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

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

30 November 1992 to 03 March 1993

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1993

Report date:

1993

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: 87/302/EWG, S.43; OECD 415 (1981)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

403-830-5

EC Name:

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

Cas Number:

89331-94-2

Molecular formula:

C35 H36 N2 O3

IUPAC Name:

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

Test material form:

solid: particulate/powder

Test animals

Species:

rat

Strain:

other: CD(SD) BR VAF/Plus strain

Details on test animals or test system and environmental conditions:

- Source:
- Age at study initiation: (P) male 6 wks, female 9 - 10 wks
- Weight at study initiation: (P) Males: 72 - 95 g; Females: 174 - 196 g
- Fasting period before study: no data
- Housing: Breeding cages, type RM-2
- Diet: Biosure laboratory animal diet no. 1, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 12 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 4°C
- Humidity (%): 45 ± 17%
- Air changes (per hr): no data
- Photoperiod: 12 hrs dark / 12 hrs light

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test substance was ground in a mortar with a small
amount of the vehicle (1 % methylcellulose) until a smooth paste was formed. The formulation was
then gradually made up to volume and mixed using a high shear homogeniser to give the concentr
ation used for the highest dosage (10.0% w/v). A sequence of suspensions was then made from
the highest concentration to give concentrations of 2.5 and 0.625% w/v for the lower dosages. A
constant dose volume of 1 ml / 100 g bodyweight was assumed throughout.
The test substance was administered as a suspension in 1% methylcellulose within four hours of pre
paration. The suspensions were stirred constantly throughout the administration period using a magn
etic stirrer. Control animals received the vehicle at the same dose volume (1 ml/100 g bodyweight).
DIET PREPARATION
- Rate of preparation of diet (frequency):
- Mixing appropriate amounts with (Type of food): n/a
- Storage temperature of food: n/a
VEHICLE
- Justification for use and choice of vehicle (if other than water): CMC, generally accepted as a
vehicle.
- Concentration in vehicle: 1%
- Amount of vehicle (if gavage): 1 ml / 100 g bodyweight
- Lot/batch no. (if required): no data
- Purity: no data
Details on mating procedure
- M/F ratio per cage: 1:1
- Length of cohabitation: 20 days
- Proof of pregnancy: vaginal smear referred to as [day 0 / day 1] of pregnancy
- After successful mating each pregnant female was caged (how): Suspended stainless steel cages
(Biotech~) equipped with solid sides and wire grid front, back, floor and top.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Apparatus and instrumentation
High performance liquid chromatograph (HPLC): As detailed below or suitable alternative.
Pump: Spectra-Physics SP 8810.
Autosampler: Waters Associates WISP 71OA.
Detector: Spectra SYSTEM UV1000.
Integrator: SP 4270.
General laboratory glassware.
Reagents
Test material: ODB-2.
Supplier: Manufacturer
Batch no.: 8B-24423-02.
Stated purity: 99.6%.
Tetrahydrofuran: Rathburn Chemicals Ltd., HPLC grade.
Acetonitrile: Merck Ltd., HiPerSolv for HPLC.
Ammonium acetate: FSA Laboratory Supplies, Analytical Reagent.
Water: Elgastat UHP-4, deionised reverse osmosis.
Sample extraction
A representative sample (approximately 1 ml) of test formulation was accurately weighed and dissolv
ed in a suitable volume of tetrahydrofuran. The extract was appropriately diluted, initially using
tetrahydrofuran and finally using mobile phase, to provide a solution containing ODB-2 in the expec
ted concentration range 4 - 8 μg/ml. The final solution was filtered (Whatman GF/F) and the concen
tration of ODB-2 was quantified by high performance liquid chromatography using ultraviolet detection
as detailed in the following section.
Typical chromatographic conditions
Analytical column: Nucleosil C18, 5 μm, 15 cm x 4.6 mm ID., Jones Chromatography Ltd.
Guard column: Aquapore RP-300, 7 μm, 3 cm x 4.6 mm ID., Applied Biosystems Inc.
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: 17 μI.
Integrator attenuation: 64.
Retention volume:6ml.
Calibration
A primary standard solution was prepared for each analytical occasion by dissolving an accura
tely weighed quantity (50 mg) of ODB-2 in tetrahydrofuran. Solutions for instrument calibration, co
ntaining ODB-2 in the concentration range 2 - 10 μg/ml, were prepared by appropriate dilution of the
primary standard using mobile phase. Calibration solutions were injected onto the HPLC, at the
beginning and end of each sample analysis sequence, using the conditions detailed above.

Duration of treatment / exposure:

The test substance was administered by intragastric intubation to rats of both sexes, once per day, prior to pairing and through to termination after weaning of the offspring.

Frequency of treatment:

Dosing regime (males): 7 days/week
Dosing regime (females): 7 days/week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

62.5 mg/kg bw/day

Remarks:

Basis:
actual ingested

Doses / concentrations 2

Dose / conc.:

250 mg/kg bw/day

Remarks:

Basis:
actual ingested

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

Basis:
actual ingested

No. of animals per sex per dose:

Number of litters per dose/conc.: O at mg/kg or mg/L

Male: 24 animals at 0 mg/kg or mg/L
Male: 24 animals at 62.5 mg/kg or mg/L
Male: 24 animals at 250 mg/kg or mg/L
Male: 24 animals at 1000 mg/kg or mg/L

Female: 24 animals 0 at mg/kg or mg/L
Female: 24 animals at 62.5 mg/kg or mg/L
Female: 24 animals at 250 mg/kg or mg/L
Female: 24 animals at 1000 mg/kg or mg/L

Control animals:

yes, concurrent no treatment

Details on study design:

Dose selection rationale: The dosages were selected based on the results of a 28-day study in the
rat performed in these laboratories (HRC report No. YKG 20/89413) in which a dosage of 1000 mg/
kg/day was without adverse effect on male or female rats.
- Rationale for animal assignment (if not random): After an acclimatisation period of 5 days, they were
weighed again and ninety-six animals of each sex assigned to four groups by computerised stratified
randomisation to give approximately equal initial group mean bodyweights.
Following allocation, the animals were earmarked to give individual identification. A further accli
matisation period of 7 days was allowed between allocation of animals to groups and commencement
of treatment.

Examinations

Fetal examinations:

Litter observations
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no
PARAMETERS EXAMINED
The following parameters were examined in F1offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weig
ht gain, physical or behavioural abnormalities
GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or
found dead.
Postmortem examinations (parental animals)
SACRIFICE
- Male animals: All surviving animals shortly after the pups had weaned.
- Maternal animals: All surviving animals shortly after the pups had weaned.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and
abdominal viscera.
HISTOPATHOLOGY / ORGAN WEIGHTS
The reproductive tissues were prepared for microscopic examination and weighed, respectively.
Postmortem examinations (offspring)
SACRIFICE
- The F1 offspring not selected as parental animals and were sacrificed at or shortly after 21 days of
age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic
examination) as follows:
adrenals
brain
epididymides, individually (identified as left or right)
heart
kidneys
liver
lungs
ovaries
pituitary
prostate (with seminal vesicles and coagulating gland)
testes, individually (identified as left or right)
thymus
GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and
abdominal viscera.
HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated above were prepared for microscopic examination and weighed, respectively.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

There were no treatment-related clinical signs observed during the study.

One female receiving 1000 mg/kg/day was found dead during week 11. Post mortem revealed moist red staining of the perioral and perinasal fur and severely congested, uncollapsed lungs but no apparent signs of intubation error. One male receiving 1000 mg/kg/day was sacrificed Week 13. Prior to sacrifice this animal appeared uncoordinate and continually rolled over, however there were no macroscopic abnormalities.

One female in the low dosage group was found dead Week 14: Post mortem findings included enlarged cervical lymph nodes; congested thumus and lungs, gaseous distension of an blood in the stomach, dark contents in the small intestines. Uterus contained recently dead foetuses and placentae; 13 pups had been born.

These mortalities were considered to be coincidental and not related to treatment. A further three animals died from accidental intubation errors.

Effect levels (maternal animals)

Key result

Effect level:

1 000 mg/kg bw/day

Basis for effect level:

other: other

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Effects on F1 generation:
Two females resorbed their single implant. This incidence was considered not to be treatment related. Among dams rearing young to weaning, mean values for implantation rates, pup survival, pup growth and associated sex ratio at birth and Day 21 post partum were similar for all groups. The macroscopic examination performed at terminal autopsy of surviving adults and offspring revealed no treatment-related changes.

Fetal abnormalities

Abnormalities:

not specified

Applicant's summary and conclusion

Conclusions:

Based on the results obtained, this study indicated that dosages of 62.5, 250 or 1000 mg/kg/day were without adverse effect on the growth and reproductive capacity of male and female rats or the development of their offspring. The dosage of ODB-2 at which no signs of toxicity were recorded is therefore considered to be NOEL 1000 mg/kg/day.