Registration Dossier

Administrative data

Description of key information

Read Across Information for subacute oral toxicity (OECD 407, OECD 422, both GLP).

For the read across source compound toxicity after repeated oral administration was investigated in two experimental studies: a subacute toxicity study with oral treatment of rats with doses of 1000, 200, and 40 mg/kg bw for 28 days and a 14 -day treatment-free recovery period according to OECD 407; and a combined repeat dose reproduction screening study with oral treatment of rats with doses of 135, 45 and 15 mg/kg bw for 42 days (males) and up to 49 days (females).

The lowest NOAEL systemic was 15 mg/kg bw/d.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Jun 13, 2006 - Feb 07, 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
other: HanRcc:WIST (SPF)
Sex:
male/female
Details on test animals and environmental conditions:
- Age at delivery: 6 weeks
- Body weight range at acclimatization: Males: 129.2 – 159.0 grams (mean 145.1 grams), Females: 114.4 – 133.6 grams (mean 124.0 grams)
- Acclimatization: For 7 days under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

HUSBANDRYCONDITIONS
- Standard Laboratory Conditions. Air-conditioned with 10-15 air changes per hour, and continuously monitored environmental conditions (temperature range: 22 ± 3 °C; relative humidity range: 30-70 %). Values outside of these ranges occasionally occurred, usually following roomcleaning. These transient variations are considered not to have any influence on the study and, therefore, these data are not reported but are retained at RCC. There was 12-hour fluorescent light/12-hour dark cycle with music during the light period.

ACCOMODATION
- In groups of five in Makrolon type-4 cages with wire mesh tops and standardized softwood bedding ('Lignocel' Schill AG, CH-4132 Muttenz/Switzerland).

DIET
- Pelleted standard Provimi Kliba 3433 (batch nos 01/06, 36/06, and 23/06) rat maintenance diet (Provimi Kliba AG, CH-4303 Kaiseraugst/ Switzerland) was available ad libitum. The feed batch was analyzed for contaminants.

WATER
- Community tap-water from Itingen was available ad libitum in water bottles. None of the contaminants analyzed in the water and diet is considered to have been present at a concentration that would have affected the validity of the results.
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
DOSE FORMULATION
The dose formulations were prepared daily from 30-June-2006 to 06-July-2006 and weekly from 07-Juli-2006 after confirmation of dose formulation stability by the PI for dose formulation analysis.The test item was pestled, weighed into a tared glass beaker on a suitable precision balance and the vehicle added (weight:volume). The mixtures were prepared using a magnetic stirrer. The mixtures were heated to 50°C to increase solubility, cooled down before application 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.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
ANALYSIS OF DOSE FORMULATIONS
Concentration, homogeneity and stability (after 2 hours and 7 days) of the dose formulations were determined in samples taken after experimental start. Analyses were performed by the Principal Investigator of the analytical phase, according to a HPLC analytical method supplied by the Sponsor. Details of the analytical method are documented in the raw data generated by the Principal Investigator (and/or his/her staff) and the Principal Investigator provided an analytical phase report. Concentration and homogeneity of the dose formulations was determined in samples taken during week 3 of the treatment. Unless otherwise specified, the dose formulations were delivered under ambient conditions. Samples of dose formulations were not discarded without the written authorization of the study director.
Duration of treatment / exposure:
28 days (groups 1-3), 6 days (group 4), 14 days (recovery group)
Frequency of treatment:
daily for 28 days
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
40 mg/kg bw/day (actual dose received)
Dose / conc.:
200 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Group 1: 0 mg/kg body weight: 10 Group 2: 40 mg/kg body weight: 5Group 3: 200 mg/kg body weight: 10 Group 4: 1000 mg/kg body weight: 10
Control animals:
yes, concurrent vehicle
Positive control:
no
Observations and examinations performed and frequency:
MORTALITY / VIABILITY
Observations for mortality/viability were recorded twice daily.

GENERAL CAGESIDE OBSERVATIONS (DAILY)
The animals were observed for clinical signs once before commencement of administration; twice daily on days 1-3; as well as once daily on days 4-28 and once daily during days 29-42 (recovery).

DETAILED CLINICAL OBSERVATIONS (WEEKLY)
The animals were observed in their home cages, outside their home cages in a standard arenaand in the hand. These observations were performed in random sequence once before commencement of administration and once weekly (weeks 1-3) thereafter.

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

BODY WEIGHTS
Body weights were recorded weekly during pretest, treatment and recovery and beforenecropsy, using an on-line electronic recording system consisting of a Mettler balanceconnected to the RCC computer.

FUNCTIONAL OBSERVATIONAL BATTERY
During week 4, relevant parameters from a modified Irwin screen test were evaluated in all animals. The results of the Functional Observational Battery are presented in the summary andindividual tables of the Detailed Clinical Observations (Weekly) under week 4. This method ofdata presentation permits a clear evaluation and assessment of weekly clinical signs observed during the study.

GRIP STRENGTH
Forelimb and hind limb grip strength measurements were performed using a push-pull straingauge (Mecmesin, AFG 25N). The animals were placed with the forepaws inside a triangulargrasping ring and with the hind paws outside a triangular grasping ring. Using one hand, theanimals were held towards the base of the tail and steadily pulled away or towards the ring untilthe grip was broken. Each measurement was repeated three times, the means were calculatedand recorded.

LOCOMOTOR ACTIVITY
Locomotor (decreased or increased) activity was measured quantitatively with AMS FöhrMedical Instruments GmbH (FMI) and DeMeTec GmbH Activity Monitor System. Animals were monitored during the fourth treatment week for a 60-minute period and the total activity of this time period was recorded.Low beams count was reported in 10-minute intervals as well as the total activity of themeasuring period.

CLINICAL LABORATORY INVESTIGATIONS
- Blood and urine sampling: after 4 weeks 28-July-2006 (Allocation A, groups 1-3 and B, group 1) after 4 weeks 18-Aug-2006 (Allocation B, group 3)after 6 weeks 11-Aug-2006 (Allocation B, group 1)after 6 weeks 01-Sept-2006 (Allocation B, group 3).
- Blood sampling:after 8 days 07-July-2006 (Allocation A and B, group 4). From group 4 animals (allocation A and B), blood samples were taken by heart puncture atnecropsy if possible. No blood for determination of coagulation parameters was collected fromthese animals. In case the amount of blood collected was not enough to determine allparameters listed (due to the bad condition of the animals), the person responsible for Clinical Laboratory Investigations decided, which parameters were determined.Blood samples for hematology and clinical biochemistry were collected from all other animalsunder light isoflurane anesthesia. The animals were fasted in metabolism cages forapproximately 18 hours before blood sampling but allowed access to water ad libitum. Blood samples were collected early in the working day to reduce biological variation caused bycircadian rhythms. Blood samples were drawn from the retro-orbital plexus using a microhematocritglass capillary tube.Urine was collected during the 18-hour fasting period into a specimen vial.The assays were performed at the testing facility under internal laboratory quality control conditions to assure reliable test results.

HEMATOLOGY
The following hematology parameters were determined: Erythrocyte coun, tHemoglobin, Hematocrit, Mean corpuscular volume, Red cell volume distribution width, Mean corpuscular hemoglobin, Mean corpuscular hemoglobin concentration, Hemoglobin concentration distribution width, Platelet (thrombocyte) count, Reticulocyte count, Reticulocyte maturity index, Methemoglobin Heinz bodies (slides were prepared but not evaluated since no changes in methemoglobin levels were noted), Total leukocyte count, Differential leukocyte count, Coagulation: Thromboplastin time, Activated partial thromboplastin time

CLINICAL BIOCHEMISTRY
The following clinical biochemistry parameters were determined: Glucose, Urea, Creatinine, Bilirubin, total Cholesterol, total Triglycerides, Phospholipids, Aspartate aminotransferase, Alanine aminotransferase, Lactate dehydrogenase, Glutamate dehydrogenase,Creatine kinase, Alkaline phosphatase, Gamma-glutamyl-transferase, Sodium, Potassium, Chloride, Calcium, Phosphorus inorganic, Protein, total Albumin, Globulin, Albumin/Globulin ratio, Bile acids

URINALYSIS
The following urinalysis parameters were determined: Volume (18 hours), Specific gravity (relative density), Color, Appearance, pH, Nitrite, Protein, Glucose, Ketones, Urobilinogen, Bilirubin, Erythrocytes, Leukocytes
Sacrifice and pathology:
NECROPSY
- Sacrifice: after 4 weeks 28-July-2006 (groups 1-3, Allocation A) after 8 days 07-July-2006 (group 4, Allocation A and B) after 6 weeks 11-Aug-2006 (group 1, Allocation B) after 6 weeks 01-Sept-2006 (group 3, Allocation B)

- All animals were weighed and necropsied. Descriptions of all macroscopic abnormalities were recorded. All animals surviving to the end of the observation period and all moribund animals were anesthetized by intraperitoneal injection of pentobarbitone and killed by exsanguination. From group 4 animals (allocation A and B), blood samples were taken by heart puncture at necropsy if possible. No blood for determination of coagulation parameters was collected from these animals. Samples of the following tissues and organs were collected from all animals at necropsy and fixed in neutral phosphate buffered 4 % formaldehyde solution (unless otherwise indicated): Adrenal glands, Aorta, Bone (sternum, femur including joint), Bone marrow (femur), Brain (at least 3 levels), Cecum, Colon, Duodenum, Epididymides (fixed in Bouin's solution), Esophagus, Eyes w/optic nerve (fixed in Davidson's solution), Harderian gland (fixed in Davidson's solution), Heart, Ileum with Peyer's patches, Jejunum with Peyer's patches, Kidneys, Larynx, Lacrimal gland exorbital, Liver, Lungs filled w/formalin at necropsy, Lymph nodes - mesenteric, mandibular, Mammary gland area, Nasal cavity, Ovaries, Pancreas, Pituitary gland, Prostate gland (incl. coagulating gland), Rectum, Salivary glands - mandibular, sublingual, Sciatic nerve, Seminal vesicles, Skeletal muscle, Skin, Spinal cord - cervical, midthoracic, lumbar, Spleen, Stomach, Testes (fixed in Bouin's solution), Thymus, Thyroid (incl. parathyroid gland, if possible), Tongue, Trachea, Urinary bladder filled w/formalin at necropsy, Uterus, Vagina, Gross lesions

Additional tissues (such as ear tattoo) were retained in accordance with standard operating procedures but were not processed or examined further.

ABSOLUTE AND RELATIVE ORGAN WEIGHTS
The following organ weights were recorded on the scheduled dates of necropsy: Brain, Thymus, Spleen, Ovaries, Heart, Kidneys, Testes, Liver, Adrenals, Epididymides. The organ to terminal body weight ratios as well as organ to brain weight ratios were determined. The determination of the terminal body weight was performed immediately prior to necropsy.

HISTOTECHNIQUE
All organ and tissue samples, as defined under Histopathology (following), were processed, embedded and cut at an approximate thickness of 2 to 4 micrometers, and stained with hematoxylin and eosin.

HISTOPATHOLOGY
Slides of all organs and tissues listed in boldface type (see Necropsy, above) that were collected at terminal sacrifice from the animals of the control and group 3 were examined by the study pathologist. Since test item-related morphologic changes were detected in organs of the high-dose (group 3) animals, those same organs (liver, adrenal glands, and testes) from the low-dose group (group 2) were examined to establish a no-effect level.The same organs of group 4 animals which were terminated in extremis after 6 days of treatment on day 8 (July, 7th, 2006) were not examined.
Statistics:
The following statistical methods were used to analyze the grip strength, locomotor activity,body weight, clinical laboratory data, organ weights, and ratios:
- The Dunnett-test (many to one t-test) based on a pooled variance estimate wereapplied if the variables could be assumed to follow a normal distribution for thecomparison 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 whenthe data can not be assumed to follow a normal distribution.
-Fisher's exact-test was applied to the macroscopic findings.
-Student’s t-test was applied to grip strength and locomotor activity data.
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
effects observed, non-treatment-related
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
Please refer to the tables attached.

MORTALITY / VIABILITY
All animals treated with 1000 mg/kg/day were killed in extremis on July 7th, 2006, after 6 days oftreatment and two more days without.One female (No. 70) treated with 200 mg/kg/day died on the first day of the recovery period.The cause of death could not be established.One female (No. 47) treated with 200 mg/kg/day died spontaneously on treatment day 21. The animal died one hour after application without showing any symptoms previously. The cause of death could not be established. One female (No. 48) treated with 200 mg/kg/day and two females of the control group (No. 37 and 39) died on the day of scheduled necropsy after blood sampling. These deaths are attributed to the blood sampling procedure.

CLINICAL SIGNS
Almost all animals treated with 1000 mg/kg/day, which were killed in extremis, showed pale and soft feces, emaciation, sedation, prostration, piloerection, and hunched posture. Slight emaciation from day 7 to 10 in one female treated with 200 mg/kg/day is considered to be test item-related. All other findings are considered not to be test item-related.

FUNCTIONAL OBSERVATIONAL BATTERY
In addition to the other clinical signs noted during daily observations, salivation was seen in one male (no. 65) treated with 200 mg/kg/day in week 4.

- Grip Strength: No test item-related differences were noted in the mean grip strength when compared with the controls.
- Locomotor Activity: Significantly decreased locomotor activity (p<0.05) was seen from 20-30 min. in males treated with 40 or 200 mg/kg/day and from 10-20 min. in females treated with 200 mg/kg/day. A statistically significant increase was seen from 40-50 min. in males treated with 200 mg/kg/day (p<0.05).These observations were inconsistent across sexes and sporadic and are therefore considered not to be test item-related.

FOOD CONSUMPTION
In animals treated with 1000 mg/kg/day, which were killed in extremis on 7-July-2006, food consumption was extremely low. Mean food consumption during the treatment period was reduced at 200 mg/kg/day. A slight decrease in absolute food consumption was also recorded in males treated with 40 mg/kg/day. A decrease in relative food consumption was noted in males at both dose levels only. The decrease was compensated during recovery.

BODY WEIGHT
Body weight and body weight gain were clearly reduced in all animals treated with 200 or 1000 mg/kg/day.

CLINICAL LABORATORY INVESTIGATIONS
- Hematology: Except for decreased hematocrit and hemoglobin in both sexes treated with 200 mg/kg/day and increased hemoglobin concentration distribution width in females treated with 200 mg/kg/day, all changes were within the range of historical data for rats of this strain and age. After recovery, these changes, together with decreased erythrocytes in both sexes (in range for males) treated with 200 mg/kg/day were still present. All other changes were within the normal range of historical data for rats of this strain and age.

- Clinical Biochemistry: After 4 weeks of treatment, the following statistically significant changes were noted:Increased urea in both sexes treated with 200 mg/kg/day (p<0.01); Decreased glucose in males treated with 200 mg/kg/day (p<0.05); Increased creatinine in males treated with 40 mg/kg/day (p<0.05) and males and females treated with 200 mg/kg/day (p<0.01 in males, p<0.05 in females); Increased bilirubin in males treated with 200 mg/kg/day (p<0.05) and females treated with 40 mg/kg/day (p<0.05); Decreased phospholipids in males treated with 40 or 200 mg/kg/day (p<0.01); Increased aspartate aminotransferase in males treated with 200 mg/kg/day (p<0.01); Increased alanine aminotransferase, lactate dehydrogenase, and glutamatedehydrogenase in both sexes treated with 200 mg/kg/day (p<0.01); Slightly increased potassium (p<0.01 in males and p<0.05 in females) and decreased calcium (p<0.01) in both sexes treated with 200 mg/kg/day. After recovery, slightly increased creatinine (p<0.05) and decreased phospholipids (p<0.01) were still found in males treated with 200 mg/kg/day. Total bilirubin was decreased in males treated with 200 mg/kg/day (p<0.05). Alanine aminotransferase was increased in males and females treated with 200 mg/kg/day (p<0.01). Alanine aminotransferase showed a clear tendency towards recovery. In females treated with 200 mg/kg/day potassium was slightly decreased. The above findings were considered to be test item-related. All other changes (even though reaching statistical significance) were within the range of historical data for rats of this strain and age and/or without dose relation and inconsistent across sexes.

- Urinalysis: In urinalysis, slightly increased leukocytes were found after 4 weeks of treatment in females treated with 200 or 40 mg/kg/day. After recovery, a trend for reversibility was noted in the affected females treated with 200 mg/kg/day. All other findings (even if reaching statistical significance) were within the normal range for rats of this strain and age.

ORGAN WEIGHTS
- After 4 Weeks: A dose related increase in absolute and relative liver weights and a dose related decrease in absolute and relative adrenal weights was found in all test item treated rats. The changes were statistically significant in all females and in males treated with 200 mg/kg/day for absolute and relative adrenal weights and liver to body weight ratio.

- After 6 WeeksAbsolute and relative adrenal weights were decreased in all animals treated with 200 mg/kg/day. In females treated with 200 mg/kg/day, increased relative thymus weights were found. In males treated with 200 mg/kg/day, decreased absolute and relative epididymis weights were noted. The liver to body weight ratio was increased in all animals treated with 200 mg/kg/day.

MACROSCOPIC / MICROSCOPIC FINDINGS
After 6 days of treatment, reddish discoloration of the testes was found in 6/10 males treated with 1000 mg/kg/day. In 8/10 males and 9/10 females treated with 1000 mg/kg/day the thymus was reduced in size. After 4 weeks of treatment, all males and 3/5 females treated with 200 mg/kg/day had enlarged livers and reduced adrenal glands. After 4 weeks of treatment and 2 weeks recovery, all males and 4/5 females treated with200 mg/kg/day had reduced adrenal glands. All other macroscopic findings were considered incidental. Microscopically, centrilobular hepatocellular hypertrophy, at minimal severity, was recorded in 4 of 5 males and 2 of 5 females treated with 200 mg/kg/day of the test item. Hepatocellular hypertrophy was not observed after the recovery period. This finding was deemed to be of metabolic nature due to the absence of any further lesion. Cortical atrophy in adrenal glands often (in 3 males and 4 females) with focal/multifocal subacute inflammation was recorded in all males and 4 of 5 females treated with 200 mg/kg/day. Cortical atrophy with focal vacuolar degeneration was recorded in one female treated with 200 mg/kg/day. These findings were also present after the recovery period. Hemangiectasis in adrenal cortices was recorded in one female treated with 200 mg/kg/day after recovery.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. The presence of focal/multifocal multinucleated spermatid giant cells is often associated with early stages of atrophic seminiferous tubules.
Key result
Dose descriptor:
NOAEL
Effect level:
40 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality
body weight and weight gain
food consumption and compound intake
haematology
clinical biochemistry
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
Key result
Critical effects observed:
no
Lowest effective dose / conc.:
200 mg/kg bw/day (actual dose received)
System:
endocrine system
Organ:
adrenal glands

Please refer to the tables attached.

Conclusions:
Based on the results of this study, 40 mg/kg body weight/day of the test item could be established as the no-observed-adverse-effect-level (NOAEL).
Executive summary:

Purpose

The purpose of this oral toxicity study was to assess the cumulative toxicity of the test item when administered daily to rats by gavage for a period of 28 days. The reversibility of treatment-related changes was assessed after a treatment-free 14-day recovery period. This study should provide a rational basis for toxicological risk assessment in man. The results of this study should indicate potential target organs and should identify chemicals with neurotoxic potential.

Study Design

In the subacute toxicity study, the test item was administered daily by oral gavage to SPF-bred Wistar rats of both sexes at dose levels of 40 and 200 mg/kg body weight/day for a period of 28 days. A control group was treated similarly with the vehicle, corn oil, only.

The groups comprised 5 animals per sex, which were sacrificed after 28 days of treatment. Additional 5 rats per sex and group were used at 0 and 200 mg/kg. These animals were treated for 28 days and then allowed a 14-day treatment-free recovery period after which they were sacrificed.

A fourth group of 10 males and 10 females was treated with 1000 mg/kg body weight/day of the test item. These animals had to be killed in extremis on day 8 after 6 days of treatment and 2 days without treatment.

Clinical signs, outside cage observation, food consumption and body weights were recorded periodically during pretest, the treatment and recovery periods. Functional observational battery, locomotor activity and grip strength were performed during week 4.

At the end of the dosing and the treatment-free recovery 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 (200 mg/kg) animals, and due to findings in the high dose group from all animals of the low dose group (40 mg/kg).

Results

Oral administration of the test item to Wistar rats at doses of 40 and 200 mg/kg/day, for 28 days resulted in 5 unscheduled deaths, only one of which was considered to be test item related.

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/day.

Decreased hematocrit and hemoglobin in both sexes treated with 200 mg/kg/day and increased hemoglobin concentration distribution width in females treated with 200 mg/kg/day were present after 4 weeks of treatment as well as after recovery. Decreased erythrocytes in both sexes (in range for males) treated with 200 mg/kg/day were also present after recovery.

In clinical biochemistry, the main changes seen were increased creatinine, decreased phospholipids and increased alanine aminotransferase with alanine aminotransferase showing a tendency towards recovery. Other affected parameters, e.g. aspartate aminotransferase were also indicating changes in the liver and kidneys.

A slightly increased number of leukocytes in the urine was found after 4 weeks of treatment in females treated with 200 or 40 mg/kg/day with a tendency towards reversibility during recovery. In the absence of macroscopic or microscopic correlating findings, this is considered test item related but non-adverse.

Histologically, alterations in the liver, adrenal glands and testes were found in animals treated with 200 mg/kg/day. 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 with focal/multifocal subacute inflammation was recorded in both sexes treated with 200 mg/kg/day.

Cortical atrophy with focal vacuolar degeneration was recorded in one female treated with 200 mg/kg/day. These findings were also present after the recovery period. Cortical atrophy corresponds to macroscopically reduced 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 two animals treated with 200 mg/kg/day. 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.

Conclusions

Based on the results of this study, 40 mg/kg body weight/day of the test item could be established as the no-observed-adverse-effect-level (NOAEL). Based on this study no classification or labelling is warranted.

Endpoint:
repeated dose toxicity: oral, other
Remarks:
OECD 422
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
other: Crl.WI (Han)
Sex:
male/female
Details on test animals 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 (e.g. ad libitum): Diet: Provimi Kliba 3433.0 ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 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
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 (12m/12f)
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).
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
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
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
Urinalysis findings:
no effects observed
Behaviour (functional findings):
effects observed, non-treatment-related
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, non-treatment-related
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
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 FOOD CONSUMPTION
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 orend of treatment period). During the pre-mating period, the females did not show statisticallysignificant differences of body weight between dose- and control group, however, body weightgain 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 statisticalsignificance) in group 3 (45 mg/kg) from day 6 onwards, and with statistical significance ingroup 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 alwaysstatistically significant. No treatment-related effects on food consumption were observed.

FOB
During the functional observational battery no treatment-related relevant changes were observedon days 0, 7, 29 (males only), or day 4 p.p. (females only) in the autonomous and sensomotoricdomains. In the neuromuscular domain, hind limb foot splay showed a slight trend of reductionin all treatment group males (15 to 135 mg/kg) on study day 29 compared to control. Group 4females (135 mg/kg) on day 4 p.p. showed decreased hind limb foot splay as well. However, theobservation was without statistical significance. In the central nervous domain a trend ofreduction of the raising number in group 4 females (135 mg/kg) on day 7, and in group 3 (45mg/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
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.

CLINICAL CHEMISTRY, HEMATOLOGY, URINALYSIS
Hematology, coagulation, clinical chemistry, and urinary parameters were measured on studyday 13. Statistically significant alterations were seen in some clinical chemistry parameters butwere all within the known internal reference interval, low in degree and therefore consideredincidental and not treatment-related.

ORGAN WEIGHTS
Adrenal weights of both sexes were absolutely and relatively reduced at 45 mg/kg and 135 mg/kg.

GROSS PATHOLOGY
At necropsy of the adult animals only spontaneous findings were observed.

HISTOPATHOLOGY
At histopathology, group 4 (135 mg/kg) showed pronounced cortical atrophy of the adrenalgland combined with endothelial cell activation and mononuclear infiltrates in both sexes. In theliver, increased centrilobular single cell necrosis was observed in eight females whereas maleswere not affected.In group 3 (45 mg/kg), cortical atrophy of the adrenal gland was slightly less pronounced than ingroup 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. Mononuclearinfiltrates were observed in 4/24 rats. In the liver, increased amounts of single cell necrosis weredetected 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-groupsincluding controls. This finding is considered to be related to the former pregnancy of those rats.
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:
clinical signs
body weight and weight gain
organ weights and organ / body weight ratios
histopathology: non-neoplastic
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:

Study design

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.

Results

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:
repeated dose toxicity: oral, other
Remarks:
OECD 422
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Justification for type of information:
For this endpoint information from a structural similar compound, 1-Methyl-4-​(4-trans-vinyl-​​[1,1'-bicyclohexyl]​​-4'-trans-yl)-​benzene, is available. The study for this similar compound were performed according to GLP and the methods applied is fully compliant OECD 422. See chapter 13 report for a more detailed justification.
Reason / purpose:
read-across source
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:
clinical signs
body weight and weight gain
organ weights and organ / body weight ratios
histopathology: non-neoplastic
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:
For this endpoint information from a structural similar compound, 1-Methyl-4-​(4-trans-vinyl-​​[1,1'-bicyclohexyl]​​-4'-trans-yl)-​benzene, is available. The study for this similar compound were performed according to GLP and the methods applied is fully compliant OECD 422. See chapter 13 report for a more detailed justification.
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Justification for type of information:
For this endpoint information from a structural similar compound, 1-Methyl-4-​(4-trans-vinyl-​​[1,1'-bicyclohexyl]​​-4'-trans-yl)-​benzene, is available. The study for this similar compound were performed according to GLP and the methods applied is fully compliant OECD 407. See chapter 13 report for a more detailed justification.
Reason / purpose:
read-across source
Key result
Dose descriptor:
NOAEL
Effect level:
40 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality
body weight and weight gain
food consumption and compound intake
haematology
clinical biochemistry
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
Key result
Critical effects observed:
no
Lowest effective dose / conc.:
200 mg/kg bw/day (actual dose received)
System:
endocrine system
Organ:
adrenal glands
Conclusions:
For this endpoint information from a structural similar compound, 1-Methyl-4-​(4-trans-vinyl-​​[1,1'-bicyclohexyl]​​-4'-trans-yl)-​benzene, is available. The study for this similar compound were performed according to GLP and the methods applied is fully compliant OECD 407. See chapter 13 report for a more detailed justification.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
15 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
GLP and Guideline conform
System:
endocrine system
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

No study for subacute oral toxicity performed with the test item ist available. However, two Guideline conform studies were conducted under GLP conditions with the structural analogue substance. For Read Across Justification please refer to section 13.

Subacute oral toxicity study (OECD 407)

Oral administration of the structural analogue substance to Wistar rats at doses of 40 and 200 mg/kg/day, for 28 days resulted in 5 unscheduled deaths, only one of which was considered to be test item-related.

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/day.

Decreased hematocrit and hemoglobin in both sexes treated with 200 mg/kg/day and increased hemoglobin concentration distribution width in females treated with 200 mg/kg/day were present after 4 weeks of treatment as well as after recovery. Decreased erythrocytes in both sexes (in range for males) treated with 200 mg/kg/day were also present after recovery.

In clinical biochemistry, the main changes seen were increased creatinine, decreased phospholipids and increased alanine aminotransferase with alanine aminotransferase showing a tendency towards recovery. Other affected parameters, e.g. aspartate aminotransferase were also indicating changes in the liver and kidneys.

A slightly increased number of leukocytes in the urine was found after 4 weeks of treatment in females treated with 200 or 40 mg/kg/day with a tendency towards reversibility during recovery.

In the absence of macroscopic or microscopic correlating findings, this is considered test itemrelated but non-adverse.

Histologically, alterations in the liver, adrenal glands and testes were found in animals treated with 200 mg/kg/day. 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 with focal/multifocal subacute inflammation was recorded in both sexes treated with 200 mg/kg/day. Cortical atrophy with focal vacuolar degeneration was recorded in one female treated with 200 mg/kg/day. These findings were also present after the recovery period. Cortical atrophy corresponds to macroscopically reduced 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 two animals treated with 200 mg/kg/day. 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 body weight/day of the structural analogue substance could be established as the no-observed-adverse-effect-level (NOAEL).

Combined repeated dose oral toxicity study with the reproduction/developmental toxicity test in rats (OECD 422)

Daily oral treatment by gavage of 15 mg/kg of the structural analogue substance to rats according to the study design was tolerated whereas 45 or 135 mg/kg 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 4 p.p. with a slight reduction of rearing time and number, plus a body temperature decrease, however, a treatment-relationship could not be defined equivocally.

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

Conclusion

As a worst case approach, the NOAEL is considered to be 15 mg/k bw/day based on the results of the OECD Guideline study 422.

Justification for classification or non-classification

Based on the available data, the substance is classified for STOT RE 2 according to Regulation (EC) No 1272/2008.