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

Endpoint summary

Description of key information

No changes were noted in the parameters and tissues examined that were considered to be related to treatment with ODB-2. The dosage level of ODB-2 at which no signs of toxicity were recorded is therefore considered to be 1000 mg / kg / day.

Key value for chemical safety assessment

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:

key study

Study period:

14 October to 11 November 1988

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study undertaken at GLP accredited laboratory to internationally accepted guidelines.

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

not specified

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl: CD(SD)BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Commercial laboratory animal supplier.
- Age at study initiation: 43 ± 1 days
- Weight at study initiation: 162 - 397 g
- Fasting period before study:
- Housing: Groups of five, according to sex, in cages
- Diet: Free access to Labsure LAD 1
- Water: ad libitum
- Acclimation period: 15 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.5 to 20.0°C
- Humidity (%): 55.0
- Air changes (per hr): 20
- Photoperiod: 12 hrs dark / 12 hrs light

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): Daily
- Mixing appropriate amounts with (Type of food): Not applicable as test substance was made into a suspension.
- Storage temperature of food: n/a

VEHICLE
- Justification for use and choice of vehicle (if other than water): tried and tested
- Concentration in vehicle: 10% w/v suspension
- Amount of vehicle (if gavage): 10 mg / kg/ day
- Lot/batch no. (if required):
- Purity:

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

1. Apparatus and instrumentation
High performance liquid chromatograph (HPLC): Typically as detailed below or suitable alternative
Pump: Waters model 510.
Autosampler:Waters WISP model 710B.
Detector: LC 871 UV-VIS. variable wavelength spectrophotometer.
Integrator: SP 4270.
General laboratory glassware.

2. Reagents
ODB-2: batch number 61-24101.
Acetonitrile: HPLC (far UV) grade.
Ammonium acetate: AR grade.
Tetrahydrofuran: HPLC grade.
Water: Glass-distilled.

3. Method of analysis
A representative sub-sample (approximately 1 ml) of test suspension was accurately weighed and dissolved in an appropriate volume of
tetrahydrofuran.
The extract was further diluted, as necessary, using mobile phase to provide a solution containing an expected concentration between
2 mcg/ml and 4 mcg/ml ODB-2.
The final solution was filtered (Whatman GF/F) and the concentration of ODB-2 quantified by high performance liquid chromatography using
ultra-violet detection as detailed in section 4, below.

4. Typical chromatographic conditions
Analytical column: Nucleosil C18, 5 mcm, 15 cm x 4.6 mm 1D, Jones Chromatography Ltd.
Guard column: Brownlee MPLC aquapore RP-300 cartridge, 10 mcm, 3 cm x 4.6 mm ID.
Mobile phase: Acetonitrile / 0.01 M aqueous ammonium acetate (90/10, v/v).
Flow rate: 1.0 ml / minute.
Detector wavelength: UV, 280 nm
Injection volume: 15 mcl.
Integrator attenuation: 64.
Retention volume: 8 mI.

5. Calibration
A primary standard solution was prepared for each analytical occasion by dissolving an accurately weighed quantity (50 mg) of ODB-2 in tetrahydrofuran. Solutions for instrument calibration in the concentration range 2, 4, 6, 8 and 10 mcg / ml were prepared by serial dilution of the above solution using mobile phase.
Calibration solutions were injected onto the HPLC at the beginning and end of each sample analysis sequence using the conditions described in section 4.

6. Determination of the chemical and physical stability of OD5-2 in 1% MC formulations
6.1 Chemical stability
Sub-samples (4 of approximately 1 ml) were taken from a freshly prepared batch of test suspension, at nominal concentrations of 6.25 mg/ml and 100 mg/ml, and dispensed into pre-weighed volumetric flasks. Duplicate sub-samples were analysed immediately and the remaining sub-samples were stored at ambient temperature in the dark for 4 hours before analysis. At each occasion, duplicate sub-samples from each suspension were analysed as detailed in section 3.

6.2 Physical stability
After sampling for chemical stability the remainder of each test suspension was thoroughly mixed by repeated inversion and then magnetically stirred. After magnetic stirring for 5 minutes, sub-samples (approximately 1 ml) were removed at points approximately one-quarter, one-half and three-quarters the depth (representing the top, middle and bottom) of the suspension and dispensed into pre-weighed volumetric flasks (0 hour). The suspension was maintained by magnetic stirring and sampling was repeated after 0.5 and 1 hour. The remaining suspension was stored at ambient temperature in the dark until resuspension and further sampling, as above, after 4 hours. At each time-point, the sub-samples removed from each suspension were analysed as detailed in section 3.

Duration of treatment / exposure:

28 days

Frequency of treatment:

Daily

Remarks:

Doses / Concentrations:
62.5 mg/ kg / day
Basis:
actual ingested

Remarks:

Doses / Concentrations:
250 mg / kg / day
Basis:
actual ingested

Remarks:

Doses / Concentrations:
1000 mg / kg / day
Basis:
actual ingested

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dosage levels administered were selected on the basis of acute oral toxicity data.
- Rationale for animal assignment (if not random):
- Rationale for selecting satellite groups:
- Post-exposure recovery period in satellite groups: None
- Section schedule rationale (if not random):

Positive control:

None

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Early each working day and late afternoon.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Early each working day and late afternoon.

BODY WEIGHT: Yes
- Time schedule for examinations: All rats were weighed prior to dosing and subsequently at weekly intervals throughout the study.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Prior to termination
- Anaesthetic used for blood collection: Yes, ether
- Animals fasted: Yes
- How many animals: All surviving rats prior to termination.
- Parameters checked in table [No.?] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Prior to termination
- Animals fasted: Yes
- How many animals: all surviving animals
- Parameters checked: Packed cell volume, haemoglobin, red blood cell count, platelet count, mean corpuscular haemoglobin concentration, mean corpuscular volume and total white blood cell count.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes see attached report

Statistics:

Statistical analyses
All statistical analyses were carried out separately for males and females. Bodyweight data were analysed using weight gains.

The following sequence of statistical tests was used for bodyweight, organ weight and clinical pathology data:

(i) 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 analysed by appropriate methods.

Otherwise:
(ii) Bartlett'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.
(iii) If no significant heterogeneity was detected (or if a satisfactory transformation was found), a one-way analysis of variance was carried
out. If significant heterogeneity of variance was present, and could not be removed by a transformation, the Kruskal-Wallis analysis of
ranks was used.
(iv) Analyses of variance were followed by Student's 't' test and Williams' 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 It' 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 bodyweight 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

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
For two male rats in the intermediate dosage group receiving ODB-2, 250 mg / kg / day, 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, receiving ODB-2, 1000 mg / kg / day and was therefore considered unlikely to be directly related to treatment with ODB-2.
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 treatment with ODB-2.

BODY WEIGHT AND WEIGHT GAIN
Bodyweight gains for rats receiving ODB-2 were similar to those of control animals during the four-week treatment period.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Food consumption for rats receiving ODB-2 was similar to that of controls throughout the study period.

HAEMATOLOGY
Haematological investigations were carried out during Week 4 for all rats. In all instances, haematological parameters for rats receiving ODB-2 were similar to those for control rats receiving 1% MC.

CLINICAL CHEMISTRY
Biochemical investigations were also carried out for all rats during Week 4. In comparison with control rats, significantly lower (P<0.01) levels of albumin were recorded for rats receiving ODB-2, 250 mg / kg / day or 1000 mg / kg / day. Chloride levels for male rats receiving ODB-2, 1000 mg/kg/day 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.
No other changes in biochemical parameters were noted that were considered to be related to treatment with ODB-2.

ORGAN WEIGHTS
Significantly lower (P<0.05) adrenal weights were recorded for female rats receiving ODB-2, 1000 mg / kg / day 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 by chance.
No other changes in organ weights were recorded for rats receiving ODB-2.

GROSS PATHOLOGY
Macroscopic abnormalities recorded for rats killed at termination were considered incidental and unrelated to treatment with ODB-2.

Following comments are made in summary:
No treatment-related changes were observed. All the histopathological findings given in the table were considered to be of no toxicological significance.

HISTOPATHOLOGY: NON-NEOPLASTIC
Microscopic examination of the full range of reserved tissues from rats previously treated with ODB-2 at 1000 mg/kg/day for 4 weeks showed no treatment-related changes.

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No changes were noted in the parameters and tissues examined that were considered to be related to treatment with ODB-2.

Critical effects observed:

not specified

Conclusions:

No changes were noted in the parameters and tissues examined that were considered to be related to treatment with ODB-2. The dosage level of ODB-2 at which no signs of toxicity were recorded is therefore considered to be 1000 mg/kg/day.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Klimisch 1

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

Justification for classification or non-classification

The result of the 28 -day repeat dose oral toxicity to rats was that no changes were noted in the parameters and tissues examined that were considered to be related to treatment with ODB-2. The dosage level of ODB-2 at which no signs of toxicity were recorded is therefore considered to be 1000 mg / kg / day.

It is on this basis that no further studies were proposed and that Black 400 (ODB-2) is not classified.