administration:
The oral route of administration was selected because this is a possible route of human exposure during manufacture, handling or use of the test item.
Vehicle:
unchanged (no vehicle)
Remarks:
Test-item treated animals were received undiluted test item and consequently, no vehicle was used.
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was administered as a solution as received. The test item has 41.4% solid matter and as such test item concentration was corrected for % solid matter and specific gravity (factor: 1.094).
Analytical verification of doses or concentrations:
no
Remarks:
The test item was used as received from the Sponsor; therefore, samples for dose formulation analysis were not collected by the Test Facility.
Details on analytical verification of doses or concentrations:
The test item was used as received from the Sponsor; therefore, samples for dose formulation analysis were not collected by the Test Facility.
Duration of treatment / exposure:
A minimum of 28 days. Males were treated for 29 days, i.e. 14 days prior to mating, during mating and up to and including the day before scheduled necropsy. Females that delivered were treated for 50-55 (most females) or 63 days (one female at 150 mg/kg), i.e. 14 days prior to mating (with the objective to cover at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and 13-15 days after delivery, up to and including the day before scheduled necropsy. Females which failed to deliver or had a total litter loss were treated for 53 or 42 days, respectively.
Frequency of treatment:
Once daily, 7 days per week. Female nos. 50 (Control), 58 and 60 (Group 2), were not dosed on one occasion as these females were littering at the time of dosing. The omission of one day of dosing over a period of several weeks was not considered to affect the toxicological evaluation.
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
150 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10/sex/dose
Control animals:
yes, sham-exposed
Details on study design:
- Dose selection rationale: Dose selection was based on the results of the dose range finder. In the range finder dose-limiting effects were observed after short-term treatment (10 days) at 1000 mg/kg [i.e. increase in mean relative liver weight of 39% compared to historical control mean, combined with body weight loss in one female, hunched posture and piloerection in all females (clinical observations) and irregular surface of the forestomach in all females (macroscopic examinations)].
- Fasting period before blood sampling for clinical biochemistry: Blood of F0-animals (except for animal nos. 17 and 66 which were sacrificed in extremis and female no. 63 with total litter loss) was collected on the day of scheduled necropsy. Samples were collected, between 7.00 and 10.30 a.m., from the retro-orbital sinus under anesthesia using isoflurane in the animal facility. Due to clotting of non-serum samples of individual animals, additional blood samples were obtained in necropsy room. After collection all samples were transferred to the appropriate laboratory for analysis. F0-males were fasted overnight with a maximum of 24 hours before blood sampling, but water was available. F0-females were not fasted overnight.
Positive control:
Not examined
Observations and examinations performed and frequency:
Five animals/sex/group were selected for functional tests, clinical pathology, collection of full list of organs/tissues at macroscopic examination, organ weights (full list) and histopathology (full list).

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily, in the morning and at the end of the working day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once daily, beginning during the first administration of the test item and lasting throughout the dosing periods up to the day prior to necropsy

BODY WEIGHT: Yes
- Time schedule for examinations: weighed on the first day of treatment (prior to dosing), and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13. Terminal body weights were recorded on the day of scheduled necropsy (fasted for males and non-fasted for females).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Not feeding study. Food consumption was quantitatively measured weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not drinking water study. Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was expected or noted at visual inspection of the water bottles.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day of scheduled necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: F0-males were fasted overnight with a maximum of 24 hours before blood sampling, but water was available. F0-females were not fasted overnight.
- How many animals: 5/sex/group

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day of scheduled necropsy
- Animals fasted: F0-males were fasted overnight with a maximum of 24 hours before blood sampling, but water was available. F0-females were not fasted overnight.
- How many animals: 5/sex/group

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Functional tests were performed on the selected 5 males during Week 4 of treatment and the selected 5 females during the last week of lactation (i.e. PND 8-11).
- Dose groups that were examined: All groups
- Battery of functions tested: sensory activity (hearing ability, pupillary reflex, static righting reflex), grip strength, motor activity

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (all animals were examined for necropsy).

HISTOPATHOLOGY: Yes
Based on treatment-related changes in tissues, the stomach of selected males and females of low and mid-dose groups (5/sex/group) were examined.

Male no. 17 (Group 2) and female no. 66 (Group 3) were euthanized as per Test Facility SOPs. These animals were deeply anaesthetized using isoflurane and subsequently exsanguinated. They underwent necropsy, and specified tissues were retained but not weighed.
Other examinations:
In addition, the following reproduction/developmental parameters were determined: mating and fertility indices, precoital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio, live litter size and early postnatal pup development (mortality, clinical signs, body weight, anogenital distance, areola/nipple retention, serum level of thyroid hormone T4 in PND 14-16 pups and macroscopy).
Statistics:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% or 5% levels.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Slight salivation after dosing, considered a physiological response to the irritant test item rather than a sign of systemic toxicity, occurred among animals treated at 150 or 500 mg/kg in a dose-related manner. Rales occurred transiently in several treated males (at all dose levels, without a dose-related trend). This respiratory symptom was attributed to the oral gavage administration of the irritant test item.
Mortality:
mortality observed, treatment-related
Description (incidence):
Two treated animals (a low-dose male and a mid-dose female) were sacrificed prematurely for humane reasons. These premature deaths were regarded to be related to the oral gavage administration of the irritant test item.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
A finding of note was the weight loss of one high-dose male (no. 39). This male lost 11% of its initial weight during treatment weeks 1-3 (no further loss in the next week). This was accompanied by hunched posture and laboured respiration on Days 7-11/10. Macroscopic and microscopic examination revealed no obvious cause of the weight loss (no. 39 showed similar gastric changes as most other high-dose males which grew normally; microscopy was limited to the stomach). The weight loss of no. 39 was considered not to reflect an adverse effect of the test item on body weight (gain) since such weight loss occurred in only a single (surviving) animal.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Statistically significant differences between treated rats and controls were limited to a lower mean corpuscular haemoglobin (MCH) value in high-dose (500 mg/kg) males (relative difference: -4%). As main red blood cell parameters (haemoglobin concentration, number of red blood cells) of high-dose males were not affected, the lower MCH was considered not to reflect an adverse effect of the test item on red blood cells.
The abnormal white blood cell values noted in low-dose female no. 59 (higher numbers of neutrophils and total white blood cells, lower number of lymphocytes) were likely to be related to a thymoma (which was unrelated to treatment).
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Test item-related changes in clinical pathology parameters consisted of increases in cholesterol and bile acids and a decrease in total protein in males treated at 500 mg/kg. In the absence of associated adverse anatomic pathology alterations, these changes were regarded as non-adverse.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Organ weight changes at 500 mg/kg consisted of increased absolute and relative weights of the liver in both sexes and increased relative weights of the thymus and kidneys in males. These organ weight changes were regarded as non-adverse as they were not associated with histopathological alterations.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Test item-related macroscopic findings were present in the stomach of one male at 150 mg/kg and most males 500 mg/kg. These findings consisted of an irregular surface of the forestomach (in 1/10 and 8/10 males at 150 and 500 mg/kg, respectively), thickened limiting ridge and/or glandular stomach (8/10 males at 500 mg/kg), and red focus/foci in the glandular stomach (5/10 males at 500 mg/kg).
The remainder of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain. These findings were therefore considered to be unrelated to treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
The microscopic correlates were inflammation and erosions in the forestomach and glandular stomach, hyperkeratosis (and a few males with squamous cell hyperplasia) in the forestomach, and submucosal edema and hemorrhages in the glandular stomach.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
Mean serum level of T4 in males treated at 500 mg/kg was 16% lower than the concurrent control mean. However, as statistical significance was not achieved and mean T4 value in 500 mg/kg treated males remained within the historical control range, this change was attributed to biological variation.
Details on results:
Treated males showed many inflammatory and correlating findings as described below.
Forestomach (non-glandular):
- Inflammation (mononuclear, epithelial and/or lymphogranulocytic, subepithelial) up to moderate degree starting at 150 mg/kg.
- Hyperkeratosis up to moderate degree starting at 50 mg/kg.
- Squamous cell hyperplasia up to slight degree at 500 mg/kg.
- Moderate erosion in a single male treated at 150 mg/kg.
- Increased vacuolation up to moderate degree at the limiting ridge starting at 150 mg/kg.
Glandular stomach:
- Increased inflammation (submucosal, eosinophilic or lymphogranulocytic) up to moderate degree starting at 150 mg/kg.
- Slight erosion in a single male at 500 mg/kg.
- Hemorrhages up to slight degree at 500 mg/kg.
- Hypertrophy of mucous cells up to slight degree starting at 50 mg/kg.
- Eosinophilic globules up to slight degree at 500 mg/kg.
- Submucosal edema up to moderate degree starting at 50 mg/kg.
Key result
Dose descriptor:
NOAEL
Remarks:
parental systemic
Effect level:
500 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios
other: clinical pathology - non-adverse
Key result
Dose descriptor:
NOAEL
Remarks:
Parental local
Effect level:
50 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
gross pathology
histopathology: non-neoplastic
Critical effects observed:
yes
Lowest effective dose / conc.:
150 mg/kg bw/day (nominal)
System:
gastrointestinal tract
Organ:
stomach
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

The gastric findings in males at 50 mg/kg (minimal hyperkeratosis at the limiting ridge, and hypertrophy of mucous cells or submucosal edema in the glandular stomach) were considered to be non-adverse based on minimal severity and lack of degenerative/proliferative changes.

Test item-related changes in clinical pathology parameters consisted of increases in cholesterol and bile acids and a decrease in total protein in males treated at 500 mg/kg. In the absence of associated adverse anatomic pathology alterations, these changes were regarded as non-adverse.

Conclusions:
Based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the following No Observed Adverse Effect levels (NOAELs) of the test substance were established:
Parental systemic NOAEL: at least 500 mg/kg.
Parental local NOAEL: 50 mg/kg, based on adverse histopathological changes in the stomach (glandular and non-glandular) in males starting at 150 mg/kg.
Executive summary:

In a combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, Wistar Han rats were treated with undiluted test substance by daily oral gavage at dose levels of 50, 150 and 500 mg solid matter/kg (10 rats/sex/dose level). Concurrent controls (10 rats/sex) received water (Elix). Males were treated for two weeks prior to mating, during mating, and up to termination (for 29 days). Females that delivered offspring were treated for two weeks prior to mating, during mating, during post-coitum, and 13-15 days of lactation (mostly for 50-55 days). A female without offspring (non-mated) was treated for 53 days and a female which had a total litter loss was treated for 42 days.

 

Two treated animals (a low-dose male and a mid-dose female) were sacrificed prematurely for humane reasons. These premature deaths were regarded to be related to the oral gavage administration of the irritant test item.

 

Slight salivation after dosing, considered a physiological response to the irritant test item rather than a sign of systemic toxicity, occurred among animals treated at 150 or 500 mg/kg in a dose-related manner. Rales occurred transiently in several treated males (at all dose levels, without a dose-related trend). This respiratory symptom was attributed to the oral gavage administration of the irritant test item.

 

Male rats showed morphological changes in the stomach (glandular and non-glandular) which were regarded as local effects resulting from the irritant properties of the test item. Although gastric changes occurred at all dose levels, in a dose-related manner, the combination of the severity and the degenerative/proliferative nature of the gastric lesions at 150 and 500 mg/kg was considered to be adverse. Main findings in the forestomach consisted of inflammation (epithelial mononuclear infiltrates and subepithelial lymphogranulocytic infiltrates), hyperkeratosis and squamous cell hyperplasia. Main findings in the glandular stomach were increased inflammation (submucosal, eosinophilic or lymphogranulocytic), mucosal hemorrhage, hypertrophy of mucous cells, eosinophilic globules and submucosal edema). Erosion in the glandular or non-glandular stomach and increased epithelial vacuolation at the limiting ridge were observed incidentally. Macroscopic changes consisted of an irregular surface of the forestomach (in one 150 mg/kg male and most 500 mg/kg males), thickened limiting ridge and/or glandular stomach (most 500 mg/kg males), and red focus/foci in the glandular stomach (half of the 500 mg/kg males). The gastric findings in males at 50 mg/kg (minimal hyperkeratosis at the limiting ridge, and hypertrophy of mucous cells or submucosal edema in the glandular stomach) were considered to be non-adverse based on minimal severity and lack of degenerative/proliferative changes.

 

Organ weight changes at 500 mg/kg consisted of increased absolute and relative weights of the liver in both sexes and increased relative weights of the thymus and kidneys in males. These organ weight changes were regarded as non-adverse as they were not associated with histopathological alterations.

 

Test item-related changes in clinical pathology parameters consisted of increases in cholesterol and bile acids and a decrease in total protein in males treated at 500 mg/kg. In the absence of associated adverse anatomic pathology alterations, these changes were regarded as non-adverse.

No reproduction or developmental toxicity was observed up to the highest dose level tested (500 mg/kg).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
500 mg/kg bw/day
Study duration:
subacute
Species:
rat

Additional information

Justification for classification or non-classification

Based on the results of combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the systemic NOAEL was established as 500 mg/kg bw/day and local NOAEL was established as 50 mg/kg bw/day, based on adverse histopathological changes in the stomach of males starting at 150 mg/kg bw/day. Therefore, according to EC 1272/2008 as amended, the test substance does not meet the criteria for STOT RE classification.