Registration Dossier

Administrative data

Description of key information

An oral 28 d toxicity study according to OECD TG 407 in the rat, revealed a NOAEL (systemic toxicity) = 40 mg/kg body weight/day (reference 7.5.1 -1)



A Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in the rat revealed an NOAEL (systemic toxicity and reproduction parameters) = 15 mg/kg body weight/day (reference 7.5.1 -2).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2012-02-06 to 2012-10-23
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)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Wistar
Remarks:
Crl.WI (Han)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River, Germany
- Age at study initiation: (P) Males 11 wks; Females 10 weeks (F1) 4 days
- Weight at study initiation: (P) Males: 313 ( 296 – 337) g; Females: 205 ( 186 – 232) g; (F1) Males: 5.89 - 6.35 g; Females: 5.65 - 6.05 g
- Fasting period before study: no
- Housing: single-housed in type III Makrolon® cages
- Diet: Provimi Kliba 3433.0 ad libitum
- Water: tap water ad libitum
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20.5 – 23.0 ºC
- Humidity: 37.8 – 64.5 %
- Air changes (per hr): 10 times
- Photoperiod (hrs dark / hrs light): 12 / 12 hours
Route of administration:
oral: gavage
Vehicle:
other: 0.25% aqueous hydroxypropyl methylcellulose (Methocel® K4M Premium)
Details on oral exposure:
- Justification for use and choice of vehicle: standard vehicle used at testing facility, without toxic properties
- Concentration in vehicle: 0, 3, 9, and 27 mg/mL
- Amount of vehicle: 5 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The quantification of the test material in the dosing formulations was performed using a HPLC method with UV detection. During the course of the study each dosing formulation (including control) of two preparation periods was sampled and analyzed at the beginning and the end of usage, resulting in 4 time points of formulation analysis.
Duration of treatment / exposure:
males: 6 weeks
females: up to 7 weeks
Frequency of treatment:
daily (7d / week)
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
15 mg/kg bw/day (actual dose received)
Dose / conc.:
45 mg/kg bw/day (actual dose received)
Dose / conc.:
135 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
24 (12 m / 12 f)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The test material has been investigated in a subacute 4-week toxicity study with daily oral administration of 40, 200, and 1000 mg/kg bw. A control group was treated similarly with the vehicle, corn oil. Recovery animals for 2 weeks were kept in the control and 200 mg/kg bw groups. All animals treated with 1000 mg/kg bw were killed in extremis after 6 days of treatment and 2 more days without treatment. One female treated with 200 mg/kg bw died spontaneously on treatment day 21, one hour after application without any previous symptoms. The cause of death could no be established. Another female of this group and 2 control females died on the day of scheduled necropsy after blood sampling. Death was attributed to the blood sampling procedure. One female treated with 200 mg/kg bw died on the first day of the recovery period, cause of death could no be established. Treatment-related findings were generally restricted to reduced food consumption and reduced body weight and body weight gain in all animals treated with 200 or 1000 mg/kg. Decreased hematocrit and hemaglobin in males and females treated with 200 mg/kg bw and increased hemaglobin concentration distribution width in females of the 200 mg/kg group were present after 4 weeks of treatment and also after recovery. Decreased erythrocyte numbers in both sexes treated with 200 mg/kg bw were also present after recovery, however, males were within internal laboratory range.In clinical chemistry, main changes were increased creatinine, decreased phospholipids and increased alanine aminotransferase with alanine aminotransferase showing a tendency of recovery. Other affected parameters included increased aspartate aminotransferase indicating changes in the liver and kidneys. A slightly increased number of leucocytes in urine was seen after 4 weeks of treatment in females at 200 or 40 mg/kg bw with a tendency of reversibility during recovery (200 mg/kg bw). In the absence of macroscopic or microscopic correlating findings, it is considered test-item related but not adverse. Histologically, alterations in the liver, adrenal glands, and testes were found in animals of the 200 mg/kg bw group. Hepatocellular hypertrophy correlated with increased liver weights and enlarged livers. It was not present after recovery and was considered to be of metabolic nature due to the absence of any further lesion. Cortical atrophy in adrenal glands often combined with focal/multifocal subacute inflammation was recorded in both sexes treated with 200 mg/kg bw. Cortical atrophy with focal vacuolar degeneration was recorded in one female treated with 200 mg/kg bw. These findings were also present after the recovery period. Cortical atrophy corresponds to macroscopically reduced size of adrenal glands and decreased adrenal weights. Findings in the adrenals are considered to be test-item related. Focal/multifocal multinucleated spermatid giant cells at minimal severity were observed in 2 animals of the 200 mg/kg bw group. This finding was not persistent after recovery. The presence of focal/multifocal multinucleated spermatid giant cells is often associated with early stages of atrophic seminiferous tubules. Based on the results of this study, 40 mg/kg bw could be established as NOAEL (no observed adverse effect level).
Positive control:
not applicable
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: checked twice daily at working days and once at off days, at the same time (s) each day

Functional observational battery: Yes
- Time schedule: pre-dose, day 7, day 29 (m) or day 4 post partum (f)

Motor Activity: yes
- Time schedule: day 29 (m) or day 4 post partum (f)

BODY WEIGHT: Yes
- Time schedule for examinations: before treatment and thereafter once a week. From the date of mating the females will be weighed daily, within 24 hours of parturition (day 0 post-partum), and on day 4 post-partum.

FOOD CONSUMPTION AND COMPOUND INTAKE: yes, once a week before mating of all animals. No food consumption was measured for the males further on. Food consumption measurements of the females were started again after the positive vaginal smears, then on days 7, 14, 21 post coitum and again on day 4 post-partum

WATER CONSUMPTION AND COMPOUND INTAKE: No

OTHER: Hematology and Clinical Chemistry undertaken on day 13
- parameters hematology: Red blood cells (erythrocytes), Hemoglobin, Hematocrit, Mean cell volume, Mean hemoglobin content, Mean hemoglobin concentration, Platelets, Reticulocytes, Absolute number of reticulocytes, White blood cells (leukocytes), Absolute number of neutrophilic granulocytes, Absolute number of lymphocytes, Absolute number of eosinophilic granulocytes, Absolute number of basophilic granulocytes, Absolute number of monocytes, Absolute number of large unstained cells, Neutrophilic granulocytes, Lymphocytes, Eosinophilic granulocytes, Basophilic granulocytes, Monocytes, Large unstained cells, Prothrombin time, Prothrombin time, Partial thromboplastin time
- parameters clinical chemistry: Sodium, Potassium, Calcium, Chloride, Inorganic phosphate, Glucose, Urea, Creatinine, Total bilirubin, Cholesterol, Triglycerides, Bile acids, Total protein, Albumin, A/G ratio, Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase, Urinalysis, pH value, Protein, Glucose, Bilirubin, Blood, Urobilinogen, Ketone, Sediment, Specific gravity
Sacrifice and pathology:
SACRIFICE
The male animals were sacrificed on day 43, the pregnant females after day 4 post-partum of the study.

GROSS NECROPSY
All adult rats were necropsied and examined for gross pathological alterations. The rats were killed by anesthesia with a carbon dioxide air mixture and exsanguination after opening of the abdominal vessels.

ORGAN WEIGHTS
Terminal body weight (after exsanguination), Heart, Liver, Kidneys (together), Spleen, Thymus,Testes (together), Prostate, Ovaries (together), Uterus, Adrenals (together after fixation), Thyroids with Parathyroids (together after fixation), Brain (after fixation), Seminal vesicles, Epididymides (together)

HISTOPATHOLOGY
(Groups 1 and 4 examined, intermediates as required), Adrenal (2), Bone with bone marrow (sternum, femur), Brain (cerebrum, cerebellum, pons, brain stem), Eye (2), Heart, Intestine, large Cecum, Colon, Rectum, Intestine, small Duodenum, Jejunum, Ileum, Kidney (2), Liver (left lateral and right medial lobe), Lung (with mainstem bronchi), Lymph nodesmandibular (2), mesenteric Mammary gland (inguinal), Muscle, skeletal (thigh), Nerve, sciaticPeyer’s Patches, Reproductive organs, female Ovary (2), Oviduct (2), Uterus (cornu/corpus/cervix), Vagina, Reproductive organs, male Epididymis (2), Prostate, Seminal vesicle, Testis (2), Spinal cord (cervical, thoracal, lumbal), Spleen, Stomach (proventricular, fundic, pyloric), Thymus, Thyroid (2), Trachea, Urinary bladder, All tissues showing abnormality
Statistics:
Statistical test
- Body weight and body weight gain: DT, 2-sided
- Food consumption (males and females): DT, 2-sided
- FOB numerical parameters: Kruskal Wallis (2-sided) + Wilcoxon
- Motor activity: Kruskal Wallis (2-sided) + Wilcoxon
- Organ weights: DT, 2-sided
- Hematological and clinical chemistry parameters: Wilcoxon (2-sided) + Bonferroni-Holm
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
All animals survived the treatment period. No treatment-related clinical signs were observed in males at any time point. In the females, no treatment-related signs were observed during the pre-mating period, during pregnancy, but increased incidences of hair loss was observed in group 4 (135 mg/kg) females.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight and body weight gain was not affected in males treated daily orally for a period of 42 days at doses of up to 135 mg/kg at any time point (including pre-mating, end of mating or end of treatment period). During the pre-mating period, the females did not show statistically significant differences of body weight between dose- and control group, however, body weight gain was decreased in group 4 (135 mg/kg) females during the 2nd week of treatment (day 7-14)compared to control. During pregnancy, body weight was decreased (without statistical significance) in group 3 (45 mg/kg) from day 6 onwards, and with statistical significance in group 4 (135 mg/kg) females from study day 8 onwards (with the exception of day 22+23) compared to control. Body weight gain was decreased until gestation day 20, not always statistically significant. No treatment-related effects on food consumption were observed.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Statistically significant alterations were seen in some clinical chemistry parameters but were all within the known internal reference interval, low in degree and therefore considered incidental and not treatment-related.
Urinalysis findings:
no effects observed
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
During the functional observational battery no treatment-related relevant changes were observation days 0, 7, 29 (males only), or day 4 p.p. (females only) in the autonomous and senso-motor domains. In the neuromuscular domain, hind limb foot splay showed a slight trend of reduction in all treatment group males (15 to 135 mg/kg) on study day 29 compared to control. Group 4 females (135 mg/kg) on day 4 p.p. showed decreased hind limb foot splay as well. However, the observation was without statistical significance. In the central nervous domain a trend of reduction of the raising number in group 4 females (135 mg/kg) on day 7, and in group 3 (45 mg/kg) and 4 (135 mg/kg) on day 4 p.p. was noted. Body temperature was slightly reduced (statistically significant) in group 4 (135 mg/kg) females on day 7 (pre-mating period) and day 4p.p. compared to control.

Motor activity (number of counts) measured on day 29 in males and on day 4 p.p. in females was unaffected at all dose levels (up to 135 mg/kg). In group 4 (135 mg/kg) females on day 4 p.p. a trend towards slightly reduced rearing numbers and rearing time was noted.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Adrenal weights of both sexes were absolutely and relatively reduced at 45 mg/kg and 135 mg/kg.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
At necropsy of the adult animals only spontaneous findings were observed.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
At histopathology, group 4 (135 mg/kg bw/d) showed pronounced cortical atrophy of the adrenal gland combined with endothelial cell activation and mononuclear infiltrates in both sexes.
In the liver, increased centrilobular single cell necrosis was observed in eight females whereas males were not affected. In group 3 (45 mg/kg bw/d), cortical atrophy of the adrenal gland was slightly less pronounced than in group 4 (135 mg/kg bw/d) in both sexes. Endothelial activation in females was comparable to group 4 (135 mg/kg bw/d) whereas in males only three animals showed endothelial activation. Mononuclear infiltrates were observed in 4/24 rats.
In the liver, increased amounts of single cell necrosis were detected in one male and three females. In group 2 (15 mg/kg), three females showed a minimal to slight degree of endothelial activation, and four females and one male showed mononuclear infiltrates. These findings are not considered to be adverse due to the slight degree and small animal number affected. In the liver three female rats had a minimal degree of increased centrilobular single cell necrosis which is also not considered to represent an adverse effect. Extramedullary hematopoiesis was observed in the adrenal gland of females of all dose-groups including controls. This finding is considered to be related to the former pregnancy of those rats.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Details on results:
Please refer to result tables attached.
Key result
Dose descriptor:
NOAEL
Effect level:
15 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical signs
histopathology: non-neoplastic
organ weights and organ / body weight ratios
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
45 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
45 mg/kg bw/day (actual dose received)
System:
endocrine system
Organ:
adrenal glands
Treatment related:
yes
Dose response relationship:
yes
Conclusions:
The No Adverse Effect Level of the test item regarding systemic toxicity was determined to be 15 mg/kg bw/d.
Executive summary:

The test item was administered orally by gavage, once daily, 7 times a week to 3 groups of male and female Crl:WI (Han) rats at doses of 15, 45 or 135 mg/kg. Males received the test item for 6 weeks, whereas females had a treatment period of up to 7 weeks.
A similarly constituted control group received the vehicle, 0.25% aqueous hydroxypropyl methylcellulose (Methocel® K4M Premium), and served to generate contemporary control data. All dose groups consisted of 12 male and 12 female rats each.
Males and females were first treated for 14 days separately, thereafter, a mating period of maximally 2 weeks started. As soon as the females showed a positive sperm result, animals were separated again. The males were treated until day 42 (6 weeks treatment duration) and the females were treated throughout birth of pups until necropsy (after day 4 post partum, approximately 7 weeks treatment duration). One control female had a late conception and was vehicle-treated for approximately 8 weeks.
The rats were single housed, except for the mating period, under conventional conditions. During the mating period of maximally 2 weeks, animals were paired (1 male and 1 female per cage) within the dose groups.

The concentration of the test material in the dosing formulations was within the predefined acceptance limits (±15 % of the nominal concentration). No test material was detected in the control formulations.

All animals survived the treatment period.
No treatment-related clinical signs were observed in males at any time point. In the females, no treatment-related signs were observed during the pre-mating period, during pregnancy, but increased incidences of hair loss was observed in group 4 (135 mg/kg) females.
Body weight and body weight gain was not affected in males treated daily orally for a period of 42 days at doses of up to 135 mg/kg at any time point (including pre-mating, end of mating or end of treatment period). During the pre-mating period, the females did not show statistically significant differences of body weight between dose- and control group, however, body weight gain was decreased in group 4 (135 mg/kg) females during the 2nd week of treatment (day 7-14) compared to control. During pregnancy, body weight was decreased (without statistical significance) in group 3 (45 mg/kg) from day 6 onwards, and with statistical significance in group 4 (135 mg/kg) females from study day 8 onwards (with the exception of day 22+23) compared to control. Body weight gain was decreased until gestation day 20, not always statistically significant. No treatment-related effects on food consumption were observed.
During the functional observational battery no treatment-related relevant changes were observed on days 0, 7, 29 (males only), or day 4 p.p. (females only) in the autonomous and sensomotoric domains. In the neuromuscular domain, hind limb foot splay showed a slight trend of reduction in all treatment group males (15 to 135 mg/kg) on study day 29 compared to control. Group 4 females (135 mg/kg) on day 4 p.p. showed decreased hind limb foot splay as well. However, the observation was without statistical significance. In the central nervous domain a trend of reduction of the raising number in group 4 females (135 mg/kg) on day 7, and in group 3 (45 mg/kg) and 4 (135 mg/kg) on day 4 p.p. was noted. Body temperature was slightly reduced (statistically significant) in group 4 (135 mg/kg) females on day 7 (pre-mating period) and day 4 p.p. compared to control.
Motor activity (number of counts) measured on day 29 in males and on day 4 p.p. in females was unaffected at all dose levels (up to 135 mg/kg). In group 4 (135 mg/kg) females on day 4 p.p. a trend towards slightly reduced rearing numbers and rearing time was noted. Overall, only single behavioral parameters within the different domains were slightly affected without statistical significance. The effect on rearing numbers and time together with the reduced body weight is probably a treatment-related clinical effect on the general condition of these females.
Hematology, coagulation, clinical chemistry, and urinary parameters were measured on study day 13. Statistically significant alterations were seen in some clinical chemistry parameters but were all within the known internal reference interval, low in degree and therefore considered incidental and not treatment-related.
At necropsy of the adult animals only spontaneous findings were observed. Female high dose rats (135 mg/kg) exhibited a terminal body weight reduction of approximately 8 %. Adrenal weights of both sexes were absolutely and relatively reduced at 45 mg/kg and 135 mg/kg. At histopathology, group 4 (135 mg/kg) showed pronounced cortical atrophy of the adrenal gland combined with endothelial cell activation and mononuclear infiltrates in both sexes. In the
liver, increased centrilobular single cell necrosis was observed in eight females whereas males were not affected.
In group 3 (45 mg/kg), cortical atrophy of the adrenal gland was slightly less pronounced than in group 4 (135 mg/kg) in both sexes. Endothelial activation in females was comparable to group 4 (135 mg/kg) whereas in males only three animals showed endothelial activation. Mononuclear infiltrates were observed in 4/24 rats. In the liver, increased amounts of single cell necrosis were detected in one male and three females.
In group 2 (15 mg/kg), three females exhibited slight degree of endothelial activation, and four females and one male showed mononuclear infiltrates. These findings are not considered to be adverse due to the slight degree and small animal number affected. In the liver three female rats had a minimal degree of increased centrilobular single cell necrosis which is also not considered to represent an adverse effect.
Extramedullary hematopoiesis was observed in the adrenal gland of females of all dose-groups including controls. This finding is considered to be related to the former pregnancy of those rats.

In conclusion, daily oral treatment by gavage of 15 mg/kg of the test item to rats according to the study design was tolerated whereas 45 or 135 mg/kg bw/d caused cortical atrophy of the adrenal glands was observed in both sexes. A statistical significant weight reduction of both absolute and relative weights correlates to this effect.
Clinically, all doses (15, 45, and 135 mg/kg) were tolerated over a treatment period of 42 days in males. The females, treated for approximately up to 7 weeks showed clinically an increased incidence of hair loss in group 4 (135 mg/kg) and statistically significant body weight decreases in group 4 (135 mg/kg) during the gestation period. No effects on clinical pathology parameters were noted in both genders of any dose group on study day 13. Behavioral parameters (FOB and
motor activity) showed some slight changes in females of group 4 (135 mg/kg) on days 7 and 4p.p. with a slight reduction of rearing time and number, plus a body temperature decrease, however, a treatment-relationship could not be defined equivocally.

Therefore, the no adverse effect level (NOAEL) regarding systemic toxicity is considered to be 15 mg/kg bw/d.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
15 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Two GLP studies performed according to OECD TG with highest reliability provide a high quality dataset for evaluation.
System:
other: glandular: adrenal gland
Organ:
adrenal glands

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Two experimental studies were performed with the test substance:

A subacute toxicity study with oral treatment of rats with doses of 1000, 200, and 40 mg/kg bw/d for 28 days and a 14 -day treatment-free recovery period according to OECD 407 and a combined repeated dose toxicity and reproduction screening study with oral treatment of rats with doses of 135, 45 and 15 mg/kg bw/d for 42 days (males) and up to 49 days (females).

Oral 28 -d repeated dose toxicity study

Administration of the test item to rats at a dose of 1000 mg/kg body weight for 28 days was not tolerated and the animals had to be killed in extremis after 6 days of treatment. Doses of 200 mg/kg/day resulted in 3 unscheduled deaths: 1 rat died due to the blood sampling procedure; 2 further rats died in the course of the study without showing any clincal symptoms or any histopathological correlates which could be attributed to the death of the animals. Therefore, the cause of these two deaths cannot be unequivocally attributed to the treatment. No treatment-related deaths were observed at doses lower than 200 mg/kg in any of the studies.

Animals treated with 200 mg/kg for 28 days showed reduced food consumption and reduced body weights; treatment with 40 mg/kg for up to 28 days did not cause any clinical effects.

In animals treated with 200 mg/kg bw for 28 days, a small but statistically significant changes in hematology (hematocrit, hemoglobin, erythrocytes) and clinical biochemistry parameters (creatinine, phospholipids, ALAT and ASAT) were observed. Doses of 135, 45 and 15 mg/kg for 42 days did not cause any changes in clinical pathology parameters in both genders.

Histologically, alterations in the liver, adrenal glands and testes were found in animals treated with 200 mg/kg/day for 28 days. Hepatocellular hypertrophy correlated with increased liver weights and enlarged livers. It was not present after recovery and was considered to be of metabolic nature based on the absence of any further lesion. Focal/multifocal multinucleated spermatid giant cells at minimal severity were observed in two animals treated with 200 mg/kg/day. This finding was not persistent after recovery and is thus not considered significant.

It should be noted that this study was already evaluated by the German BAUA during the registration under ChemG. BAUA agreed with the scientific interpretation of the results presented.

Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test

Doses of 15, 45, and 135 mg/kg were tolerated over a treatment period of 42 days in males. The females, treated for approximately up to 7 weeks showed an increased incidence of hair loss and a statistically significant body weight decreases at 135 mg/kg during the gestation period. Behavioral parameters (FOB and motor activity) showed some slight variations in females treated with 135 mg/kg, however, a treatment-relationship could not be defined equivocally.

Increased centrilobular single cell necrosis was observed in eight females whereas males were not affected after 6 weeks treatment with 135 mg/kg. Cortical atrophy in adrenal glands with focal/multifocal subacute inflammation or with focal vacuolar degeneration (1 animal) was recorded in animals treated with 200 mg/kg/day. Cortical atrophy of the adrenal gland combined with endothelial cell activation and mononuclear infiltrates was observed in rats of both sexes at doses of 135 and 45 mg/kg after 6 weeks of treatment. Cortical atrophy corresponds to macroscopically reduced adrenal glands and decreased adrenal weights.

In the low dose groups of both studies, ie. 40 mg/kg for 4 weeks or 15 mg/kg for 6 weeks, no adverse findings were identified.

Conclusion
In conclusion, the no adverse effect level (NOAEL) regarding systemic toxicity is considered to be 15 mg/kg after 6 weeks treatment. In both well documented studies, clear dose-response relationships could be established with no adverse effects at the low dose, moderate sub-lethal effects at the mid dose and clear toxic effects at the high dose. A comprehensive picture of the sub-lethal effects of the test item was obtained and sound NOAELs were derived.

The no adverse effect level (NOAEL) regarding systemic toxicity is considered to be 15 mg/kg after 6 weeks treatment.

Both studies are considered adequate for Classification and Labelling as well as for robust risk assessment and risk management.


Justification for classification or non-classification

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

 

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008.

Test item related toxicological effects after repeated oral dosing to rats were observed in the combined subacute reproduction screening study at daily doses of 45 mg/kg bw/d. 15 mg/kg bw was identified as NOAEL. At 45 mg/kg bw, changes in adrenal glands were found which are considered to be of relevance for classification and labelling according to CLP. According to CLP, a classification with specific target organ toxicity after repeated exposure is required in case relevant adverse effects were found at doses in the range 10 -100 mg/kg bw/d (oral dose, rat, category 2) after a 90 day exposure. For studies with exposure duration of 28 days the ranges should be increased by a factor of 3 (i.e. 30 -300 mg/kg bw/d). The LOAEL of the combined subacute reproduction screening study was 45 mg/kg bw/d and in the range for STOT RE category 2. In addition, this LOAEL also covers very sensitive life stages (i.e. reproduction and embryo-fetal-development) and the exposure duration was 42 days up to 7 weeks and, thus, distinctly longer than 28 days.

Consequently, and based on the available data, the test item has to be classified and labelled with STOT RE Category 2 (adrenal glands) according to Regulation (EC) No 1272/2008 (CLP), as amended for the twelfth time in Regulation (EU) 2019/521.