2,4-diethyl-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:

08 December 1988 to 31 January 1989

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1989

Report date:

1989

Materials and methods

Test guideline

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

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-(propan-2-yl)-9H-thioxanthen-9-one

Test material form:

solid: particulate/powder

Details on test material:

- Appearance:yellow powder

Test animals

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.

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

10 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 3

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.

Examinations

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.

Results and discussion

Results of examinations

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.

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.

Effect levels

1

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

2

Key result

Dose descriptor:

NOAEL

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

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study, the No Effect Level observed was 10 mg/kg/day but as the changes seen were suggestive of an adaptive response the No Adverse Effect Level was 1000 mg/kg/day in both sexes.

Executive summary:

The repeated dose oral toxicity of the test material 2-Isopropylthioxanthone (CAS 5495-84-1, EC 226-827-9) 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 (cholesterol, total protein and GGT in both sexes; triglycerides, albumin globulin ratio in males only, albumin and total bilirubin in females only). These findings indicated that the test material had an effect on liver function. In addition small differences were seen in creatinine levels 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. 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 alteration and periportal cytoplasmic rarefaction was present in the liver of most High dose male main study animals. These findings were not seen in females. The changes were very mild and the cytoplasmic enlargement was indicative of an adaptive response to treatment.

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 typical of an adaptive response to treatment. All these findings showed some, if not total, resolution after two weeks recovery.

Under the conditions of this study, the No Effect Level observed was 10 mg/kg/day but as the changes seen were suggestive of an adaptive response the No Adverse Effect Level was 1000 mg/kg/day in both sexes.