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Diss Factsheets

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12.09.2016 - 08.08.2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Benzophenone-3,3':4,4'-tetracarboxylic dianhydride
EC Number:
219-348-1
EC Name:
Benzophenone-3,3':4,4'-tetracarboxylic dianhydride
Cas Number:
2421-28-5
Molecular formula:
C17H6O7
IUPAC Name:
5-(1,3-dioxo-1,3-dihydro-2-benzofuran-5-carbonyl)-1,3-dihydro-2-benzofuran-1,3-dione
Test material form:
solid

Test animals

Species:
rat
Strain:
other: CD® / Crl:CD(SD)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS:
- Strain: Rat / CD® / Crl:CD(SD)
- Number and sex: 100 animals (50 males and 50 females)
- Breeder: Charles River Laboratories
- Body weigth: Males: 272.0 g - 303.8 g; Females: 197.2 g - 226.3 g
- Age: 60 days at 1st administration
- Adaptation period: 6 days

ENVIRONMENTAL CONDITIONS:
- Diet: a certified commercial diet (ssniff® R/M-H V1534) ad libitum
- Water: tab water ad libitum
- Housing individually in MAKROLON cages (type III plus)
- Temperature: 22°C ± 3°C
- Rel. Humidity: 55% ± 15%
- Light/dark period: 12/12h

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
Frequency of administration: Once daily for 91 consecutive days.
Vehicle:
corn oil
Details on oral exposure:
Dose levels 100 / 300 / 1000 mg/kg b.w./day
Administration volume 2 mL/kg b.w./day
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentration immediately after preparation and at study termination: 99.7% - 110.9%
Stability (left at room temperature for 8 h or 24 h): 97.3% - 111.9%
Homogeneity at start of administration, during administration; and before administration to the last animal: 100.2% - 113.5%

The results indicate that all test item formulations were correctly prepared, were very homogeneous, and were stable for at least 24 hours.
Duration of treatment / exposure:
Duration of study:
6 adaptation days
91 test days
28 recovery days for scheduled animals
Frequency of treatment:
Once daily for 91 consecutive days
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
vehicle control
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
low dose
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
intermediate dose
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
high dose
No. of animals per sex per dose:
50
Control animals:
yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:
CLINICAL SIGNS:
The animals were observed individually before and after dosing at each time of dosing for any signs of behavioural changes, reaction to treatment or illness, and additionally regularly throughout the working day. Cageside observations included skin/fur, eyes, mucous membranes, respiratory and circulatory systems, somatomotor activity and behaviour patterns.

NEUROLOGICAL SCREENING:
At the end of the treatment period, screening of sensory reactivity to stimuli of different types (e.g. auditory, visual and proprioceptive stimuli; based on Gad5), as well as the assessment of grip strength (Meyer6) and motor activity assessment was conducted for all animals.
In detail the test battery included testing of righting reflex, body temperature, salivation, startle response, respiration, mouth breathing, urination, convulsions, piloerection, diarrhoea, pupil size, pupil response, lacrimation, impaired gait, stereotypy, toe pinch, tail pinch, wire manoeuvre, hind leg splay, positional passivity, tremors, positive geotropism, limb rotation, auditory function.

MORTALITY:
Checks on mortality were made early in the morning and again in the afternoon of each day to look for dead or moribund animals.

BODY WEIGTH:
The weight of each rat was recorded at group allocation (test day -7), on test day 1 (before first administration), and once a week thereafter always on the same day of the week throughout the experimental period (test days 8, 15, 22, 29, 36, 43, 50, 57, 64, 71, 78, 85 and 90).

FOOD CONSUMPTION:
The quantity of food left by individual animals was recorded on a weekly basis throughout the experimental period. The food intake per animal (g/animal/week) was calculated using the total amount of food given to and left by each rat in each group on completion of a treatment week.

DRINKING WATER CONSUMPTION:
The drinking water consumption was recorded weekly by weighing the water bottles when filled and the residues upon removal at the end of the test week. Any residue was discarded. Weekly mean values per animal were calculated.

LABORATORY EXAMINATIONS:
Blood samples were taken from the retrobulbar venous plexus under isoflurane anaesthesia from animals fasted overnight. The blood samples were collected at the end of test week 13 (on test day 91, before necropsy) and were used for haematological investigations, coagulation tests, clinical chemistry tests, and for bile acid determination.

HAEMATOLOGICAL PARAMETERS:
Haemoglobin content (HGB), erythrocytes (RBC), leucocytes (WBC), reticulocytes (Reti), platelets (PLT), haematocrit value (HCT), absolute and relative differential blood count, mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC).

COAGULATION PARAMETERS:
Thromboplastin time (TPT), activated partial thromboplastin time (aPTT).

CLINICAL CHEMISTRY PARAMETERS:
Albumin, globulin, albumin/globulin ratio, bile acids, bilirubin (total), cholesterol, creatinine, glucose, protein, triglycerides, urea (in blood), calcium, chloride, potassium, sodium, alanine amino-transferase (ALAT), alkaline phosphatase (aP), aspartate aminotransferase (ASAT), lactate dehydrogenase (LDH)

URINALYSIS:
Urine samples were collected from all animals fasted overnight on test day 91 (test week 13). Investigated parameters were volume, pH and specific gravity. The analytes of qualitative urinalyis were protein, glucose, bilirubin, urobilinogen, ketones, heamoglobin and nitrite. A microscopic examination of urine samples was carried out by centrifuging samples and spreading the resulting deposit on a microscope slide. The deposit was examined for the presence of epithelial cells, leucocytes, erythrocytes, organisms, further constituents (i.e. sperm, cassts), crystalluria.

OPHTHALMOLOGICAL AND AUDITORY EXAMINATIONS:
Examinations were performed pre-dose (test day 1, prior to first dosing) and in test week 13 (end of the treatment period, test day 90). The eyes were examined with a HEINE ophthalmoscope. Prior to examination, mydriasis was produced after instillation of STULLIN®10 eye drops into the conjunctival sacs.
The following ocular structures were examined:
• Adnexa oculi (i.e. lids, lacrimal apparatus), conjunctiva
• Cornea, anterior chamber
• Lens, vitreous body, fundus (retina, optic disc)
The auditory acuity was checked with a simple noise test.
Sacrifice and pathology:
SACRIFICE:
Starting on test day 92 (approx. 24 hours after the last administration), the animals were dissected following a randomisation scheme. Animals not dissected on test day 92, were dosed again on test day 92 and dissected on test day 93. Necropsy of all animals scheduled for the recovery period was performed on test day 120.The animals were euthanized by carbon dioxide (CO2), exsanguinated by cutting the aorta abdominalis, weighed, dissected, and inspected macroscopically.The weights of the following organs of all animals were determined before fixation: adrenal gland, kidney, spleen, uterus (incl. cervix), brain, liver, testicle, prostate and seminal vesicles (with coagulating glands as a whole), epididymis, ovary, thymus, heart, pancreas. The paired organs (adrenal glands, gonads and kidneys) were identified as left or
right and weighed individually.
The organs or parts of organs of all animals were fixed in 7% buffered formalin. Eyes were preserved in Davidson's solution and testes in Bouin's solution for optimum fixation.

HISTOPATHOLOGY:
The organs listed below of all animals of groups 1 and 4 (control and high dose) were examined histologically after preparation of paraffin sections and haematoxylin-eosin (H & E) staining:
Adrenal gland (2)
Aorta abdominalis
Bone (os femoris with joint)
Bone marrow (os femoris)
Brain (cerebrum, cerebellum, brain stem)
Epididymis (2)
Eye with optic nerve (2)
Gross lesions observed
Heart (left and right ventricle, septum)
Intestine, small (duodenum, jejunum, ileum, incl. Peyer's patches, Swiss roll method)
Intestine, large (colon, rectum)
Kidney and ureter (2)
Liver
Lungs (with mainstem bronchi and bronchioles, preserved by inflation with fixative and then immersion)
Lymph node (1, cervical)
Lymph node (1, mesenteric)
Mammary gland
Muscle (skeletal, leg)
Nerve (sciatic)
Oesophagus
Ovary and oviducts (2)
Pancreas
Pituitary
Prostate and seminal vesicles with coagulating glands
Salivary glands (mandibular, parotid, sublingual)
Skin (left flank)
Spinal cord (3 sections)
Spleen
Stomach
Testicle (1)
Thymus
Thyroid (2) (incl. parathyroids)
Tissue masses or tumours (including regional lymph nodes)
Trachea (incl. larynx)
Urinary bladder
Uterus (incl. cervix)
Vagina

In addition, frozen sections of the heart, liver and one kidney were prepared, stained with Oil Red O, and examined microscopically, and the stomach and the kidneys (2) of the animals of group 2 (low dose) and group 3 (intermediate dose) were examined microscopically following H & E) staining.
Furthermore, a detailed histomorphological examination was performed on one testicle and one epididymis of all male animals of groups 1 to 4 (control and all dose groups) following H & E and PAS staining (with special emphasis on the qualitative stages of the spermatogenesis and the histopathology of the interstitial testicular structure). Sperm count an viabilty was assessed and sperm morphology was examined for all male animals.
Statistics:
Data for toxicology and pathology were captured, as far as possible, using the departmental computerized systems (Provantis® Integrated preclinical software, version 9.4.0, Instem LSS Ltd., Stone, Staffordshire ST15 0SD, United Kingdom). Raw data not fully compatible with the computerized systems were maintained on paper according to appropriate SOPs. The test item-treated groups 2 to 4 were compared with the control group 1.
The following statistical methods were used for the data captured with the Provantis system: Multiple t-test based on DUNNETT, Exact Test of R.A. Fisher.

Homogeneity of variances and normality of distribution were tested using BARTLETT's test and the SHAPIRO-WILKS test. In case of heterogeneity and/or non-normality of distribution, stepwise transformation of the values into logarithmic or rank values was performed prior to ANOVA. If the ANOVA yielded a significant effect (p ≤ 0.05), intergroup comparisons with the control group were made by DUNNETT's test (see above).

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
None of the male and female rats treated with 100, 300 or 1000 mg BTDA/kg b.w./day by oral administration for 91 days revealed any test item-related changes in behaviour, external appearance, or consistency of faeces, and no test itemrelated changes were noted during the 28-day treatment-free recovery period.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
All male and female animals treated with 100 or 300 mg BTDA/kg b.w./day survived until their scheduled sacrifice on test day 92 or 93.Two of 30 animals treated with 1000 mg BTDA/kg b.w./day died or had to be sacrificed prematurely. The deaths of both animals are considered not to be test item-related; one was due to a gavage error and the other was considered to be incidental.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
No test item-related influence was observed on the body weight and the body weight gain of the male animals treated with 100, 300 or 1000 mg BTDA/kg b.w./day and of the female animals treated with 100 or 300 mg BTDA/kg b.w./day by oral administration for 91 days compared to the control animals throughout the treatment period. No test item-related differences were noted for the body weight at autopsy between the test item-treated animals and the control animals.

The body weight of the female animals treated with 1000 mg BTDA/kg b.w./day was slightly reduced by up to 9% compared to the control animals as of test day 57 (statistically significant at p ≤ 0.05 on test days 64 and 71). Accordingly, the body weight gain was reduced by approx. 20% compared to the control animals. The body weight at autopsy appeared not to be influenced.

The body weight of the female animals previously treated with 1000 mg BTDA/kg b.w./day had normalized during the 28-day treatment-free recovery period.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No test item-related influence was observed on the relative and absolute food consumption of the male and female animals treated with 100 or 300 mg BTDA/kg b.w./day by oral administration compared to the control animals throughout the 91-day treatment period. The relative food consumption of the male and female animals treated with 1000 mg BTDA/kg b.w./day by oral administration was increased by up to 12% for the males and by up to 25% for the females compared to the control animals starting in test weeks 1 or 3 for the male and female animals, respectively (statistically significant at p ≤ 0.05 or p ≤ 0.01 in test weeks 1 to 7, 9 to 10 and 13 for the males and in test weeks 3, 5, 6, and 8 to 13 for the females). The absolute food consumption of the male and female animals treated with 1000 mg BTDA/kg b.w./day by oral administration was increased by up to 15% compared to the control animals starting in test weeks 1 or 8 for the male and female animals, respectively (statistically significant at p ≤ 0.05 or p ≤ 0.01 in test weeks 1 to 7, 9, 10 and 13 for the males and in test week 13 for the
females).
Since the increased food and drinking water usage was not accompanied by body weigth increases, it may have been caused by the animals by water or feed spoilage to neutralise a bitter or unpleasant taste at the high dose level.

The relative food consumption of the female animals previously treated with 1000 mg BTDA/kg b.w./day was still increased by 10% to 21% during the 28-day treatment-free recovery period (statistically significant at p ≤ 0.05). The absolute food consumption of the female animals of the high dose group had normalized. The food consumption of the male animals previously treated with 1000 mg BTDA/kg b.w./day had normalized and the values were within the range of the control group at the end of the 28-day treatment-free recovery period.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
No test item-related influence was observed on the drinking water consumption of the male and female animals treated with 100 or 300 mg BTDA/kg b.w./day by oral administration compared to the control animals throughout the 91-day treatment period. The drinking water consumption (by weekly quantitative assessment) of the male and female animals treated with 1000 mg BTDA/kg b.w./day by oral administration was increased by up to 38% for the males and by up to 36% for the females compared to the control animals starting in test week 1 (statistically
significant at p ≤ 0.01 in test weeks 1 to 13 for both sexes). The drinking water consumption of the male and female animals previously treated with 1000 mg BTDA/kg b.w./day was still increased by 15% to 26% during the first two weeks of the 28-day treatment-free recovery period (statistically significant at p ≤ 0.05 for the females in test week 14). Afterwards the values of the drinking water consumption were within the range of the control group.

Since the increased food and drinking water usage was not accompanied by body weigth increases, it may have been caused by the animals by water or feed spoilage to neutralise a bitter or unpleasant taste at the high dose level.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
The ophthalmological examination did not reveal any test item-related changes of the eyes and the optic region in any animal after repeated oral treatment with 100, 300 or 1000 mg BTDA/kg b.w./day in test week 13 (test day 90) and test week 17 (test day 119).
No test item-related pathological changes were noted on the adnexa oculi (i.e. lids, lacrimal apparatus), conjunctiva, cornea, anterior chamber, lens, vitreous body, and fundus (retina, optic disc). There was no indication of any impairment to auditory acuity.
Haematological findings:
no effects observed
Description (incidence and severity):
No test item-related influence was observed on the haematological parameters for the male and female animals treated with 100, 300 or 1000 mg BTDA/kg b.w./day by oral administration for 91 days compared to the control animals at the end of the treatment period (test day 88, only the main study animals) and at the end of the recovery period (test day 120).
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No test item-related influence was observed on the biochemical parameters for the male and female animals treated with 100, 300 or 1000 mg BTDA/kg b.w./day by oral administration for 91 days compared to the control animals at the end of the treatment period (test day 88, only the main study animals) and at the end of the recovery period (test day 120).
Urinalysis findings:
no effects observed
Description (incidence and severity):
No test item-related influence was observed on the urinary parameters for the male and female animals treated with 100, 300 or 1000 mg BTDA/kg b.w./day by oral administration for 91 days compared to the control animals at the end of the treatment period on test day 88.
Behaviour (functional findings):
no effects observed
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No test item related changes were noted for the relative and absolute organ weights of the animals treated with 100 or 300 or 1000 mg BTDA/kg b.w./day by repeated oral administration at terminal sacrifice on test day 92 or 93 or at recovery sacrifice on test day 120.
Gross pathological findings:
no effects observed
Description (incidence and severity):
The macroscopic inspection at necropsy did not reveal any changes in the organs and tissues of the animals treated with 100, 300 or 1000 mg BTDA/kg b.w./day by repeated oral administration after terminal sacrifice at the end of the treatment period (test day 92 or 93) and at the end of the recovery period (test day 120).
Neuropathological findings:
effects observed, treatment-related
Description (incidence and severity):
No test item-related influence was noted on any of the parameters examined during the functional observation tests, on the fore- and hind limb grip strength, or on the spontaneous motility for any of the male and female animals after repeated oral treatment with 100 or 300 mg BTDA/kg b.w./day in test week 13. The male and female animals treated with the high dose of 1000 mg BTDA/kg b.w./day revealed a slightly decreased hindlimb grip strength by 13% for the males and by 17% for the females compared to the control group (statistically significant at p ≤ 0.05 for the males and by p ≤ 0.01 for the females).

No test item-related influence was noted on any of the parameters examined during the functional observation tests, on the fore- and hind limb grip strength, or on the spontaneous motility for any of the male and female animals previously treated with 1000 mg BTDA/kg b.w./day in test week 17.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
The histomorphological examination of a variety of organs and tissues was restricted to the control group 1 and the high dose group 4 treated with 1000 mg BTDA/kg b.w./day. No significant changes were observed. All macroscopic and microscopic changes observed were not considered to be toxicologically relevant and fall within the normal background findings of rat of this age and strain.
No test item-related effects were noted for the testes, with special emphasis on the qualitative stages of the spermatogenesis, and the epididymides of the male animals in the high dose group.

In summary, based on the macroscopic and microscopic observations, the findings did not indicate a test item-related effect for both sexes when given BTDA by oral administration via gavage daily for 91 days at 1000 mg/kg. Moreover, after a 28-day recovery period, no test item-related changes were observed in the recovery animals.
Histopathological findings: neoplastic:
no effects observed

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
neuropathology
Dose descriptor:
LOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
neuropathology

Target system / organ toxicity

open allclose all
Critical effects observed:
no
Lowest effective dose / conc.:
1 000 mg/kg bw/day (actual dose received)
System:
nervous system
Critical effects observed:
no
Lowest effective dose / conc.:
1 000 mg/kg bw/day (actual dose received)
System:
other: body weigth

Any other information on results incl. tables

No influence was noted on the sperm count (number of ultrasound-resistant spermatids per gram testicular tissue) and on the sperm motility (determined as percentage of motile spermatozoa in the epididymal cauda of the males) for any of the test item-treated groups in comparison to the control group in test week 13 (end of treatment) and in test week 17 (end of recovery). Furthermore, the examination of the sperm morphology did not reveal any test item-related differences between the control group and the test item-treated groups in test week 13 (end of treatment) and in test week 17 (end of recovery).

The results of the sperm viability determination and the sperm morphology examination is supported by the fact that the microscopic evaluation did not reveal any test item related effects on the male reproductive organs at the histomorphological level.

Applicant's summary and conclusion

Conclusions:
In conclusion, adverse test item-related effects were noted at 1000 mg BTDA/kg b.w./day in form of a reduced body weight in females, an increased food and drinking water consumption, and a decreased hindlimb grip strength compared to the control group.
The experimental no-observed-adverse-effect level (NOAEL) was 300 mg 3,3 ',4,4' -benzophenonetetracarboxylic dianhydride (BTDA)/kg b. w. by daily oral administration for 91 days.
Executive summary:

The aim of this repeated dose toxicity study was to obtain information on the toxicity of 3,3',4,4'-benzophenonetetracarboxylic dianhydride (BTDA) administered daily by oral administration to rats for 91 consecutive days and to assess the reversibility of any effects at the end of a 28-day recovery period. The rats were treated with 100, 300 or 1000 mg BTDA/kg b.w./day. The control animals received the vehicle (corn oil). No test item-related deaths occurred. Treatment with 1000 mg BTDA/kg b.w./day led to a slightly reduced body weight and body weight gain for the female animals as of test week 9. In addition, neurological screening revealed a slightly decreased hindlimb grip strength in the male and female animals. An increased food usage was observed starting in test weeks 1 or 3 for the male and female animals, respectively, and an increased drinking water usage in the male and female animals as of test week 1 at the high dose level of 1000 mg BTDA/kg b.w./day compared to the control group. This increased usage may have been caused by the animals by water or feed spoilage to neutralise a bitter or unpleasant taste at the high dose level. No test item-related changes were observed for the behaviour or external appearance of the animals, the detailed clinical observations, for any of the haematological, clinical chemical and urinary parameters, the eyes or optic region, the auditory acuity, the relative and absolute organ weights, the sperm viability and morphology, and at macroscopic inspection at necropsy at any dose level. The histopathological examination did not reveal any test item-related morphological changes. No test item-related effects were noted for the testes, with special emphasis on the qualitative stages of the spermatogenesis, and the epididymides of the male animals in the high dose group. The relative food consumption of the female animals previously treated with 1000 mg BTDA/kg b.w./day was still increased during the 28-day treatment-free recovery period. All further changes observed during the main study had completely subsided in all male and female previously high-dosed animals at the end of the recovery period. Histopathology of the previously high dosed recovery animals did not reveal any test item-related changes.