2-isopropyl-9H-thioxanthen-9-one

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 April 2006 to 06 June 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Version / remarks:

1996

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

1995

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3050

Version / remarks:

2000

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Health, Labor and Welfare (No 1121002) , Ministry of Economy, Trade and Industry (No 2) and Ministry of Environment (No 031121002).

Version / remarks:

2003

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2-isopropyl-9H-thioxanthen-9-one

EC Number:

226-827-9

EC Name:

2-isopropyl-9H-thioxanthen-9-one

Cas Number:

5495-84-1

Molecular formula:

C16H14OS

IUPAC Name:

2-isopropyl-9H-thioxanthen-9-one

Test material form:

solid: particulate/powder

Details on test material:

- Storage: At room temperature in the dark
- Appearance: Pale-yellow powder

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl:(WI) BR (outbred, SPF-Quality)

Details on species / strain selection:

Recognised by international guidelines as the recommended test system (e.g. EPA, FDA, OECD, EC).

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Females nulliparous and non-pregnant: Yes
- Age at study initiation:
Approximately 6 weeks.
- Weight at study initiation:
Males: 246 – 255 g; females 179 – 182 g
- Fasting period before study:
No. The animals were fasted overnight (with a maximum of 20 hours) before blood sampling, but water was provided.
- Housing:
Group housing of 5 animals per sex in Macrolon plastic cages (MIV type, height 18 cm; during overnight activity monitoring individual housing in Miii type; height 15 cm.) with sterilised sawdust as bedding material.
- Diet:
Free access to pelleted rodent diet.
- Water:
Free access to tap water.
- Acclimation period:
Acclimatisation period was at least 5 days before start of treatment under laboratory conditions.

DETAILS OF FOOD AND WATER QUALITY:

- Diet: SM R/M-Z from SSNIFF® Spezialdiaten GmbH, Soest, Germany. Each batch is analysed for nutrients and contaminants are analysed on a regular basis. Results are examined and archived.
- Water: Certificates of analysis (performed quarterly) were examined and archived.
- Results of analysis for ingredients and/or contaminants of diet, bedding, paper and water were assessed and did not reveal any findings that were considered to have affected study integrity.

ENVIRONMENTAL CONDITIONS
- Temperature (°C):
21.0 ± 3.0 °C (actual range: 20.2 - 23.7 °C).
- Humidity (%):
Relative humidity of 30 - 70 % (actual range: 39 - 92 %).
- Air changes (per hr):
15 air changes per hour.
- Photoperiod (hrs dark / hrs light):
12 hours artificial fluorescent light and 12 hours darkness per day.

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

Oral gavage, using a stainless steel stomach tube.
Formulations were placed on a magnetic stirrer during dosing.

Vehicle:

polyethylene glycol

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 4 hours prior to dosing and were homogenised to a visually acceptable level. Adjustment was made for specific gravity of the vehicle (prolyplene glycol, specific gravity 1.036).

VEHICLE
- Justification for use and choice of vehicle: Based on trial formulations and information from the Sponsor.
- Amount: 5 mL/kg body weight. Actual dose volumes were calculated weekly according to the latest body weight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Samples of formulations prepared for use on day 2 were analysed to check stability over 5 hours, homogeneity (highest and lowest concentration) and accuracy of preparations (all concentrations). The analytical method used was based on the results of a separate project for the development and validation of the analytical method.

SAMPLES
- Accuracy, homogeneity and stability were determined for formulations prepared for use during treatment.
- Duplicate samples (approximately 500 mg), which were taken from the formulations using a pipette, were accurately weighed into volumetric flasks. For determination of accuracy, samples were taken at 50 % height, or at 90 %, 50 % and 10 % height. The latter set of samples was also used for the determination of the homogeneity of the formulations. For determination of stability, additional samples at t = 5 hours were taken at 50 % height. Because at t = 0 samples were taken at 10 %, 50 % and 90 % height, the mean concentration of these samples was used as t = 0 value for calculation.
- The flasks were filled up to the mark with ethylacetate. The solutions were further diluted with ethylacetate to obtain concentrations within the calibration range.

ANALYTICAL CONDITIONS
Instrument: Gaschromotograph 6890N (Agilent Technologies, Palo Alto, CA, USA).
Injector: Mass selective detector (MSD) 5973N (Agilent Technologies).
Column: DB-5; 25 m x 320 µm i.d., df = 0.25 µm (J&W Scientific, Folsom, CA, USA).
Carrier gas: Helium
Carrier gas flow: 1.0 mL/min
Injection: Programmed temperature vapouriser (PTV)
PTV Mode: Split
Split flow: 10 mL/min
Injection volume 1 µL

PTV temperature program:
Initial temperature: 65 °C
Initial time: 0.1 min
Rate: 720 °C/min
Final temperature: 300 °C
Hold time: 10 min

Oven temperature program:
Initial temperature: 90 °C
Initial time: 1 min
Rate 1: 50 °C/min
Temperature 1: 200 °C
Hold time: 0 min
Rate 2: 15 °C/min
Final temperature: 300 °C
Hold time: 2 min

MS Detection
Ionisation source: El+
MS Interface temperature: 280 °C
Data acquisition: Selected ion monitoring (SIM) at m/z 239 and 254 amu
Quantitation on mass: m/z 254 amu
Quantifier ions: m/z 239 amu

PREPARATION OF THE CALIBRATION SOLUTIONS
Stock and spiking solutions
- Stock and / or spiking solutions of the test material were prepared in ethylacetate at concentration of 926 – 4996 mg/L.
Calibration solutions
- Calibration solutions in the concentration range 0.100 – 2.00 mg/L were prepared from two stock solutions. The end solution of the calibration solutions was ethylacetate.
Procedural recovery samples
- Blank vehicle (approximately 500 mg) was spiked with the test substance at a nominal concentration of approximately 1 or 200 mg/g using a spike solution of pure test substance respectively. The accuracy samples were treated as described above.
- Nominal concentrations were not corrected for spiking volume.

SAMPLE INJECTIONS
- Calibration solutions were injected in duplicate. Test samples and procedural recovery samples were analysed by single injection.

ELECTRONIC DATA CAPTURE
- System control, data acquisition and data processing were performed using the following programme: Enhanced chemstation version D.00.00.38 (Agilent Technologies, Wilmington, DE, USA).

FORMULAS
Response (R): Peak area test substance [units]

Calibration curve: R = aCN + b

Where:
CN = nominal concentration [mg/L]
a = slope [units x L/mg]
b = intercept [units]

Analysed concentration (CA) = CA = ((R – b) / a)) x ((V x d) / w)) [mg/g]

Where:
w = weight sample [mg]
V = volume volumetric flask [mL]
d = dilution factor

Recovery: (CA / CN) x 100 %

Where
CN = nominal concentration [m/g]

Accuracy: (CA / CT) x 100 %

Where:
CT = target concentration [mg/g]

Relative difference (relative diff.): ((Mean conc. t = 5 – mean conc. t = 0) / mean concentration t = 0) x 100 %

Where:
T = time of sampling [hours]

SPECIFICATIONS
- Preparation of formulations was considered acceptable if the measured concentration levels were between 90 % and 110 % of the target concentrations and if the coefficient of variation was < 10 %. Additionally, formulations were considered to be stable in the relative difference between the t = 0 and t = 5 hour samples was < 10 %.

RESULTS
CALIBRATION CURVES
- A calibration curve was constructed using four concentrations. For each concentration, two responses were used. Linear regression analysis was performed using the least squares method with a 1/concentration^2 weighting factor. The coefficient of correlation (r) was > 0.99.

PROCEDURAL RECOVERY SAMPLES
- Mean recoveries of the procedural recovery samples were 95 % and 101 %. Because the criterion that mean recoveries should be between 70 % and 100 % was met the results for the test samples were accepted.

TEST SAMPLES
- In Group 1 formulations, no test material was detected.
- The concentrations analysed in formulations of Group 2, Group 3 and Group 4 were between 95 % and 105 % of target.
- The formulations of Group 2 and Group 4 were homogeneous (2.6 % and 2.2 % relative standard deviation, respectively).
- Analysis of Group 2 and Group 4 formulations after 5 hours of storage at room temperature yielded a relative difference of -0.88 % and -5.7 %, respectively. Therefore, the samples were considered stable at room temperature for at least 5 hours.

Duration of treatment / exposure:

For at least 28 days, 7 days per week, approximately the same time each day with a maximum of 4 hours difference between the earliest and latest dose. Animals were dosed up to the day prior to necropsy of the Main group animals.

Frequency of treatment:

Once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

50 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

150 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

Five per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Rationale for animal assignment: Randomisation. Prior to commencement of treatment, by computer-generated random algorithm according to bodyweight, with all animals within ± 20 % of the sex mean.
- Post-exposure recovery period in satellite groups: 14 days

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily. Time of death was recorded as precisely as possible.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily (except, inadvertently, on day 3 of the recovery period), detailed clinical observations were made in all animals. Once prior to start of treatment and on a weekly basis thereafter, this was also performed outside the home cage in a standard arena. The symptoms were graded according to fixed scales and the time of onset, degree and duration were recorded:
Maximum grade 1; grade 0 = absent, grade 1 = present
Maximum grade 3 or 4; grade 1 = slight, grade 2 = moderate, grade 3 = severe, grade 4 = very severe

BODY WEIGHT: Yes
- Time schedule for examinations:
Treatment period: On days 1, 8, 15, 22 and 28.
Recovery period: On days 1, 8 and 14.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Weekly

FOOD EFFICIENCY: No

WATER CONSUMPTION: Yes
- Time schedule for examinations: Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were collected between 7.00 and 10.30 a.m, immediately prior to scheduled post mortem examination at the end of the treatment and recovery period. The blood samples were drawn from the retro-orbital sinus and collected into tubes (Greiner Bio-One) prepared with EDTA for haematological parameters (0.5 mL), and with citrate for clotting tests (0.9 mL).
- Anaesthetic used for blood collection: Yes iso-flurane anaesthesia.
- Animals fasted: Yes, the animals were fasted overnight (with a maximum of 20 hours) before blood sampling, but water was provided.
- How many animals: All surviving animals.
- Parameters examined: White blood cells, differential leucocyte count (neutrophils, lymphocytes, monocytes, eosinophils, basophils), red blood cells, reticulocytes, red blood cell distribution width, haemoglobin, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, platelets, prothrombin time, activated partial thromboplastin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were collected between 7.00 and 10.30 a.m, immediately prior to scheduled post mortem examination at the end of the treatment and recovery period. The blood samples were drawn from the retro-orbital sinus and collected into tubes (Greiner Bio-One) prepared with Li-heparin treated tubes for clinical biochemistry parameters (0.5 mL).
- Animals fasted: Yes, the animals were fasted overnight (with a maximum of 20 hours) before blood sampling, but water was provided.
- How many animals: All surviving animals.
- Parameters examined: Alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, total protein, albumin, total bilirubin, urea, creatinine, glucose, cholesterol, sodium, potassium, chloride, calcium, inorganic phosphate.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: During week 4 of treatment, the following tests were performed on all animals:
Hearing ability, pupillary reflex, static righting reflex and grip strength (score 0 = normal/present, score 1 = abnormal/absent).
Motor activity test (recording period: 12 hours during overnight for individual animals, using a computerised monitoring system, Pearson Technical Services, Debenham, Stowmarket, England).

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- All animals surviving to the end of the observation period were deeply anaesthetised using iso­ flurane vapour and subsequently exsanguinated. All animals assigned to the study were necropsied and descriptions of all macroscopic abnormalities recorded. Samples of the following tissues and organs were collected from all animals at necropsy and fixed in a neutral phosphate buffered 4 % formaldehyde solution:
Adrenal glands, aorta, brain [cerebellum, mid-brain, cortex], caecum, cervix, (clitoral gland), colon, duodenum, epididymides, (eyes with optic nerve [if detectable] and Harderian gland), (female mammary gland area), (femur including joint), heart, ileum, jejunum, kidneys, (larynx), (lacrimal gland, exorbital), liver, lung infused with formalin, lymph nodes - mandibular, mesenteric, (nasopharynx), oesophagus, ovaries, pancreas, peyer's patches [jejunum, ileum] if detectable, pituitary gland, (preputial gland), prostate gland, rectum, (salivary glands - mandibular, sublingual), sciatic nerve, seminal vesicles, (skeletal muscle), (skin), spinal cord -cervical, mid-thoracic, lumbar Spleen, Sternum with bone marrow, Stomach, Testes, Thymus, Thyroid including parathyroid [if detectable], (Tongue), Trachea, urinary bladder, uterus, vagina, all gross lesions.
- Tissues mentioned within brackets were not examined microscopically as there were no signs of toxicity or target organ involvement.
- Identification marks: Not processed.

ORGAN WEIGHTS:
- The following organ weights (and terminal body weight) were recorded from the surviving animals on the scheduled day of necropsy:
Adrenal glands, epididymides, kidneys, ovaries, testes, brain, heart, liver, spleen, thymus.

HISTOPATHOLOGY: Yes.
- All organs and tissue samples listed below were processed, embedded and cut at a thickness of 2-4 micrometers and stained with haematoxylin and eosin.
- Representative tissue samples of the following organs were preserved in 4 % phosphate buffered neutral formaldehyde solution (10 % formalin).
- Following fixation, organs (except those listed in brackets) from Group 1 and 4 rats of the main study, one unscheduled death and all organs with macroscopic abnormalities as well as the liver from all rats were trimmed, processed and embedded in paraffin wax. Sections were cut at a thickness of 2 – 4 micrometers and stained with haematoxylin and eosin. Numbers of sections made are given in parentheses:
Adrenal glands (2), aorta (1), bone – sternum (1) [and femur including joint]; bone marrow – sternal (1), brain (3), [clitoral glands], epididymides (4), esophagus (1), [eyes with optic nerve and Harderian glands]; heart (1), kidneys (2), [lacrimal glands – exorbital], large intestine (3) – cecum, colon and rectum; [larynx], liver (2), lungs (2), lymph nodes – mandibular (2) and mesenteric (1); [female mammary gland area], [nasopharynx], ovaries (2), pancreas (1), pituitary gland (1), [preputial glands], prostate gland (1), [salivary glands – mandibular and sublingual]; sciatic nerve (1), seminal vesicles (2), [skeletal muscle], [skin], small intestine (3) – duodenum, jejunum and ileum with Peyer’s patches; spinal cord (3) – cervical, mid-thoracic and lumbar; spleen (1), stomach (1), testes (2), thymus (1), thyroid glands (2) with parathyroid glands (2), [tongue], trachea (1), urinary bladder (1), uterus with uterine cervix (3), vagina (1) and all organs or tissues with macroscopic abnormalities.

- Following examination of the above tissues, sections were prepared and examined from kidneys (males), stomach and thyroid glands from rats of the intermediate dose Groups 2 and 3 and rats of the recovery allocation.
- The sections were examined by light microscopy.

Statistics:

The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to­one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied when the data could not be assumed to follow a normal distribution.
- The exact Fisher-test was applied to frequency data.
- All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. No statistical analysis was performed on histopathology findings. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no clinical signs of toxicity noted during the observation period.

Incidental findings that were noted among the animals during the treatment phase included alopecia, scabs, wounds and/or rales. These findings are occasionally noted in rats of this age and strain and housed and treated under the conditions in this study and considered of no toxicological significance. In addition one high dose female showed swelling of the right periorbital region, chromodacryorrhoea of the right eye and ptosis of the right eye during the recovery phase. Findings in this female were considered to be related to the blood sampling procedure at the end of the treatment phase. Apart from these findings in a single high dose female and the incidental findings described above, no further clinical signs were noted in any of the other animals.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No mortality related to treatment with the test substance occurred.
One male (no.26, receiving 1 000 mg/kg/day) died at blood sampling at the end of the treatment period, which was considered to be an accidental death.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Mean body weights and body weight gain of treated animals remained in the same range as controls over the 4-week study period.

A slightly low body weight gain was noted for one female at 1 000 mg/kg/day (no. 57). Since other animals of this dose group showed normal body weight gain, this was considered to be of no toxicological significance.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption before or after allowance for body weight was similar between treated and control animals.

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):

No toxicologically relevant changes were noted in haematological parameters.

Any statistically significant changes in haematology parameters were considered to be of no toxicological significance since means remained well within the normal range for rats of this age and strain, changes at the end of the recovery period were absent at the end of the treatment period, and/or a dose-related response was absent. These changes included increased reticulocyte counts and mean corpuscular volume (MCV) in males at 1 000 mg/kg/day at the end of the treatment phase, decreased mean corpuscular haemoglobin concentration (MCHC) in males at 1 000 mg/kg/day at the end of the recovery period, decreased haemoglobin concentration and haematocrit level in females at 150 mg/kg/day, and decreased partial thromboplastin time (APTT) in females at 1 000 mg/kg/day at the end of the treatment period.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The following statistically significant changes in clinical biochemistry parameters distinguished treated from control animals at the end of the treatment period:
- Increased total protein, albumin and cholesterol levels in males and females at 50 mg/kg/day and higher (increase in cholesterol levels not statistically significant for females at 150 mg/kg/day);
- increased creatinine levels in males and females at 1 000 mg/kg/day;
- other, less pronounced but statistically significant changes at the end of treatment included: Reduced sodium and chloride levels and increased potassium and calcium levels in females at 1 000 mg/kg/day, reduced total bilirubin levels in males at 1 000 mg/kg/day, and increased calcium levels in males at 50 and 1 000 mg/kg/day. These changes were within the normal range encountered for rats of this age and strain. Means were similar to those measured at the end of the recovery period (but without achieving statistical significance).

At the end of the recovery phase, these changes had recovered to control levels. However, other statistically significant changes emerging at the end of the recovery period at 1 000 mg/kg/day included increased inorganic phosphate levels in males and females, increased sodium levels in males, and reduced total bilirubin levels in females.

The lower aspartate aminotransferase activity (ASAT) of females at 150 mg/kg/day occurred in the absence of a dose-related distribution and was therefore considered to be of no toxicological relevance.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all animals. The variation in motor activity did not indicate a relation with treatment.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Liver weight and liver to body weight ratio were increased in males and females at 50 mg/kg/day and higher, achieving a level of statistical significance at each dose level. At the end of the recovery period, liver weights remained slightly increased in males and females, without achieving a level of statistical significance.

The statistically significant higher testes and epididymides to body weight ratio of males at 1 000 mg/kg/day at the end of the recovery period was absent at the end of the treatment period, and means were considered normal for rats of this age and strain. Other statistically significant changes in organ weights and organ to body weight ratios at 50 and 150 mg/kg/day occurred in the absence of a treatment related distribution and means were within the normal range for rats of this age and strain. These changes were confined to adrenals, spleen, testes, epididymides and kidneys. No toxicological significance was ascribed to these changes.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Macroscopic observations at necropsy did not reveal any alterations that were considered toxicologically significant.

Incidental findings among control and/or treated animals included pelvic dilation in the kidneys, nodules or foci on the thymus, red discolouration of the thymus, enlarged and/or discoloured mandibular lymph nodes, scab formation on the skin, reduced size of the papillary process of the liver with greenish discolouration, gelatinous lacrimal glands, fluid in the uterus and discoloration of harderian glands. These findings are occasionally seen among rats used in these types of studies. In the absence of correlated microscopic findings these were considered changes of no toxicological significance. No macroscopic abnormalities were noted in the high dose male that died at blood sampling.

Recorded macroscopic findings were considered to be spontaneous in nature and did not distinguish treated animals from the controls.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

In the stomach hyperplasia of the forestomach squamous epithelium at a minimal or slight degree was recorded in three males and two females of Group 4 (1 000 mg/kg/day). Diffuse midzonal/centrilobular hypertrophy was seen in the liver of three males and three females of Group 3 (150 mg/kg/day) at minimal severity, in three males of Group 4 also at minimal severity and in all five females of Group 4 at minimal to slight severity. The minimal degree of this finding may be regarded as an adaptive physiological response rather than an indicator of frank toxicity.

In the kidneys of male rats there was a slight increase in the incidence and severity of corticomedullary tubular basophilia in Group 4 - three minimal and one slight degree compared to two minimal in controls (Group 1). This finding was associated with an increase in the severity of cortical hyaline droplets to moderate in four group 4 males.

There was a slight increase in the severity of diffuse follicular hypertrophy/ hyperplasia in the thyroid glands to slight in two Group 4 males.

Following the recovery period corticomedullary tubular basophilia in the kidneys remained slightly increased in Group 4 males - three minimal and two slight compared to three minimal in control males. Liver and thyroid microscopic changes were no longer observed at the end of the recovery period.

All other microscopic findings were within the range of background pathology encountered in Wistar rats of this age and strain and occurred at similar incidences and severity in both control and treated rats.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Details on results:

- Microscopic findings related to treatment were noted in stomach, liver, kidneys, and thyroid glands at the end of the main study.
- In the stomach, hyperplasia of the forestomach squamous epitheium at a minimal or slight degree was recorded in three males and two females of Group 4. Diffuse midzonal/certilobular hypertrophy was seen in the liver of three males and three females of Group 3 (150 mg/kg/day) at minimal severity, in three males of Group 4 also at minimal severity and in all five females of Group 3 at minimal to slight severity.
- In the kidneys of male rats there was a slight increase in the incidence and severity of corticomedullary tubular basophilia in Group 4 – three minimal and one slight degree compared to two minimal in controls (Group 1). This finding was associated with an increase in the severity of cortical hyaline droplets to moderate in four Group 4 males. An unusual finding of unknown aetiology, diffuse basophilia of the outer stripe of the medulla was noted at moderate or severe degree in two Group 1, one Group 2 (50 mg/kg/day) and one Group 4 male rats and was therefore regarded as not related to treatment.
There was a slight increase in the severity of diffuse follicular hypertrophy/hyperplasia in the thyroid glands to slight in two Group 4 males.
- Following a recovery period corticomedullary tubular basophilia in the kidneys remained slightly increased in Group 4 males – three minimal and two slight compared to the three minimal in control males.
- The remainder of the recorded microscopic findings at both sacrifices, were within the range of background pathology encountered in Wistar rats of this age and occurred at similar incidences and severity in both control and treated rats.
- In conclusion, morphologic alterations following the administration of the test material by once daily gavage to Wistar rats were present in the stomach, liver, kidneys and thyroid glands.
- In the stomach hyperplasia of the forestomach squamous epithelium at minimal or slight degree were recorded in three males and two females of Group 4 (1 000 mg/kg/day). Diffuse midzonal/centrilobular hypertrophy was seen in the liver of three males and three females of Group 3 (150 mg/kg/day) at minimal severity, in three males of Group 4, also at minimal severity and in all five females of Group 4 at minimal to slight severity. The minimal degree of this finding may be regarded as an adaptive physiological response rather than an indicator of frank toxicity.
- In the kidneys of male rats there was a light increase in the incidence and severity of cotricomedullary tubula basophilia in Group 4 – three minimal and one slight degree compared to two minimal in controls (Group 1). This finding was associated with an increase in the severity of cortical hyaline droplets to moderate in four Group 4 males.
- There was a slight increase in the severity of diffuse follicular hypertrophy / hyperplasia in the thyroid glands to slight in two Group 4 males.
- Following the recovery period corticomedullary tubublar basophilia in the kidneys remained slightly increased in Group 4 males – three minimal and two slight compared to three minimal in control males.
- Based on the above findings the NOAEL may be regarded as 150 mg/kg/day.

Effect levels

Dose descriptor:

NOAEL

Effect level:

150 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:

no

Any other information on results incl. tables

Analysis of Dose Preparations

Test substance formulations in propylene glycol were noted as stable for at least 5 hours and formed a homogeneous suspension at the concentrations tested. Analysis of the accuracy of dose preparations revealed values within the range of 90 % to 110 % of nominal, which was considered to represent an acceptable level of accuracy for formulations of this type.

Applicant's summary and conclusion

Conclusions:

Under the conditions of the test, treatment with the test material at the limit dose of 1 000 mg/kg/day resulted in treatment­related effects in the liver, thyroid, kidneys and stomach. At 50 and 150 mg/kg/day, findings were confined to the liver. At 150 mg/kg/day, clinical biochemistry changes and higher liver weights were associated with slight histopathological effects in the liver. At 50 mg/kg/day, clinical biochemistry changes and higher liver weights were not associated with any histopathological changes. Therefore, these effects observed at 50 mg/kg/day were considered to be of an adaptive rather than toxic nature, and were considered not to constitute evidence of impaired organ function. Also, since these effects were fully reversible at the highest dose, a definitive No Observed Adverse Effect Level (NOAEL) for the test material of 50 mg/kg/day was established. Since effects observed at 150 mg/kg/day were only slightly more pronounced than at 50 mg/kg/day, and the observed liver hypertrophy at 150 mg/kg/day was of a minimal nature and appeared fully reversible at the highest dose, a NOAEL of 150 mg/kg/day may be considered

Executive summary:

A repeated dose 28-day oral toxicity study was conducted by daily oral gavage in the rat followed by a 14-day recovery period according to EU Method B.7, OECD Guideline 407, Japanese MHLW (No 1121002), METI (No 2) and ME (No 031121002) and EPA OPPTS 870.3050 and in compliance with the principles of GLP.

Based on the results of a 5-day range-finding study, the dose levels for the 28-day study were selected to be 0, 50, 150 and 1 000 mg/kg/day.

The test material was administered daily for 28 days by oral gavage to SPF-bred Wistar rat. One control group and three treated groups were tested, each consisting of 5 males and 5 females. An extra 5 animals per sex in the control and high dose groups were allowed 14 days of recovery.

Chemical analyses of formulations were conducted once during the study to assess accuracy, homogeneity and stability over 5 hours.

The following parameters were evaluated: Clinical signs daily; functional observation tests, body weight and food consumption weekly; clinical pathology and macroscopy at termination; organ weights and histopathology on a selection of tissues.

Accuracy, homogeneity and stability over 5 hours of formulations of test material in propylene glycol were demonstrated by analyses.

In two males at 1 000 mg/kg/day, there was a slight increase in the severity of diffuse follicular hypertrophy/hyperplasia in the thyroid glands. Additionally, in the kidneys of males at 1 000 mg/kg/day, a slight increase in the incidence and severity of corticomedullary tubular basophilia was observed, in association with an increase in the severity of cortical hyaline droplets to moderate in most high dose males. Following the recovery period corticomedullary tubular basophilia in the kidneys remained slightly increased in males at 1 000 mg/kg/day.

In the stomach, hyperplasia of the forestomach squamous epithelium at a minimal or slight degree was recorded in some animals at 1 000 mg/kg/day.

At 50 mg/kg/day and higher, total protein, albumin and cholesterol levels were increased along with increased liver weights. At 150 and 1 000 mg/kg/day, these findings occurred with diffuse midzonal/centrilobular hypertrophy of the liver. These findings had resolved at the end of the recovery period.

Statistically significant clinical biochemistry changes emerging at the end of the recovery period at 1 000 mg/kg/day were absent at the end of the treatment period. These included increased inorganic phosphate levels in males and females, increased sodium levels in males, and reduced total bilirubin levels in females.

There were no changes at determination of clinical appearance, performance of functional observations, body weight and food consumption measurements, or alterations during haematological investigations and macroscopic examination that were considered to be an effect of treatment.

Hyperplasia of the forestomach squamous epithelium recorded in some animals at 1 000 mg/kg/day was considered to be a local adaptive response to presence of the test substance in the stomach.

Diffuse midzonal/centrilobular hypertrophy seen in the liver of most animals at 150 and 1 000 mg/kg/day, supported the slightly increased liver weights and increased total protein, albumin and cholesterol levels at 150 and 1 000 mg/kg/day. At 50 mg/kg/day similar clinical biochemistry changes were apparent, but apart from an increased liver weight no morphological correlates were present to support these changes. Also, these clinical biochemistry changes occurred in the absence of a clear dose-related response and were absent at the end of the recovery phase. The minimal degree of diffuse midzonal/centrilobular hypertrophy was suggestive of adaptive hepatic changes in response to xenobiotic administration, and had resolved at the end of the recovery phase.

Clinical biochemistry changes at 1 000 mg/kg/day other than increased total protein, albumin and cholesterol levels were within the normal range expected for rats of this age and strain. Due to the minor degree of these changes, these could not with any certainty be associated with any of the above morphological changes. At the end of the recovery phase, these changes had recovered to control levels.

General health condition based on clinical signs, body weights, food intake and functional observation tests was unaffected at dose levels up to 1 000 mg/kg/day. Also no clinical signs of toxicity were apparent at the end of the recovery period.

Treatment with the test material at a dose of 1 000 mg/kg/day resulted in treatment­related effects in the liver, thyroid, kidneys and stomach. At 50 and 150 mg/kg/day, findings were confined to the liver. At 150 mg/kg/day, clinical biochemistry changes and higher liver weights were associated with slight histopathological effects in the liver. At 50 mg/kg/day, clinical biochemistry changes and higher liver weights were not associated with histopathological changes. Therefore, these effects observed at 50 mg/kg/day were considered to be of an adaptive rather than toxic nature, and were considered not to constitute evidence of impaired organ function. Also, since these effects were fully reversible at the highest dose, a definitive No Observed Adverse Effect Level (NOAEL) for the test material of 50 mg/kg/day was established. Since effects observed at 150 mg/kg/day were only slightly more pronounced than at 50 mg/kg/day, and the observed liver hypertrophy at 150 mg/kg/day was of a minimal nature and appeared fully reversible at the highest dose, a NOAEL of 150 mg/kg/day may be considered.