Registration Dossier

Diss Factsheets

Administrative data

Description of key information

In a 28 day study (Harlan, 2012), hepatocellular hypertrophy was observed histologically in males dosed with 500 mg/kg bw/day, which was considered to be an adaptive change due to increased metabolic load. Increased incidence and severity of tubular basophilia in the kidneys were observed in females at 500 mg/kg bw/day. The NOAEL was considered to be 250 mg/kg bw/day for females and 500 mg/kg bw/day for males. In a subchronic toxicity study with a structural analogue, an adaptive liver enlargement at doses of 450 - 500 mg/kg bw was observed. The NOEL was 125 mg/kg bw.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
see attached justification document
Reason / purpose for cross-reference:
read-across source
Dose descriptor:
NOAEL
Effect level:
125 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Key result
Critical effects observed:
no
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24.04.2012. - 07.6.2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
GLP compliance:
not specified
Remarks:
Test laboratory for analysis in the fields of clinical chemistry, hematology, blood-coagulation and urine diagnostics in accordance with the Standard ISO/IEC 17025 under accreditation number STS 085 by the Swiss Accreditation Service.
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories B.V., Kreuzelweg 53, 5961 NM Horst / Netherlands
- Age at study initiation: 7 weeks
- Weight at study initiation: 176-221 g (males), 116-149 f /females)
- Housing: In groups of five in Makrolon type-4 cages with wire mesh tops and standard softwood bedding (J. Rettenmaier & Söhne GmbH & Co. KG, 73494 Rosenberg / Germany, imported by Provimi Kliba AG, 4303 Kaiseraugst / Switzerland) including paper enrichment (Enviro-dri from Lillico, Biotechnology, Surrey, UK)
- Diet (e.g. ad libitum): Pelleted standard Harlan Teklad 2914C (batch no. 73/11) rodent maintenance diet (Provimi Kliba AG, 4303 Kaiseraugst / Switzerland) was available ad libitum.
- Water (e.g. ad libitum): Community tap-water from Itingen was available ad libitum in water bottles.
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Remarks:
PEG300
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test article was weighed into a glass beaker on a tared Mettler balance and thereafter the vehicle added. The mixtures were stirred using a magnetic stirrer and stored at room temperature (15 - 25 °C). Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer. The dose formulations were prepared daily.

VEHICLE
- Concentration in vehicle: 25, 50 and 100 mg/ml
- Amount of vehicle (if gavage): 5 ml/kg body weight
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analysis was performed by Harlan Laboratories Ltd. using an HPLC method provided by the Sponsor. After experimental start and during week 3, duplicate samples of the control group as well as three samples (top, middle and bottom) of about 2 g of each concentration were taken prior to dosing for analysis of homogeneity and concentration. Duplicate samples of about 2 g of each concentration were taken to confirm stability (4 hour and 8 days). The samples were delivered to the analytical department (Harlan Laboratories Ltd., Itingen / Switzerland) and stored frozen (-15 - -25 °C) until analysis. The test item was used as analytical standard.
Duration of treatment / exposure:
28 days
Frequency of treatment:
daily
Dose / conc.:
125 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected based on the results of a 28-day study with a structurally similar compound.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Observations for viability / mortality were recorded twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before commencement of administration as well as daily on days 1 - 28 (twice daily during days 1 - 3) during the treatment period.

BODY WEIGHT: Yes
- Time schedule for examinations: weekly during acclimatization, treatment period and before necropsy.

FOOD CONSUMPTION
The food consumption was recorded once during the acclimatization period and weekly thereafter, using an on-line electronic recording system consisting of a Mettler balance connected to the Harlan Laboratories computer.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: after 4 weeks
- Anaesthetic used for blood collection: Yes, isoflurane
- Animals fasted: Yes, 18 hours
- How many animals: all animals
- Parameters checked: Erythrocyte count, Hemoglobin, Hematocrit, Mean corpuscular volume, Red cell volume distribution width, Mean corpuscular hemoglobin, Mean corpuscular hemoglobin concentration, Hemoglobin concentration distribution width, Reticulocyte count, Reticulocyte maturity index (low medium, high fluorescence), Leukocyte count, total, Differential leukocyte count: Neutrophils, Eosinophils, Basophils, Lymphocytes, Monocytes, Large unstained cells, Platelet count.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: after 4 weeks
- Animals fasted: Yes, 18 hours
- How many animals: all animals
- Parameters checked: Glucose, Urea, Creatinine, Bilirubin, total, Cholesterol, total, Triglycerides, Phospholipids, Aspartate aminotransferase, Alanine aminotransferase, Lactate dehydrogenase, Alkaline phosphatase, Bile acids, Gamma-glutamyl-transferase, Creatine kinase, Sodium, Potassium, Chloride, Calcium, Phosphorus, Protein, total, Albumin, Globulin, Albumin/Globulin ratio.

URINALYSIS: Yes / No / No data
- Time schedule for collection of urine:
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: Urine volume (18 hour), Specific gravity (relative density), Color, Appearance, pH value, Nitrite, Protein, Glucose, Ketones, Urobilinogen, Bilirubin, Erythrocytes, Leukocytes.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
For details see below.
Statistics:
The following statistical methods were used to analyze food consumption, body weight, clinical laboratory data, organ weights and ratios as well as macroscopic findings:
• The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
• Fisher's exact-test
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
At all doses increased salivation was noted after administration, generally from week 3 of treatment onwards, which was transient and occurred at low incidence at 125 and 250 mg/kg bw/day, and at a higher incidence at 500 mg/kg bw/day. Increased salivation was noted once in one male of the control group and red salivation was occasionally seen in two males at 125 mg/kg bw/day and in one male at 250 mg/kg bw/day. No other test item-related clinical signs of toxicological relevance were noted during the treatment period.
At all dose levels, pale feces was noted in treatment weeks 3 and 4 in males and females. This finding was considered to result from the test item (yellowish liquid) dosed at relatively high doses, rather than any systemic effect. Soft feces was seen in all groups, including controls, and considered to be an effect of the vehicle.
At 500 mg/kg bw/day, breathing noises were noted in one male on day 18 of treatment and due to the isolated incidence considered not to be an effect of the test item.
Mortality:
no mortality observed
Description (incidence):
There were no test item-related effects upon mortality. All animals survived the scheduled treatment period.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weight development in males and females was not affected by treatment with the test item.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Daily food consumption was not affected by treatment with the test item. Minor fluctuations in food consumption were similar between test item-treated groups and controls.
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 differences between the hematology parameters of the control and treated males or females.
In females treated with 500 mg/kg bw/day and 250 mg/kg bw/day, a statistically significant difference was noted in hemoglobin. This finding was considered to be biological variation due to the isolated incidence and absence of dose-dependency, although slightly below the range of the historical control values. In case of the remaining statistically significant differences noted (higher hemoglobin vales in males at 250 mg/kg bw/day; lower hematocrit values in males at 125 mg/kg bw/day and in males and females at 500 mg/kg bw/day; higher MCHC values in males at 500 mg/kg bw/day; lower RDW vales in females at 250 mg/kg bw/day; lower reticulocyte counts in females at 250 and 500 mg/kg bw/day; lower platelet counts in females at 250 mg/kg bw/day) the values were within the ranges of the historical control values and considered to be incidental and of no toxicological significance.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related differences between the clinical biochemistry parameters of the control and treated males or females.
Marginal but significantly significantly elevated potassium concentrations were recorded at 500 mg/kg bw/day in males and females and at 250 mg/kg bw/day in females. A marginal elevated albumin/globulin ratio was recorded at 500 mg/kg bw/day and 250 mg/kg bw/day in males, and elevated globulin levels were recorded at 500 mg/kg bw/day in females. Females treated with 250 and 500 mg/kg bw/day had higher creatinine levels, and lower inorganic phosphate concentrations were present in females at 125 and 500 mg/kg bw./day, in both cases without dose-dependency. All differences were within the range of the historical control values and considered to be incidental. None of these differences were considered to be related to the treatment with the test item or to be of toxicological significance.
Urinalysis findings:
no effects observed
Description (incidence and severity):
There were no test item-related differences between the urine parameters of the control and treated males or females.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
No compound-related effects were observed.
Liver to body weight ratios were statistically significantly higher in males of all dose groups. Since there was no dose-dependency, and liver weights of females were comparable to control values at all dose levels, these differences were considered not to be compound-related. The same applies to the statistically significantly higher heart to body weight ratios in males at 125 mg/kg bw/day, since there was no dose-dependency.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no macroscopical findings considered to be related to treatment.
Dilation of the renal pelvis, noted unilaterally in one male at 125 mg/kg bw/day, two males at 250 mg/kg bw/day and two males at 500 mg/kg bw/day, was not considered an effect of the test item as there was no microscopic correlate. This was not noted in the females.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Liver: In 4/5 males at 500 mg/kg bw/day a minimal to slight centrilobular to diffuse hepatocellular hypertrophy was observed. In females, a slight decrease in glycogen deposits was present but was considered to be still within the biological range of this finding.
Kidneys: In females, the age-related and often normally present basophilic tubules were slightly increased in incidence and severity in kidneys at 500 mg/kg bw/day, in one female associated with minimal multifocal simple tubular dilation. The basophilic tubules occurred in control females only unilaterally whereas in high dose females the finding was present bilaterally in 4/5 affected animals.
Other Findings: The remaining microscopical findings recorded in this study were within the range of normal background lesions which may be recorded in animals of this strain and age.
Histopathological findings: neoplastic:
not examined
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Critical effects observed:
not specified
Conclusions:
Based on the results of this study, the No Observed Adverse Effect Level (NOAEL) was considered to be 250 mg/kg bw/day for females and 500 mg/kg bw/day for males.
Executive summary:

In this subacute toxicity study, the test material was administered daily by oral gavage to-bred Wistar rats of both sexes at dose levels of 125, 250 and 500 mg/kg body weight/day for a period of 28 days. A control group was treated similarly with the vehicle, PEG300, only. The groups comprised 5 animals per sex which were sacrificed after 28 days of treatment. Clinical signs, outside cage observation, food consumption and body weights were recorded periodically during pretest and the treatment period. At the end of the dosing period, blood samples were withdrawn for hematology and plasma chemistry analyses. Urine samples were collected for urinalyses. All animals were killed, necropsied and examined post mortem. Histological examinations were performed on organs and tissues from all control and high dose animals, and all gross lesions from all animals. Livers and kidneys were examined histologically in all groups. All animals survived the scheduled treatment period. Increased salivation was noted after test item administration, generally from week 3 of treatment onwards, with the highest incidence at 500 mg/kg bw/day. No other test item-related clinical signs of toxicological relevance were noted during daily observations. No test item-related effects on food consumption and body weights were noted. There were no test item-related differences between hematology, clinical biochemistry and the urine parameters of the control and treated males or females. No compound-related effects on oragen weights were observed. All macroscopical findings were considered to be unrelated to treatment. Microscopical findings noted at 500 mg/kg bw/day included minimal to slight centrilobular to diffuse hepatocellular hypertrophy in four of five males and slightly increased incidence and severity of tubular basophilia in the kidney of females were noted, accompanied in one female with minimal tubular dilation. Based on the results of this study, the No Observed Adverse Effect Level (NOAEL) was considered to be 250 mg/kg bw/day for females and 500 mg/kg bw/day for males.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
125 mg/kg bw/day
Study duration:
subchronic
Species:
rat
System:
hepatobiliary
Organ:
liver

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

Subacute Toxicity Data

In a subacute toxicity study following OECD guideline 407, the test material was administered daily by oral gavage to 5 Wistar rats per sex at dose levels of 125, 250 and 500 mg/kg body weight/day for a period of 28 days. A control group was treated similarly with the vehicle, PEG300, only. All animals survived the scheduled treatment period. Increased salivation was noted after test item administration, generally from week 3 of treatment onwards, with the highest incidence at 500 mg/kg bw/day. No other test item-related clinical signs of toxicological relevance were noted during daily observations. No test item-related effects on food consumption and body weights were noted. There were no test item-related differences between hematology, clinical biochemistry and the urine parameters of the control and treated males or females. No compound-related effects on organ weights were observed. All macroscopical findings were considered to be unrelated to treatment. Microscopical findings noted at 500 mg/kg bw/day included minimal to slight centrilobular to diffuse hepatocellular hypertrophy in four of five males which was considered to be an adaptive change due to increased metabolic load. In females, the age-related and often normally present basophilic tubules were slightly increased in incidence and severity in kidneys at 500 mg/kg bw/day, in one female associated with minimal multifocal simple tubular dilation. Based on the results of this study, the No Observed Adverse Effect Level (NOAEL) was considered to be 250 mg/kg bw/day for females and 500 mg/kg bw/day for males.

Subchronic Toxicity Data

This information is taken from a structural analogue (see attached document for read across justification). In a GLP-compliant repeated dose toxicity study following OECD guideline 408, the structural analogue was administered by gavage to groups of 10 male and 10 female Wistar rats at dose levels of 0 (test group 0), 25 (test group 1), 125 (test group 2) and 400 mg/kg bw/d (test group 3) over a period of 3 months (2015). In addition to the required examinations special attention was given to the reproductive organs of male and female animals. Immediately after necropsy and organ weight determination the right testis and cauda epididymis were taken from all male animals for sperm examinations. Signs of general systemic toxicity were not observed even at a dose level of 400 mg/kg bw/d. In addition, no test substance-related effects on estrous cycle length and the number of cycles were obtained. In male and female rats of test group 3 (400 mg/kg bw/d) a slight anemia was indicated by decreased hemoglobin, hematocrit and red blood cell (RBC) counts (only in females). In males of the mentioned test group an effect of the coagulation could be assumed by a prolonged prothrombin time and in females some chronic stress or inflammation was expressed by higher absolute lymphocyte counts. Liver cell swelling led to higher alkaline phosphatase activities in male and female rats of test group 3 (400 mg/kg bw/d) and the liver cell metabolism seemed to be affected because of lower cholesterol levels in males and higher glucose levels in females of this test group. Lower serum creatinine levels may be due to a decreased synthesis of creatin as precursor of creatinine in the liver cells. An increased excretion of creatinine via the kidneys may also be possible. In the livers of male and female animals in test groups 2 and 3 (125 and 400 mg/kg bw/d) a diffuse or centrilobular hypertrophy in a dose-depended manner was observed. This type of finding also corresponded to the liver weight increase in this test groups. Females of test group 3 (400 mg/kg bw/d) revealed in addition a light to middle brown pigment in the liver. The character of the pigment could not further be determined, but it was negative for special stains for iron, bile and lipofuscin. In combination with deviations in clinical chemistry the above mentioned findings were regarded to be treatment-related and adverse for animals of test group 3 (400 mg/kg bw/d). In test group 2 (125 mg/kg bw/d) there was still an increase in liver weight and liver hypertrophy was observed in some males and females. Due to the fact that there were no other additional histopathologic findings and not more than one clinical chemistry parameter was changed, these findings were regarded to be treatment-related but not adverse in nature (Hall et al., 2012).

As a possible secondary effect to the liver hypertrophy, males and females of test groups 2 and 3 (125 and 400 mg/kg bw/d) showed a hypertrophy/hyperplasia in the thyroid gland. This could have been caused by the liver cell hypertrophy (most likely enzyme induction). It was regarded to be a secondary finding, treatment-related but not relevant for humans. In the kidneys of male animals in test groups 2 and 3 (125 and 400 mg/kg bw/d) eosinophilic droplets were observed. These droplets were immunohistologically stained positive for alpha 2u globulin. Eosinophilic droplets in the proximal convoluted tubules represent alpha 2u globulin, a poorly hydrolysable, low molecular weight protein characteristic for male rats. The increase of eosinophilic droplets was regarded to be treatment-related, but is not of human relevance. All other 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. In conclusion, the administration of the test article by gavage to male and female Wistar rats for 3 months caused test substance-related, adverse signs of systemic toxicity at a dose level of 400 mg/kg bw/d. Therefore, under the conditions of the present study the no observed adverse effect level (NOAEL) was 125 mg/kg bw/d for male and female Wistar rats.

Justification for classification or non-classification

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

The available experimental test data are reliable and suitable for the purpose of classification under Regulation (EC) No.1272/2008. Based on the data, classification for repeated dose toxicity is not warranted under Regulation (EC) No.1272/2008.