Benzophenone-3,3':4,4'-tetracarboxylic dianhydride

Endpoint summary

Description of key information

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.

For systemic effects following administration via inhalation, the LOAEC is above 136 mg/m3 as dust. For local effects, the NOAEC is 55 mg/m3 as dust.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

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

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)

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

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

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

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).

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

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

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

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.

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: experimental result, precedes establishment of guideline and GLP. Documentation is limited.

Qualifier:

no guideline available

Principles of method if other than guideline:

Study precedes establishment of guideline protocols and GLP. Guinea pigs are exposed (full body) to dust of BTDA for 6 hours/day, 5 days per week for 14 days. Animals are euthanized and organs are examined. Specific attention is paid to the immune response in lung and lymph tissue.

GLP compliance:

no

Remarks:

preceeds establishment of GLP

Limit test:

no

Species:

guinea pig

Strain:

other: English short-hair

Sex:

not specified

Details on test animals or test system and environmental conditions:

200-300 g body weight.
1530 liter exposure chamber, full body exposure.
The animals were group-housed in metal cages suspended above the droppings. Food and water were available ad libitum.

Route of administration:

inhalation: dust

Type of inhalation exposure:

whole body

Vehicle:

not specified

Remarks on MMAD:

MMAD / GSD: no data

Details on inhalation exposure:

Exposure occurred in a 1530 liter stainless steel inhalation chamber. The total air flow through the system was 50 liters per minute.

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

No attempt was made to determine the actual chamber concentration during the exposure. The mean theoretical concentration was calculated from the air flow and net loss of material.

Duration of treatment / exposure:

6 hours/day

Frequency of treatment:

daily for 5 days/week, for two continuous weeks.

Dose / conc.:

136 mg/m³ air (nominal)

Remarks:

Theoretical, as calculated. Ranges up to 251 mg/m3

No. of animals per sex per dose:

10

Control animals:

not specified

Details on study design:

During the 6 hour exposure period, all animals were continuously observed for mortality and toxic effects. Following exposures, the animals were observed daily for possible latent effects. After sacrifice, the animals underwent a necropsy.

Observations and examinations performed and frequency:

clinical signs, signs of toxicity, death

Sacrifice and pathology:

necropsy

Statistics:

Theoretical doses were calculated from air flow and net loss of material.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

2 of 10 animals showed marked dyspnea and cyanosis, and later died; survivors displayed sneezing and muscle spasms.

Mortality:

mortality observed, treatment-related

Description (incidence):

2 of 10 animals showed marked dyspnea and cyanosis, and later died; survivors displayed sneezing and muscle spasms.

Body weight and weight changes:

not specified

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not examined

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Marked to severe but reversible granulomatous pneumonia and hyperplasia of mediastinal lymph nodes. Also moderate diffuse granulomatous mediastinitis.

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not examined

Details on results:

Survivors displayed sneezing, muscle spasms and slight dyspnea. Two of ten animals showed marked dyspnea and cyanosis and later died. Animals showed marked to severe but reversible granulomatous pneumonia and hyperplasia of mediastinal lymph nodes. Moderate diffuse granulomatous mediastinitis was also observed.

Key result

Dose descriptor:

LOAEC

Effect level:

136 mg/m³ air (nominal)

Based on:

test mat.

Sex:

not specified

Basis for effect level:

other: Death and toxicity observed. Doses ranged from 136 to 251 mg/m3.

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

136 mg/L air (nominal)

System:

other: granulomatous pneumonia and hyperplasia of mediastinal lymph nodes

Organ:

lungs

lymph node

Treatment related:

not specified

Dose response relationship:

not specified

Relevant for humans:

not specified

Conclusions:

Guinea pigs were exposed by inhalation (whole body) to the dust of 1,3-Isobenzofurandione, 5,5'-carbonylbis- (BTDA), 6 hours per day, five days per week (whole body) for two continuous weeks. Two of twenty animals died, and severe but reversible granulomatous pneumonia, moderate diffuse granulomatous mediastinitis and hyperplasia of lymph nodes was found on necropsy. No NOAEL could be determined, but is presumed lower than 136 mg/m3, (LOAEC), under conditions of this study.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEC

135 mg/m³

Study duration:

subacute

Species:

guinea pig

Quality of whole database:

poor

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Reliable scientific study, by GLP. Precedes establishment of guideline

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Deviations:

yes

Remarks:

; minimal pathology review was performed

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

40 albino rats (Cr-1, Sprague-Dawley descended, COB CD [SD] BR, 25 males and 25 females were received from Charles River Breeding Laboratories Inc., Wilmington, MA, on 7/23/80. Animals were acclimated 7 days prior to study initiation, to a diurnal cycle of 12 hours light and 12 hours darkness by automatic timer. Exposures were conducted during the light phase.

Route of administration:

inhalation: dust

Type of inhalation exposure:

whole body

Vehicle:

clean air

Remarks on MMAD:

MMAD / GSD: The average mass median diameter (µ) ranged from 2.65 to 3.24, with a geometric standard deviation (sigma m) ranging from 1.74 to 3.31.

Details on inhalation exposure:

Animals were exposed (whole body) to test material in a 100-L glass and stainless steel inhalation chamber operated dynamically with dry filtered air. Total airflow into chambers was monitored by flowraters and held at a constant 20 L per minute. Exposure was maintained for 6 hours in duration. Mean temperature and relative humidity for the entire study was 25.5 degrees C (± 1.0) and 46% (± 6.4).
BTDA was generated as a dust using a Wright Dust Feeds. The dust-laden air was mixed with an accessory airflow and passed into the chamber. Wright Dust Feed Gear ratios were adjusted to achieve the desired concentration.
The average mass median diameter (µ) ranged from 2.65 to 3.24, with a geometric standard deviation (sigma m) ranging from 1.74 to 3.31.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Chamber monitoring: Gravimetric concentration. Three daily gravimetric filter (Gelman DM-450) samples were obtained from each of the test chambers during exposures. The filters were preweighed and the gravimetric concentration was calculated as the weight of material collected per total volume of air sampled. The overall mean concentrations were within 30% of the target levels (see Table 1) for Group 2, 2% for Group 3 and 10% for Group 4.
Aerodynamic particle size measurements of BTDA dust were taken prior to animal exposures with an Andersen 8-stage Impactor operating at a flowrate of 28.4 L/min. Measurements were recorded during exposures with an Andersen 4-stage Mini-Impactor. The average mass median diameter (µ) ranged from 2.65 to 3.24, with a geometric standard deviation (sigma m) ranging from 1.74 to 3.31. Sampling durations were 5 minutes for Group 2, 3 minutes for Group 3 and 1 minute for Group 4. Particle weight gains on each stage and back-up filters were converted to percentages of the total weight, and back-up filters were analyzed to indicate that the majority of the particles sampled were within the respirable range (<5.0 microns) for all test groups.

Duration of treatment / exposure:

6 hours/day

Frequency of treatment:

5 days

Dose / conc.:

1 mg/m³ air (analytical)

Dose / conc.:

55 mg/m³ air (analytical)

Dose / conc.:

3 000 mg/m³ air (analytical)

No. of animals per sex per dose:

10/dose group, 5 males and 5 females

Control animals:

yes, concurrent vehicle

Details on study design:

Animals were exposed (whole body) to test material in a 100-L glass and stainless steel inhalation chamber operated dynamically with dry filtered air. Groups 1-4 animals were observed prior to, at hourly intervals during, and following each daily exposure for evidence of unusual appearance and/or behavior. Observations were recorded. Individual pre- and postexposure weights were recorded for all animals.
On the day following the last administration of the test and control substances, all animals received a single intraperitoneal injection of colchicine (2.0 mg/kg-5.0 ml/kg body weight) for cytogenetics analysis. Approximately two hours after injection of colchicine, the animals were sacrificed by CO2 asphyxiation. The femurs were removed from the animals and bone marrow was obtained by aspiration and processed for cytogenetic analysis.

Positive control:

Triethylene melamine (TEM), 0.4mg/kg body weight, given by a single intraperitoneal injection24 hours prior to sacrifice, as a positive control for genotoxic effects in the bone marrow.

Observations and examinations performed and frequency:

Groups 1-4 animals were observed prior to, at hourly intervals during, and following each daily exposure for evidence of unusual appearance and/or behavior. Observations were recorded. Individual pre- and postexposure weights were recorded for all animals.

Sacrifice and pathology:

Approximately two hours after injection of colchicine, the animals were sacrificed by CO2 asphyxiation.

Other examinations:

Cytogenetic analysis for chromosomal aberrations in bone marrow cells.

Statistics:

The mean changes in body weights, the mean mitotic indices and mean modal numbers were analyzed using Bartlett's test for equality of variance (Bartlett, 1937), the one-way classification analysis of variance (Snedecor and Cochran, 1967), and (if a significant ANOVA was reported) Scheffe's multiple F Test procedure (Scheffe, 1953). Significance was established at p ≤ 0.05.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Five of ten animals died during the 5 day exposure to 3000 mg/m3.

Mortality:

mortality observed, treatment-related

Description (incidence):

Five of ten animals died during the 5 day exposure to 3000 mg/m3.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Significant (p ≤ 0.05) decreased terminal body weight and body weight gain the high dose group when compared to control animals. All exposed groups showed significantly decreased weight gain when compared to control animals (p ≤ 0.05).

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Agitation in high dose group

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Details on results:

All animals in the control group exhibited normal appearance and behavior throughout the study. Animals in the low dose group (1 mg/m3) exhibited normal appearance and behavior throughout the study with the exception of notable sniffing and preening, which occurred approximately one minute after initiation of the Day 5 exposure. Animals in the mid dose group (55 mg/m3) exhibited normal animal appearance and behavior, except for sniffing, preening, and sneezing (3/10 animals) approximately one minute after initiation of the final exposure. Five animals in the high dose group (3000 mg/m3) expired during the exposure periods (three on Day 1, and one each on days 4 and 5). Signs of eye irritation, respiratory distress, agitation, diarrhea, and bloody eye, nose and mouth discharges were recorded at various intervals throughout the remainder of the exposures.

Dose descriptor:

NOEC

Effect level:

> 55 - < 3 000 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse behavioural effects noted in the 55 mg/m3 group. Five of ten animals died in the group exposed to 3000 mg/m3.

Critical effects observed:

not specified

Table 3: Pre- and Post-Exposure Body Weights for Dose Groups

Dose Group (mg/m^3)	Initial Body Weights (grams,± sd)		Terminal Body Weights (grams, ± sd)		Body Weight Changes (grams, ± sd)
0	232.5	10.3	254.5	17.1	22.0	8.8
1	231.5	10.8	229.2	15.0	-2.3	14.3
55	233.4	8.5	237.9	10.9	4.5	6.4
3000	235.0	8.5	166.6	14.5	-64.6	12.4

 

Conclusions:

An in vivo repeated dose cytogenicity study was undertaken in CD rats using the inhalation route of exposure to 1,3-Isobenzofurandione, 5,5'-carbonylbis- (BTDA, 6 hours per day for 5 consecutive days). Doses were 1, 55, and 3000 mg/m3 BTDA, generated as dust (MMAD 3 microns) in a whole-body exposure chamber. Five of ten animals died in the 3000 mg/m3 group; there were no notable pharmacotoxic or behavioural effects in the lower dose groups. No pathology examinations were undertaken on any animal. The NOEL for toxicity associated with repeated dose inhalatory exposure to BTDA, under the conditions of this study, is 55 mg /m3.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

55 mg/m³

Study duration:

subacute

Species:

guinea pig

Quality of whole database:

poor

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

Fourteen-day repeated dose inhalation toxicity studies are available for 1,3-Isobenzofurandione, 5,5'-carbonylbis- (BTDA as dust, 77-0160-FKT). The studies were undertaken in guinea pigs for elucidation of respiratory sensitisation effects. The systemic LOAEC is 135 mg/m3 for 6 hours/day, 5 days/week for 14 days. For the local inhalatory NOAEC of 55 mg/m3, a study is available (81-0274-FKT) where exposure took place for 6 hours per day, for 5 days.

In a 90 -day oral repeat-dose study in rats (2016 -0012 -DGT), 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 BTDA/kg b. w. by daily oral administration for 91 days.

Justification for classification or non-classification

No specific toxic effect for repeated dose systemic toxicity is noted from these studies. Respiratory irritation is addressed using harmonized classifications according to Regulation EC No. 1272/2008.