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Administrative data

Description of key information

Two 28-day repeated toxicity studies are available on 2-isopropyl-9H-thioxanthen-9-one. According to the results obtained in the Key study (van Otterdijk, 2006), the NOAEL was 150 mg/kg/day.


 

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

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:
supporting study
Study period:
08 December 1988 to 31 January 1989
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: 4 weeks old on arrival
- Weight at study initiation: Male ca 85 g, female ca 60 g on arrival
- Housing: Rats were housed in a barrier maintained animal room. Rats were housed either 2 or 3 of one sex per cage in suspended polypropylene cages (overall dimensions ca 420 x 270 x 200 mm) with stainless steel wire grid tops and bottoms. Beneath each cage was suspended a polypropylene tray containing absorbent paper. Tray paper was changed as required during the study. Each cage had a poly­propylene water bottle (total capacity 300 mL) with rubber washer and melamine cap.
- Diet: Ad libitum
- Water: Ad libitum
- Acclimation period: 12 days


ENVIRONMENTAL CONDITIONS
- Temperature: 20 ± 2 °C
- Humidity: 55 ± 10 %
- Air changes: 15 to 20 air changes per hour
- Photoperiod: a 12 h light/dark cycle was controlled by a time switch, light hours being 0700 to 1900 h.
Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Remarks:
(PEG 400)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
- Dosing solutions were prepared daily using polyethylene glycol (PEG) 400 as vehicle.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Test material preparations were analysed on the first and fourth week of the study.
- On week 1 for the nominal concentrations of: 0, 2, 20 and 200 mg/mL the mean found concentrations were: 0, 2.07, 20.16 and 187.5 mg/mL.
- On week 4 for the nominal concentrations of: 0, 2, 20 and 200 mg/mL the mean found concentrations were: 0, 1.9, 20.03 and 195.77 mg/mL.
- The maximum % difference from the nominal values was 6.3 %.
- Analysis for all the samples were within ± 10 % of the theoretical values.
Duration of treatment / exposure:
4 weeks
Frequency of treatment:
Once daily
Dose / conc.:
10 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5 animals per sex per dose for the main study.
5 animals per sex at 0 and 1000 mg/kg doses in the recovery study.
Control animals:
yes, concurrent vehicle
Details on study design:
- Post-exposure recovery period in satellite groups: The recovery study rats were allowed to recover from dosing for 2 weeks before they were killed.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Viability was checked once each morning and once as late as practicable on each day. All animals were examined for reaction to treatment during each day. The onset, intensity and duration of these signs were recorded.

DETAILED CLINICAL OBSERVATIONS: Yes
- All animals received a detailed clinical examination once each week.

BODY WEIGHT: Yes
- The weight of each animal was recorded at weekly intervals commencing one week before the start of treatment up until the end of the study.

FOOD CONSUMPTION: Yes
- The quantity of food consumed by each cage of animals (g.rat^-1.week^-1) was recorded once each week, commencing one week before the start of treatment up until the end of the study.

WATER CONSUMPTION: Yes
- Water consumption was monitored by visual inspection throughout the study period.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Samples were taken from all main study rats from each group during Week 4 and from all surviving recovery study animals during Week 6. Blood samples were collected from the orbital sinus under light ether anaesthesia.
- The following parameters were measured on whole blood taken into tubes containing EDTA: Haemoglobin, Total red blood cell count, Total white blood cell count, Differential white cell count, Haematocrit, Calculations of absolute indices, Hepato Quick test (on sample obtained by tailsnip without anaesthesia) and Platelet count.

CLINICAL CHEMISTRY: Yes
- The following parameters were measured on plasma from whole blood taken into tubes containing heparin: Blood urea nitrogen, Glucose, Aspartate aminotransferase, Alanine aminotransferase, Sodium, Potassium, Calcium, Chloride, Total protein, Albumin, Albumin-globulin ratio, Alkaline phosphatase, Creatinine, Phosphate, Total bilirubin, Triglycerides, Cholesterol and Gamma glutamyl transferase.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- All animals were killed and necropsied. Method of killing was by carbon dioxide asphyxiation followed by exsanguination. The gross dissection and necropsy were performed under the guidance of a pathologist. Premature decedents were also necropsied.
- The following organs were weighed: adrenals, brain, heart, kidneys, liver, ovaries, pituitary, spleen and testes (plus epididymides).
- The following tissues were examined in situ and fixed: Adrenals, Any abnormal tissue, Bladder, Bone marrow (Femur), Brain, Eyes, Heart, Kidneys, Liver, Ovaries, Pituitary, Spleen, Stomach (glandular and non­glandular), Testes (plus epididymides) and Thyroids (with parathyroids).
- Samples of the above tissues were taken from all animals and placed in 10 % neutral buffered formalin (except eyes which were preserved in Davidson's fluid).
- Tissues were trimmed to a maximum thickness of 3 mm for processing. Parenchymal organs, e.g. liver, were trimmed to allow the largest surface area possible for examination. Mid-transverse sections through the entire cortex and medulla of each kidney were submitted. Tissues were cut at 4-6 µm thickness and stained with haematoxylin and eosin (H and E).

HISTOPATHOLOGY: Yes
- Liver, heart, kidneys, spleen and adrenals were examined from all Control and High dose animals of the main and recovery studies.
Statistics:
- Haematology, clinical chemistry, organ weight and body weight data were statistically analysed for homogeneity of variance using the 'F-max' test. If the group variances appeared homogeneous a parametric ANOVA was used and pairwise comparisons made via Student' t-test using Fisher's F-protected LSD. If the variances were heterogeneous, log or square root transformations were used in an attempt to stabilise the variances. If the variances were still heterogeneous then a non-parametric test such as Kruskal-Wallis ANOVA was used and pairwise comparisons made via Dunn Z test where considered appropriate.
- Organ weights were also analysed conditional on body weight (i.e. analysis of covariance).
- Histopathology data were analysed using Fisher's Exact Probability test.
Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs that could be attributed to administration of the test material.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
- There were 3 unscheduled deaths, one Control dose group male and 2 High dose group males. None of these deaths could be definitely attributed to dosing with the test material.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
There were no notable intergroup differences in either sex, the reduction in the female High dose group (10 %) seen after the recovery period was considered to be due to chance, as after 4 weeks of receiving the test material this group had a comparable body weight gain to their Controls.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There were no notable intergroup differences in either sex.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Visual assessment of the water bottles showed no notable differences.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
MALES
- After 4 weeks of dosing haemoglobin was slightly reduced at the High dose level (8 %, P < 0.01). There was a consequent slight reduction in red blood cell counts and haematocrit (3 and 6 % respectively, both not statistically significant) and MCH (4 %, P < 0.05). After the 2 week recovery period MCH was still slightly reduced (3 % P < 0.05) and MCV was slightly reduced (5 %, P < 0.05).
- The small magnitude of the changes suggests these are chance effects.
- There were no other notable intergroup differences at either timepoint.

FEMALES
- After 4 weeks the only statistically significant findings were a slight reduction in MCHC in the Intermediate (2 %, P < 0.05) and High dose (3 %, P < 0.01) dose groups, these findings were not considered to be due to treatment as no other red blood cell parameters were affected.
- After the recovery period there were no notable intergroup differences.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
MALES
- After 4 weeks dosing, cholesterol levels were increased in all groups receiving the test material, reaching statistical significance in the Intermediate (45 %, P < 0.01) and High (73 %, P < 0.001) dose groups. Triglycerides were statistically significantly decreased in the same groups (24 %, P < 0.05 and 52 %, P < 0.001 respectively). Total protein was increased in both these groups (both 8 %, P < 0.01) with a consequent reduction in AG-R (9 %, P < 0.05 in the Intermediate dose group and 9 %, P < 0.01 in the High dose group). AG-R was also reduced in the Low dose group (10 %, P<0.05).
- GGT was notably increased in the High dose group.
- In addition, there was a slight increase seen in creatinine in the Intermediate (5 %, P < 0.05) and High (9 %, P < 0.01) dose groups.
- Chloride was slightly reduced in the High (3 %, P < 0.01) dose group and sodium was slightly increased in the Intermediate (1 %, P < 0.05) dose group but these were considered chance effects due to the small magnitude of the changes.
- After the 2 week recovery period none of these changes were evident between the Control and High dose groups.

FEMALES
- After 4 weeks dosing, cholesterol levels were statistically significantly increased in the Intermediate (73 %, P < 0.001) and High 114 %, P < 0.001) dose groups. Total protein was also increased in these groups (9 %, P < 0.01 and 12 %, P < 0.001 respectively), while albumin was increased in all groups: Low dose (6 %, P < 0.05), Intermediate dose (11 %, P < 0.001) and High dose (14 %, P < 0.001). AG-R was not affected. Total bilirubin was also slightly increased in the Intermediate and High dose groups (both 88 %, P < 0.01).
- In addition, creatinine was slightly increased in the High (11 %, P < 0.01) dose group when compared to Controls.
- In the High dose group glucose and chloride were reduced (10 %, P < 0.05 and 3 %, P < 0.05 respectively), but these were considered to be chance effects.
- GGT was increased for animals in the Intermediate and High dose groups.
- After the 2 week recovery period there was no evidence of the changes between the Control and High dose groups seen at Week 4. Other instances of minor statistical significance were attributed to chance since there was no evidence of an effect after 4 weeks dosing.
Endocrine findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
MALES
- After 4 weeks dosing, absolute liver weight was increased in the Intermediate (17 %, not statistically significant) and High (58 %, P < 0.001) dose groups. Following adjustment for terminal body weight (covariance analysis), the increases were 32 %, (P < 0.001) and 69 %, (P < 0.001) respectively. Slight increases were still present in absolute weight and after covariance analysis following the recovery period but, the differences were reduced in magnitude and did not achieve statistical significance. There were no other notable intergroup differences.

FEMALES
- After 4 weeks of dosing with the test material absolute liver weight was increased in the Intermediate (19 %, not statistically significant) and High (60 %, P < 0.001) dose groups. After covariance analysis the increases were still apparent and reached statistical significance in both groups (38 %, P < 0.001 and 74 %, P < 0.001 respectively). The increase in the High dose group was still apparent after the 2 week recovery period reaching statistical significance only after covariance analysis (24 %, P < 0.01).
- The High dose group also showed increased absolute ovaries weight (29 %, P < 0.01) and after covariance analysis (32 %, P < 0.01). The Low dose group also showed an increase in ovaries weight (absolute -19 %, P < 0.05, covariance analysis -21 %, P < 0.05). Ovaries weight in the Intermediate dose group were similar to Controls. After the recovery period there were no notable differences between the Control and High dose groups.
- After the recovery period, spleen weight was increased in the High dose group after covariance analysis (22 %, P < 0.05). This is thought to be a chance as there was no effect after 4 weeks dosing.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
- One male and one female High dose animal had an enlarged liver.
- Other findings recorded were those commonly seen in rats of this age and strain.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no histopathological findings which provided evidence of impaired hepatic function; clinical chemistry and liver weight findings are often more sensitive indicators of altered liver function than histopathological findings, however. The effects on lipid and protein metabolism are not necessarily indicative of a harmful effect on the liver, although an increase in GGT is often an indicator of liver damage. In addition, creatinine levels were increased at 100 and 1000 mg.kg^-1.day^-1 in males and at 1000 mg.kg^-1.day^-1 in females.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Details on results:
Dosing Sprague-Dawley rats for 4 weeks with the test material produced changes in clinical chemistry parameters at 100 and 1000 mg. kg^-1.day^-1 in males and at 10, 100, and 1000 mg. kg^-1.day^-1 in females, liver weight increases in both sexes at 1000 mg. kg^-1.day^-1 and in females only at 100 mg. kg^-1.day^-1 and liver pathology changes typical of an adaptive response to treatment. All these findings showed some, if not total, resolution after two weeks recovery.
Key result
Dose descriptor:
NOEL
Effect level:
10 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical biochemistry
gross pathology
organ weights and organ / body weight ratios
Key result
Dose descriptor:
LOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical biochemistry
gross pathology
organ weights and organ / body weight ratios
Critical effects observed:
not specified
Conclusions:
Under the conditions of this study, the No Effect Level observed was 10 mg/kg/day and the Low Adverse Effect Level was 100 mg/kg/day in both sexes.
Executive summary:

The repeated dose oral toxicity of the test material was investigated in a study similar in design to OECD 407 under GLP conditions.


Groups of 5 male and 5 female Sprague-Dawley rats were dosed daily for 4 weeks with the test material, at dose levels of 0, 10, 100 and 1000 mg/kg/day, via a steel dosing cannula. A further 5 males and 5 females were included in the groups receiving 0 or 1000 mg/kg/day to allow the extent of recovery from treatment to be assessed. Blood samples were taken for haematology and clinical chemistry investigations from all main study animals during Week 4. After 4 weeks of dosing all main study animals were killed and necropsied. The recovery study animals were maintained for a further 2 weeks. Blood samples for haematology and clinical chemistry investigations were taken from these animals during Week 6. After 6 weeks all the recovery group animals were killed and necropsied. A limited histopathological examination was carried out on all Control and High dose animals from the main and recovery studies.


There were 3 unscheduled deaths: One Control dose group male and two High dose group males. None of these deaths could definitely be attributed to administration of the test material. There were no clinical signs which could be attributed to administration of the test material. There were no notable intergroup differences in either sex in the animal body weights and food or water consumption.


In the haematology investigations there were no intergroup differences that could be attributed to treatment with the test material. After 4 weeks dosing there were some differences in clinical chemistry results between Controls and those groups receiving the test material (increased cholesterol, total protein and GGT in both sexes; decreased triglycerides, albumin globulin ratio in males only, albumin and total bilirubin in females only). These findings indicated that the test material had an adverse effect on liver function, starting at 100 mg/kg/day and clearly evident at 1000 mg/kg/day. In addition small differences were seen in creatinine levels (increased) in males (Intermediate and High dose) and females (High dose). There were no notable intergroup differences after the recovery period.


Liver weight was increased after 4 weeks dosing in Intermediate and High dose males and females, with significant increases recorded for the relative to body weight values in the Intermediate dose animals, and marked, significant increases recorded in the High dose animals. This effect showed some resolution after the recovery period in both sexes. Ovary weight was increased in High dose females after 4 weeks dosing only. There were no notable intergroup differences in the gross pathology.


Centrilobular hepatocyte hypertrophy accompanied by degenerative changes (i.e. periportal hepatocyte rarefaction and focal or single hepatocyte necrosis) was present in the liver of most High dose male main study animals. These findings were not seen in females.


Dosing Sprague-Dawley rats for 4 weeks with the test material produced changes in clinical chemistry parameters at 100 and 1000 mg/kg/day in males and at 10, 100, and 1000 mg/kg/day in females, liver weight increases in both sexes at 1000 mg/kg/day and in females only at 100 mg/kg/day and liver pathology changes.


Under the conditions of this study, the No Effect Level observed was 10 mg/kg/day; the Low Adverse Effect Level was 100 mg/kg/day in both sexes.

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
Qualifier:
according to guideline
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Version / remarks:
1996
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Version / remarks:
1995
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EPA OPPTS 870.3050
Version / remarks:
2000
Deviations:
no
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
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.
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
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
150 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:
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
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.
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.
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
Critical effects observed:
no

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.

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.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
150 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The key study is considered to be reliable with a klimisch score of 1.

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

Oral - Key Study: van Otterdijk (2006)


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. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).


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.


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, and no clinical signs of toxicity were apparent also during or at the end of the recovery period.


There were no changes at haematological investigations and macroscopic examination that were considered to be an effect of treatment.


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. 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.


Statistically significant clinical biochemistry changes noted 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.


In the stomach, hyperplasia of the forestomach squamous epithelium at a minimal or slight degree was recorded in some animals of both sexes at 1 000 mg/kg/day. This was considered to be a local adaptive response to the presence of the test substance in the stomach, at a high concentration and given as a bolus.


Diffuse midzonal/centrilobular hypertrophy was seen in the liver of most animals at 150 and 1 000 mg/kg/day, supporting 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.


At 1 000 mg/kg/day, there was a slight increase in the severity of diffuse follicular hypertrophy/hyperplasia in the thyroid glands which was reported as slight for two animals. Additionally, in the kidneys of males at 1 000 mg/kg/day, a slight increase in the incidence (4 males vs. 2 control males) 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, no changes were detected in the thyroid gland of any animal. Corticomedullary tubular basophilia in the kidneys remained slightly increased in recovery males at 1 000 mg/kg/day. The incidence of corticomedullary tubular basophilia in female rats was comparable to the control group at termination of the treatment period, and absent at the end of the recovery period.


Treatment with the test material at a dose of 1 000 mg/kg/day resulted in treatment related 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 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 in the report. 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 as a conservative measure.


The increased liver weight at 1 000 mg/kg bw/day recovered in males and tended to recovery in females. In addition, the diffuse midzonal/centrilobular hypertrophy observed at the end of dosing period, as well as the diffuse follicular cell hypertrophy/hyperplasia in thyroids were no longer apparent at the end of two weeks recovery period at this dose level, supporting the changes in the liver at 1 000 mg/kg bw/day, and the secondary effects on thyroid, may well represent an adaptive response to the administration of a high dose of a xenobiotic rather than substance-specific adverse effects. The diffuse follicular hypertrophy/ hyperplasia in thyroids in males is likely to be the rat-specific secondary effect to the hepatic enzyme induction following the administration of test substance. The renal lesions observed in male but not female rats in the form of a hyaline droplet nephropathy is likely to be mediated by alpha-2u-globulin that is not relevant for human and hence to be disregard for the human health risk assessment.


 


Oral - Supporting Study: Atkinson (1989)


The repeated dose oral toxicity of the test material was investigated in a study similar in design to OECD 407 under GLP conditions. The study was awarded a reliability score of 2 in accordance with the criteria set forth by Klimisch et al. (1997) mainly because parameters now required are missing (e.g. neurological end points) and because histopathology was very limited and not performed in groups other than the high dose and controls.


 


Groups of 5 male and 5 female Sprague-Dawley rats were dosed daily for 4 weeks with the test material, at dose levels of 0, 10, 100 and 1 000 mg/kg/day, via a steel dosing cannula. A further 5 males and 5 females were included in the groups receiving 0 or 1 000 mg/kg/day to allow the extent of recovery from treatment to be assessed. Blood samples were taken for haematology and clinical chemistry investigations from all main study animals during Week 4. After 4 weeks of dosing all main study animals were killed and necropsied. The recovery study animals were maintained for a further 2 weeks. Blood samples for haematology and clinical chemistry investigations were taken from these animals during Week 6. After 6 weeks all the recovery group animals were killed and necropsied. A limited histopathological examination was carried out on all Control and High dose animals from the main and recovery groups.


There were 3 unscheduled deaths: One Control dose group male and two High dose group males. None of these deaths could definitely be attributed to administration of the test material. There were no clinical signs which could be attributed to administration of the test material. There were no notable intergroup differences in either sex in the animal body weights and food or water consumption.


In the haematology investigations there were no intergroup differences that could be attributed to treatment with the test material. After 4 weeks dosing there were some differences in clinical chemistry results between Controls and those groups receiving the test material (increased cholesterol, total protein and GGT in both sexes; decreased triglycerides, albumin globulin ratio in males only, albumin and total bilirubin in females only almost exclusively at the Intermediate and High doses). These findings indicated that the test material affected the liver functions, starting at 100 mg/kg/day and clearly at 1 000 mg/kg/day. In addition, small differences were seen in creatinine levels in males (increased at Intermediate and High doses) and females (High dose). There were no notable intergroup differences after the recovery period.


Liver weight was increased after 4 weeks dosing in Intermediate and High dose males and females, with significant increases recorded for the relative to body weight values in the Intermediate dose animals, and marked, significant increases recorded in the High dose animals. This effect showed some resolution after the recovery period in both sexes. Ovary weight was increased in High dose females after 4 weeks dosing only. There were no notable intergroup differences in the gross pathology.


Centrilobular hepatocyte hypertrophy accompanied by degenerative changes (i.e. periportal hepatocyte rarefaction and focal or single hepatocyte necrosis) was present in the liver of most High dose male main study animals. These findings were not seen in females or in recovery animals.


Dosing Sprague-Dawley rats for 4 weeks with the test material produced changes in clinical chemistry parameters at 100 and 1 000 mg/kg/day in males and at 10, 100, and 1 000 mg/kg/day in females, liver weight increases in both sexes at 1000 mg/kg/day and in females only at 100 mg/kg/day and liver pathology changes.


Under the conditions of this study, the No Effect Level observed was 10 mg/kg/day; the Low Adverse Effect Level was 100 mg/kg/day in both sexes. It should be noted that the study report concluded the changes in the liver at 1 000 mg/kg bw/day represented an adaptive response to the treatment which showed almost complete recovery after 2 weeks~.


 


Overall Conclusion on Repeated Dose Oral Toxicity


Two studies are available on the repeated dose toxicity of the test material. The key study (van Otterdijk, 2006) was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997). It is a GLP compliant study conducted according to internationally recognised guidelines and included neurobehavioural parameters as well as sufficient histopathology. The supporting study (Atkinson, 1989) is an older study awarded a reliability score of 2 due to minor methodological deficiencies, in particular limited histopathology.


In both studies there were no clinical signs or adverse effects on body weight that could be attributed to administration of the test material. Both studies demonstrated increases in liver weights. While the supporting study reported histopathological findings that provided evidence of liver damage, in the key study histopathology revealed only midzonal/centrilobular diffuse hypertrophy, without any degenerative changes. Both studies independently interpreted the findings in the liver as adaptive responses to the treatment with the substance, as commonly observable with xenobiotics. The effects on clinical parameters did not reveal a linear dose response relationship, as the difference were often marginal. Given that the changes observed at 150 mg/kg/day were so similar to those at 50 mg/kg/day, the NOAEL of the key study is 150 mg/ kg/day.


Effects were also noted in the kidney in the key study however, these were observed almost exclusively in males and were not accompanied by any supportive chemical chemistry changes. That histopathological kidney findings were observed only in male Wistar rats (high-dose group, van Otterdijk, 2006) might be due to a slightly different sensitivity of the strains. These effects included the detection of increased hyaline droplets and may be the alpha-2u-globulin-mediated male rat specific effect, not relevant for human health risk assessment (Borghoff et al., 1990; Read et al., 1988; Swenberg et al., 1989). Although a slightly higher incidence of corticomedullary tubular basophilia was also noted in low-dose males (3 vs. 2 in controls), this was not considered an adverse effect of the substance or possibly even unrelated to treatment, because the mid-dose showed no difference to the concurrent control group.


Both studies also indicate the observed effects are reversible in nature, and most likely representing adaptive responses of the liver starting at 50 mg/kg bw/day in Wistar rats. No other human relevant findings were noted up to 1 000 mg/kg bw/day; even for the limit dose of 1 000 mg/kg bw/day the conclusion by van Otterdijk was that overall the general health conditions of the animals appeared unaffected based on clinical signs, body weight, food intake and functional observations. Furthermore, the severity of the effects noted within the wide dose range tested (factor 10 to 20) is rather low and an almost complete recovery for the high dose group (1 000 mg/kg bw/day) is noted. The diffuse follicular hypertrophy/ hyperplasia in thyroids in males at 1 000 mg/kg bw/day is likely to be the rat-specific secondary effect to the hepatic enzyme induction following the administration of the test substance. Corroborative changes were seen in the reproduction/developmental toxicity screening test in rats, where a reduction in T4 level and an increase in the thyroid weight were seen (OECD TG 421; Shivakumar, 2018). It is well known that xenobiotics can act indirectly on the thyroid gland as inducers of liver microsomal enzymes and, in the rat, such induction can accelerate the removal of T4 as seen in the reproduction/developmental toxicity screening test (OECD TG 421; Shivakumar, 2018) by increasing the metabolism and excretion of T4 (McClain, 1989). This would cause stimulation of the normal feedback control of the thyroid (Richardson and Klaasen, 2010), resulting in an enhanced TSH release by the anterior pituitary and thereby stimulation of the thyroid gland for compensation of the increased metabolic clearance of T4. The continual stimulation results in thyroid activation, manifested by follicular cell hypertrophy and/or hyperplasia. Also these effects completely recovered after the 14-day recovery period.


In conclusion, as the effects in the liver were reversible, and from the entire clinical and histopathological picture representing an adaptive response commonly seen with xenobiotics rather than toxic effects, classification with STOT RE 2 for liver effects is not required.


 


Reference


- Borghoff SJ, Short BG, Swenberg JA (1990) Biochemical mechanisms and pathobiology of alpha 2u-globulin nephropathy. Annual review of pharmacology and toxicology 30:349-367.


- McClain MR (1989) The Significance of Hepatic Microsomal Enzyme Induction and Altered Thyroid Function in Rats: Implications for Thyroid Gland Neoplasia. Toxicologic pathology, 17, 294-306.


Toxicol Sci. 2010 Aug;116(2):413-21. doi: 10.1093/toxsci/kfq125. Epub 2010 Apr 26.


- Read NG, Astbury PJ, Morgan RJ, Parsons DN, Port CJ (1988) Induction and exacerbation of hyaline droplet formation in the proximal tubular cells of the kidneys from male rats receiving a variety of pharmacological agents. Toxicology 52(1-2):81-101.


- Richardson TA and Klaassen CD (2010) Role of UDP-glucuronosyltransferase (UGT) 2B2 in metabolism of triiodothyronine: effect of microsomal enzyme inducers in Sprague Dawley and UGT2B2-deficient Fischer 344 rats. Toxicological Sciences 116¿2):413-421.


- Swenberg JA, Short B, Borghoff S, Strasser J, Charbonneau M (1989) The comparative pathobiology of alpha 2u-globulin nephropathy. Toxicology and applied pharmacology. 97(1):35-46.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to repeated dose toxicity.