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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From December 21,2009 to 1 February,2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well described GLP compliant study conducted to recognized international test guidelines

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report):1,2,3,6, Tetrahydrophtalic Anhydride (THPA)
- Molecular formula :C8H8O3
- Molecular weight : 152.1473 g/mol
- Smiles notation : O=C1OC(=O)C2C1C/C=C\C2
- InChl : InChI=1/C8H8O3/c9-7-5-3-1-2-4-6(5)8(10)11-7/h1-2,5-6H,3-4H2
- Physical state:solid , white scales
- Analytical purity: no data
- Lot/batch No.:SAT4209215
- Expiration date of the lot/batch:03/06/2010
- Storage condition of test material:room temperature in a desiccator

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:Harlan Italy S.r.l.
- Age at study initiation:27-29 days
- Weight at study initiation:75-99 g
- Justification for choice:The Sprague Dawley rat was the species and strain of choice because it is accepted by many regulatory authorities and there is ample experience and background data on this species and strain.
- Housing:The animals were housed in a limited access rodent facility.The animals were housed 5 of one sex to a cage, in clear polycarbonate cages measuring 59x38.5x20 cm with a stainless steel mesh lid and floor.Each cage tray held absorbent paper which was inspected and changed at least 3 times a week.
- Diet :A commercially available laboratory rodent diet (4 RF 21) was offered ad libitum throughout the study.
- Water :Drinking water was supplied ad libitum to each cage via water bottles
- Acclimation period:7 days
- Health check: during the acclimatisation period

ENVIRONMENTAL CONDITIONS
- Temperature (°C):22 +/- 2°C
- Humidity (%):55 +/- 15 %
- Air changes (per hr):15 to 25 air changes per hour
- Photoperiod : 12 hour dark/light by artificial light

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle: Test substance is unstable in aqueous media
- Concentration in vehicle:The test item was mixed with corn oil to give the required concentrations of 10, 25 and 60 mg/ml.
- Amount of vehicle :dose volume of 10 ml/kg body weight.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Formulations of the test item were prepared as suspensions in corn oil. Concentration and homogeneity of the low and high dose levels were assessed by taking six analytical aliquots (approximately lg) in different positions. For the intermediate level only concentration was assessed by taking two different analytical aliquots (approximately 1g). Each analytical aliquot was analysed separately. Concentration was evaluated as the mean of the single determinations and homogencity was evaluated as the coefficient of variation of the sextuplicate set.
Duration of treatment / exposure:
Administered daily to rats by oral route for 4 consecutive weeks and to investigate a possible recovery from any treatment-related effects, during a 2 week recovery period.
Frequency of treatment:
All animals were dosed once a day, 7 days a week, for a minimum of 4 consecutive weeks followed by a recovery period of 2 weeks for 5 males and 5 females from groups 1 and 4. All animals from the main phase groups were dosed up until the day before necropsy.
No treatment was given during the recovery period.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0 mg/kg bw
Basis:
actual ingested
Remarks:
Doses / Concentrations:
100 mg/kg bw
Basis:
actual ingested
Remarks:
Doses / Concentrations:
250 mg/kg bw
Basis:
actual ingested
Remarks:
Doses / Concentrations:
600 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
0 mg/kg/day (control) – 20 males, 20 females
100 mg/kg/day – 10 males, 10 females
250 mg/kg/day – 10 males, 10 females
600 mg/kg/day – 20 males, 20 females
Control animals:
yes
Details on study design:
- Dose selection rationale: Available (literature) information on similar cyclic anhydrides
- Rationale for animal assignment (if not random): Random
- Rationale for selecting satellite groups: Control and high dose groups for investigation of recovery from any effects of treatment
- Post-exposure recovery period in satellite groups: 2 weeks

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: End of treatment and recovery periods
- Anaesthetic used for blood collection: Yes (isofluorane)
- Animals fasted: Yes
- How many animals: All
- Parameters examined. Haematocrit, Haemoglobin, Red blood cell count, Reticulocyte count, Mean red blood cell volume, Mean corpuscular haemoglobin, Mean corpuscular haemoglobin concentration, White blood cell count, Differential leucocyte count, Platelets, Prothrombin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: End of treatment and recovery periods
- Anaesthetic used for blood collection: Yes (isofluorane)
- Animals fasted: Yes
- How many animals: All
- Parameters examined. Alkaline phosphatase, Alanine aminotransferase, Aspartate aminotransferase, Gamma glutamyltransferase, Urea, Creatinine, Glucose, Triglycerides, Bile acids, Phosphorus, Total bilirubin, Total cholesterol, Total protein, Albumin, Globulin, A/G Ratio, Sodium, Potassium, Calcium, Chloride

URINALYSIS: Yes
- Time schedule for collection of urine: End of treatment and recovery periods
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined. Appearance, Volume, Specific gravity, pH, Protein, Total reducing substances, Glucose, Ketones, Bilirubin, Urobilinogen, Blood. The sediment, obtained from centrifugation at approximately 3000 rpm for 10 minutes, was examined microscopically for: Epithelial cells, Leucocytes, Erythrocytes, Crystals, Spermatozoa and precursors, Other abnormal components

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: End of treatment and recovery periods
- Dose groups that were examined: All
- Battery of functions tested: sensory activity / grip strength / motor activity
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Statistics:
For continuous variables the significance of the differences amongst groups was assessed by analysis of variance. Differences between each treated group and the control group were assessed by Dunnett’s test. The homogeneity of the data was verified by Bartlett’s test before Dunnett’s test.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
but normal after recovery
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Stomach lesions at 250 and 600 mg/kg/day
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Stomach lesions at 250 and 600 mg/kg/day
Details on results:
CLINICAL SIGNS AND MORTALITY:No mortality occurred during the study. No treatment-related clinical signs were observed.

BODY WEIGHT AND WEIGHT GAIN:No changes were seen in body weight growth and in terminal body weight between treated and control animals, during the treatment and recovery periods.

FOOD CONSUMPTION:No treatment-related changes were observed in food consumption in either sex during the experimental period.

HAEMATOLOGY:No changes of toxicological significance were recorded.
Some statistically significant findings were observed, such as leucopenia in males treated with 250 mg/kg/day, a decrement of haemoglobin in females from the same group, an increment of mean corpuscular haemoglobin concentration in those treated with 600 mg/kg/day and leucocytosis in females receiving 250 and 600 mg/kg/day. However, such changes were of low magnitude and/or not dose- related, therefore they were considered to be of no toxicological importance.

CLINICAL CHEMISTRY:
Dosing Phase:
Phosphorus was increased in all treated females, with no dose-relation.
Fluctuations of other biochemical parameters were recorded, such as a decrement of triglycerides, cholesterol and potassium and an increment of chloride and sodium in the males, an increase in cholesterol and bile acids as well as a decrease in potassium in the females.
These changes were of low magnitude and mostly observed in the intermediate groups only, therefore they were considered to be of no toxicological significance.
Recovery Phase
Calcium, phosphorus, sodium and potassium were re-evaluated at the end of recovery.
At the end of the recovery period, the changes described for parameters during the dosing phase (calcium, phosphorus, sodium and potassium) were completely recovered.
Phosphorus in males and sodium in females were lower than controls. However, these findings were not observed for both sexes during the dosing phase, therefore they were considered to be incidental.

URINALYSIS:
Dosing Phase
An increment of specific gravity and a decrement of pH were recorded in some treated animals, with no clear dose-relation.
Males treated with 600 mg/kg/day showed an additional protein increment.
Recovery Phase:
All urine parameters were re-evaluated at the end of recovery.
After 2 weeks of recovery, data from treated animals were comparable with controls.

ORGAN WEIGHTS:No changes of toxicological significance were reported in the weight of the organs.

GROSS PATHOLOGY:Detailed macroscopic observations were reported for individual animals in all main and recovery phase groups.

Final sacrifice
Potentially treatment-related changes were detected at post mortem examination in the stomach of males and females receiving the test item at 250 and 600 mg/kg/day. These findings consisted of thickening of the forestomach mucosa (non-glandular region), in some instances associated with oedema and/or multiple, pale, raised and firm areas with central depression.
The remaining minor findings, seen in control and treated animals, were considered as incidental or spontaneous in origin.
Recovery sacrifice:
The sporadic changes reported at post mortem examination in both control and treated animals were considered to be incidental or spontaneous in origin.
Findings concerning ovaries and uterus, noted in control and treated females, were considered to be related to the physiologic oestrus cycle.


HISTOPATHOLOGY:
In the first instance, the histopathological evaluation was performed on all males and females from the control and high dose groups receiving the test item at 600 mg/kg/day, sacrificed at the end of treatment, as well as on all the abnormalities detected during post mortem observations in the remaining treated main phase animals.
On the basis of the histopathological results, the evaluation was then extended to the stomach of all males and females from the remaining groups in the main and recovery phases (2 and 3 of the main phase and 1 and 4 of the recovery phase).
For ease and clarity of reporting, only tissues with diagnoses are reported in the incidence tables.

Final sacrifice
Potentially treatment-related changes were seen in the stomach of males and few females receiving the test item at 600 mg/kg/day, as well as in 2 males and 1 female receiving the test item at 250 mg/kg/day. The findings noted in the stomach were epithelial hyperplasia of the forestomach mucosa (non-glandular region), often associated with inflammatory cell infiltration and oedema in the submucosa. Two males (No. 79420034 receiving 250 mg/kg/day and No. 79420042 receiving 600 mg/kg/day) also showed erosions of the gastric mucosa (forestomach).
The hyperplastic epithelium of the forestomach mucosa did not show any evidence of dysplasia or proliferation of the epithelial cells deep into the submucosa and no lesion was noted in the glandular region of the stomach. Furthermore, the relevance of changes in rodent forestomach is disputed, since a squamous lining is lacking in the human gastric mucosa.
A range of lesions seen in control and/or treated animals, such as inflammatory cell foci in the liver and nephropathy in the kidney, were considered to be an expression of spontaneous pathology normally seen in this species under our experimental conditions.

Recovery sacrifice
The gastric lesions observed in the animals receiving the test item at 600 mg/kg/day during the main phase were completely recovered after a 2 week recovery period


Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
600 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Systemic effects
Dose descriptor:
NOEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Local (irritant) effects in the stomach

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
No signs of toxicity were observed during the “in-life” phase of the study. No significant variations were observed in haematological and clinical chemistry parameters. Therefore, the high dose of 600 mg/kg/day and the low dose of 100 mg/kg/day are considered to be the No Observed Adverse Effect Level (NOAEL) and the No Observed Effect Level (NOEL), respectively, for the test item after daily oral administration to rats over a period of 4 weeks.


Executive summary:

The oral toxicity of tetrahydrophthalic Anhydride (THPA) over an exposure period of 4 consecutive weeks and recovery from any potential treatment-related effects over a period of 2 consecutive weeks has been investigated. Methods were in accordance with OECD/EU test guidelines.No clinical signs of toxicity were observed and no significant variations were observed in haematology and clinical chemistry parameters. The high dose of 600 mg/kg/day was considered to be the No Observed Adverse Effect Level (NOAEL) for systemic toxicity and the low dose of 100 mg/kg/day was considered to be the No Observed Adverse Effect Level (NOAEL) for local effects.