6'-(dibutylamino)-3'-methyl-2'-(phenylamino)spiro[isobenzofuran-1(3H),9-(9H)-xanthen]-3-one

Endpoint summary

Description of key information

A NOAEL of 1000 mg/kg bw/d has been determined in a 90 d repeated dose toxicity study (CETA, 2015).

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1988-10-14 to 1988-11-11

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Remarks:

CD (SD) BR strain

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 28 days
- Weight at study initiation: 65 - 79 g
- Housing: groups of five according to sex
- Diet: Labsure LAD 1 Diet, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 25 days

DETAILS OF FOOD AND WATER QUALITY:
Analyses were made on all batches of diet used to establish levels of basic nutrients and of specified substances and micro-organisms likely to be present in feed components and which, if in excess of specified amounts, might have had an undesirable effect on the test system. Results of the routine physical and chemical examination of drinking water at source (Grafham Final Water) as conducted usually weekly by the supplier, Anglian Water Authority, were made available to HRC as quarterly summaries.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 20.5
- Humidity (%): 55.0 - 61.1
- Air changes (per hr): 20
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Details on route of administration:

The test substance was administered by oral gavage.

Vehicle:

methylcellulose

Remarks:

1 %

Details on oral exposure:

VEHICLE
- Amount of vehicle: 10 mL/kg bw/d

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

28 days

Frequency of treatment:

Animals were treated once daily, seven days per week for four weeks.

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Dose / conc.:

62.5 mg/kg bw/day (actual dose received)

Dose / conc.:

250 mg/kg bw/day (actual dose received)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed daily for signs of ill health, behavioural changes or toxicosis. Any observed changes were recorded.

BODY WEIGHT: Yes, all rats were weighed prior to dosing and subsequently at weekly intervals throughout the study.

FOOD CONSUMPTION: Yes, the quantity of food consumed in each cage was measured at weekly intervals throughout the study.

HAEMATOLOGY: Yes, food was withdrawn overnight prior to collection of samples. Blood was withdrawn under light ether anaesthesia from the orbital sinus of all surviving rats prior to termination (Week 4).
Following Parameter were analysed:
Packed cell volume (PCV) %
Haemoglobin (Hb) g/dL
Red blood cell count (RBC) x10^6/mm³
Platelt count (Plts) x10^3/mm³
Absolute indices:
Mean corpuscular haemoglobin concentration (MCHC)
[Calculated: Hb (g/dL)/ PCV (%)] x 100 %
Mean corpuscular volume (MCV)
Calculated: [PCV (%) x 10] / RBC (x10^6/mm³) fL
Total white blood cell count (WBC) x10^3/mm³
Differential white blood cell count (Diff) x10^3/mm³
Thrombotest (TT) Method of Owren, P.A. (Lancet, 1959, i i , 754)

CLINICAL CHEMISTRY: Yes
Following Parameter were analysed:
Glucose - using BCL Test Kit (hexokinase mediated), mg/dL
Glutamic-pyruvic transaminase (GPT), also known as 'alanine aminotransferase' - using BCL Test kit Reaction temperature 30 °C, mU/mL
Glutamic-oxaloacetic transaminase (GOT), also known as 'aspartate aminotransferase' - using BCL Test Kit Reaction temperature 30 °C, mU/mL
Alkaline Phosphatase (AP) Reaction temperature 30 °C, mU/mL
Total bilirubin, mg/dL
Cholesterol (Chol), mg/dL
Urea nitrogen (Urea Nitr), mg/dL
Total protein, g/dL
Albumin (Alb), g/dL
Globulin (Glob) - by subtraction: Total protein (g/dL) minus Albumin (g/dL) g/dL
Albumin/Globulin ratio (A/G) - by calculation from Total protein and Albumin concentrations
Sodium (Na), mEq/L
Potassium (K), mEq/L
Calcium (Ca), mEq/L
Chloride (Cl), mEq/L
Inorganic phosphorus (P), mEq/L
Creatinine, mg/dL

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, organs examined: adrenals, heart, kidneys, liver, spleen, any other macroscopically abnormal tissue

HISTOPATHOLOGY: Yes,
Samples of the following tissues from all rats in the control and high dosage groups were preserved in 10 % buffered formalin for subsequent histological examination:
adrenals, liver, heart, spieen, kidneys, any other macroscopically abnormal tissue

The following additional tissues were preserved:
adrenals+
aorta
brain (medullary, cerebellar and cortical sections)
caecum
Colon
duodenum
eyes (Davidson's fluid as fixative)
heart+
ileum
jejunum
kidneys+
larynx
liver+
lungs
lymph nodes (cervical and mesenteric)
mammary gland
oesophagus
ovaries
pancreas
pharynx
pituitary
prostate
salivary gland
sciatic nerve
seminal vesicles
skeletal muscle
spleen+
sternum (for bone and marrow section)
stomach
testes (including epididymis)
thymus (where present)
thyroid (with parathyroid)
tongue
trachea
urinary bladder
uterus
vagina
any other macroscopically abnormal tissue+
+ Intermediate and low dosage groups

Statistics:

The following sequence of statistical tests was used for bodyweight, organ weight and clinical pathology data: If the data consisted predominantly of one particular value (relative frequency of the mode exceeded 75 %), the proportion of values different from the mode was analyzed by appropriate methods. Otherwise: Bartlestt’s test was applied to test for heterogeneity of variance between treatments. Where significant (at the 1 % level) heterogeneity was found, a logarithmic transformation was tried to see if a more stable variance structure could be obtained. If no significant heterogeneity was detected (Or if a satisfactory transformation was found), a one-way analysis was present, and could not be removed by a transformation, the Kruskal-Wallis analysis of ranks was used. Analyses of variance were followed by Student’s ‘t’ test and Wiliams’ test for a dose related response, although only the one thought more appropriate for the response pattern observed was reported. The Kruskal-Wallis analyses were followed by the non-parametric equivalents of the ‘t’ test and Williams’ test (Shirley’s test). For organ weight data, where appropriate, analysis of covariance was used in place of analysis of variance in the above sequence. The final body weight was used as covariate in an attempt to allow for differences in bodyweight which might have influenced the organ weights.

Clinical signs:

no effects observed

Description (incidence and severity):

For two male rats in the intermediate dosage group noisy respiration was observed on Days 5 and 6 or on Days 6 and 12 to 16. However, this clinical finding was not observed for rats in the high dosage group and was therefore considered unlikely to be directly related to treatment. On Days 27 and 28, pallor was recorded for all rats. This was considered to be the direct result of the blood sampling procedure carried out on Day 27 and unrelated to the treatment.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female rat died on day 14. Macroscopic examination revealed clear serous fluid and pale gelatinous material in the thoracic cavity. At microscopic examination vascular congestion, pleural inflammation, adhesions and bronchial epithelial hyperplasia were noted in the lungs. Congestion of the ovaries was also observed. It is considered that the death of this animal occurred as a result of a dosing intubation error.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Similar to those of control animals.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Similar to those of control animals.

Food efficiency:

not examined

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

no effects observed

Description (incidence and severity):

Similar to those of control animals.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

Similar to those of control animals.

Haematological findings:

no effects observed

Description (incidence and severity):

Similar to those of control animals.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

In comparison with control rats, significantly lower (P<0.01) levels of albumin were recorded for rats receiving 250 mg/kg bw/d or 1000 mg/kg bw/d. Chloride levels for male rats receiving 1000 mg/kg bw/d were significantly higher (P<0.05) than those for control rats. These changes in albumin and chloride levels were, however, very low in magnitude and were considered unlikely to be of toxicological importance.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Significant lower adrenal weights were recorded for female rats receiving the test substance at 1000 mg/kg bw/d in comparison with controls. This apparent shift to lower adrenal weights was not recorded for treated male rats and individual adrenal weights for treated female rats were generally within the expected weight range for this organ (adrenal weights, female rat: 5 percentile 44 mg, median 61 mg, 95 percentile 79 mg). In the absence of any other treatment related finding, the lower adrenal weights recorded for female rats in the high dosage group were therefore considered likely to have arisen spontaneously.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No treatment-related changes were observed.

Histopathological findings: neoplastic:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects were observed up to the highest dose

Key result

Critical effects observed:

no

Conclusions:

The short term repeated dose study did not reveal any adverse findings. A NOAEL of above 1000 mg/kg bw/d was determined.

Executive summary:

In a sub-acute toxicity study according to EU method B.7 (Huntingdon, 1989), the test substance was administered to 5 Sprague Dawley rats/sex/dose by gavage at dose levels of 62.5, 250 and 1000 mg/kg bw/d. Animals were treated 7 days/week for 28 days.

There were no compound related effects in mortality, clinical signs, body weight, food consumption, hematology, clinical chemistry, organ weights, or gross and histologic pathology.

Clinical signs were only observed in the high dose animals during the first 10 treatment days. Animals showed piloerection and a hunched posture. One female rat died on day 14, but it was considered that the death of this animal occurred as a result of a dosing intubation error.

Albumin was slightly reduced in animals of the mid dose group. A slight increase of the chloride values was observed in males of the high dose group. This effect was not regarded as treatment related.

Significant lower adrenal weights were recorded for female rats receiving the test substance at 1000 mg/kg bw/d in comparison with controls. This apparent shift to lower adrenal weights was not recorded for treated male rats and individual adrenal weights for treated female rats were generally within the expected weight range for this organ (adrenal weights, female rat: 5 percentile 44 mg, median 61 mg, 95 percentile 79 mg). In the absence of any other treatment related finding, the lower adrenal weights recorded for female rats in the high dosage group were therefore considered likely to have arisen spontaneously.

Macroscopic abnormalities recorded for rats killed at termination were considered incidental and unrelated to treatment with the test substance.

A NOAEL of above 1000 mg/kg bw/d was determined in this study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

GLP and guideline compliant study

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Key study

The test substance was tested for subchronic toxicity according to OECD Test Guideline No. 408. Wistar rats of SPF quality were used for testing. The test substance was administered in a vehicle (1% water solution of methylcellulose) by stomach tube; oral application to rats occurred daily. The study included four main groups and two satellite groups of animals. Each main group consisted of 10 males and 10 females; each satellite group consisted of 6 males and 6 females. Main groups contained 3 treated groups (doses 62.5, 250, 1000 mg/kg of body weight/day) and one control group (vehicle only). The satellite groups contained one control group (vehicle only) and one treated group (1000 mg/kg/day). The administration period lasted 90 days. Animals in the main groups were then sacrificed while satellite animals were observed for the next 28 days without treatment. During the 90-day study, clinical observation and health status control were performed daily. The body weight, food consumption were measured weekly and the detailed clinical observation was carried out in the same time interval. Water consumption was measured twice a week. Ophthalmologic examination was performed at the beginning and at the end of the study. Before the end of study, the functional observations were performed. The study was completed by urinalysis, haematological and biochemical analysis, and gross necropsy of animals. The organs selected for weighing and histopathological examination were removed. These parameters (except clinical observation during the recovery period) were also checked for the satellite groups of animals.

The test substance treatment did not produce changes detected in health condition control, opthalmological observation, daily clinical observation after application and functional observation of animals. Food and water consumptions were not negatively influenced by the test substance administration. The test substance did not interfere with normal growth of treated animals. Body weight and body weight increments of treated animals were not affected by the test substance administration. During haematological examination, changes in red blood components of both sexes were recorded: decreased total erythrocyte count and related decreased concentration of haemoglobin and haematocrit (statistically significant decreases in dose levels 250 and 1000 mg/kg/day). This change was irreversible and dependent on dose level. In females, platelet count was statistically significantly decreased at the dose levels 250 and 1000 mg/kg/day. This decrease was also irreversible. This irreversible effect was not accompanied by any significant clinical changes or pathomorphological findings of related tissues and organs (spleen, bone marrow). Upon histological examination, there were no changes that indicated an increase in erythrocyte damage in the spleen or a failure of hematopoiesis in the bone marrow. Therefore, these findings are considered to be without significant toxicological importance. During biochemical examination no significant changes related to the test substance treatment were recorded in males. An unbalanced concentration of ions was detected. Statistically significant decreases in the concentration of sodium and potassium ions and increased concentration of chloride ions were recorded in treated females. Also concentration of phosphorus was significantly increased in all treated groups of females compared to control group. All changes were reversible. During biometry of organs, significant changes in organ weights related to test substance administration were not detected. Toxicologically significant microscopical changes in all examined organs were not found. Urine parameters were not influenced by test substance administration - only a decrease in the value of urine pH in males of the dose level 250 mg/kg/day was recorded and this finding can be considered to be incidental, because it was not dependent on the dose levels. (Many factors can influenced pH of urine - e.g. age and sex, so this minor finding has no toxicological importance). No macroscopical changes related to test substance administration were recorded. Microscopical evaluation showed that the test substance orally administered at the dose of 1000 mg/kg/day (the highest dose level) did not cause histopathological changes indicative of a toxic effect in any examined organs.

The NOAEL (No Observed Adverse Effect Level) value in male and female rats was determined as 1000 mg/kg/day.

Supporting study

In a sub-acute toxicity study according to EU method B.7 (Huntingdon, 1989), the test substance was administered to 5 Sprague Dawley rats/sex/dose by gavage at dose levels of 62.5, 250 and 1000 mg/kg bw/d. Animals were treated 7 days/week for 28 days.

There were no compound related effects in mortality, clinical signs, body weight, food consumption, hematology, clinical chemistry, organ weights, or gross and histologic pathology.

Clinical signs were only observed in the high dose animals during the first 10 treatment days. Animals showed piloerection and a hunched posture. One female rat died on day 14, but it was considered that the death of this animal occurred as a result of a dosing intubation error.

Albumin was slightly reduced in animals of the mid dose group. A slight increase of the chloride values was observed in males of the high dose group. This effect was not regarded as treatment related.

Significant lower adrenal weights were recorded for female rats receiving the test substance at 1000 mg/kg/d in comparison with controls. This apparent shift to lower adrenal weights was not recorded for treated male rats and individual adrenal weights for treated female rats were generally within the expected weight range for this organ (adrenal weights, female rat: 5 percentile 44 mg, median 61 mg, 95 percentile 79 mg). In the absence of any other treatment related finding, the lower adrenal weights recorded for female rats in the high dosage group were therefore considered likely to have arisen spontaneously.

Macroscopic abnormalities recorded for rats killed at termination were considered incidental and unrelated to treatment with the test substance.

A NOAEL of above 1000 mg/kg bw/d was determined in this study.

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on repeated dose toxicity, the test item is not classified according to Regulation (EC) No 1272/2008 (CLP).