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

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

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 1000 mg/kg/day.

Link to relevant study records

Reference

Endpoint:

one-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 November 1992 to 03 March 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

other: 87/302/EEC, 30 May 1988

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl: CD(SD) BR VAF/Plus strain

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- 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

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 concentration 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 preparation. The suspensions were stirred constantly throughout the administration period using a magnetic 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:

ANALYTICAL PROCEDURE

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 dissolved 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 expected concentration range 4 - 8 µg/ml. The final solution was filtered (Whatman GF/F) and the concentration 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 accurately weighed quantity (50 mg) of ODB-2 in tetrahydrofuran. Solutions for instrument calibration, containing 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:

Daily

Details on study schedule:

- Age at mating of the mated animals in the study: 9 weeks

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:

24

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 acclimatisation period of 7 days was allowed between allocation of animals to groups and commencement of treatment.
- Other:

Positive control:

None

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily to weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Dams that littered were weighed on Days 0, 7, 14 and 21 post partum.

OTHER:

Oestrous cyclicity (parental animals):

Cages of males were interspersed amongst those holding females to promote development of regular oestrous cycles.

Sperm parameters (parental animals):

No data

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, weight 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.

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not specified

Reproductive performance:

not specified

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
There were no treatment-related clinical signs observed during the study.

One female receiving 1000 mg/kg/day (no. 186) was found dead during Week II (Day 3 of pairing) having shown no previous clinical signs; post mortem examination revealed moist red staining of the perioral and perinasal fur and severely congested, uncollapsed lungs but no apparent signs of intubation error. Male no. 93, also receiving 1000 mg/kg/day, was sacrificed Week 13 (Day 19 of pairing) after having previously been observed continually leaning to one side for nine days. Prior to sacrifice this animal appeared uncoordinated and continually rolled over; however, no abnormalities were detected at macroscopic post mortem examination.

One female in the low dose group (no. 134) was found dead Week 14 (Day I post partum) with red salivation and vaginal discharge. No previous clinical signs were recorded. Post mortem findings included enlarged cervical lymph nodes, congested thymus and lungs, gaseous distension of, and blood in, the stomach, dark contents in the small intestine. The uterus contained recently dead foetuses and placentae; thirteen pups had been born, all were cold and unfed when the dam was found.

As there were no trends or consistent pathology findings among these animals, the mortalities were considered to be coincidental and not related to treatment.

A further three female rats (no. 113 from control and nos. 169 and 191, 1000 mg/kg/day) were either found dead or were killed on the second day of treatment following loss of bodytone and laboured respiration. Post mortem examination revealed all three to have a perforated oesophagus. The clinical signs and necropsy findings were consistent with accidental intubation errors occurring during the dosing procedure. As one of the animals (no. 169) receiving 1000 mg/kg/day was found dead within 24 hours of the first dose, a replacement animal (no. 193) was selected for the duration of the study and dosed from this day. Data relating to no. 169 are not reported.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Weekly bodyweight gain for males and females was generally similar for all groups (P>0.05).

Bodyweight change for females during pregnancy and lactation was unaffected by treatment (P>0.05).

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
The test substance was administered as a suspension in 1% methylcellulose within four hours of preparation. The suspensions were stirred constantly throughout the administration period using a magnetic stirrer. Control animals received the vehicle at the same dose volume (1 ml/100 g bodyweight).

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.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
No data


REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
No data

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Mating performance, as assessed by the incidence and distribution of successful matings, the median pre-coital time and the type of smear recorded on the day of conception, was not adversely affected by treatment. The pregnancy rate was 100% for all groups with most females conceiving within the first four days after the start of pairing, corresponding with the expected length of an oestrous cycle.

The duration of pregnancy was similar for all groups (P>0.05).

ORGAN WEIGHTS (PARENTAL ANIMALS)
Intergroup differences in organ weights, adjusted for final bodyweight as appropriate, were only slight (P>0.05) and revealed no clear or consistent changes in either sex which could be attributed to treatment.

GROSS PATHOLOGY (PARENTAL ANIMALS)
The macroscopic examination performed at terminal autopsy of surviving adults and offspring revealed no treatment-related changes.

HISTOPATHOLOGY (PARENTAL ANIMALS)

OTHER FINDINGS (PARENTAL ANIMALS)
No treatment-related findings were detected in the tissues examined.

No microscopic findings were detected which might have been associated with the failure of a small number of male/female pairings amongst rats receiving 62.5 or 250 mg/kg/day to produce offspring.

The few microscopic findings seen in the adult rats examined were considered to be spontaneous in origin and of no toxicological importance.

Key result

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 adverse effects were observed on the growth and reproductive capacity of male and female rats or the development of their offspring.

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

VIABILITY (OFFSPRING)
No data

CLINICAL SIGNS (OFFSPRING)
Mean ages of attainment of surface and air righting reflexes, startle response and presence of the pupil reflex at Day 20 post partum were similar for treated and control groups (P> 0.05).


BODY WEIGHT (OFFSPRING)
No data


SEXUAL MATURATION (OFFSPRING)
Offspring did not reach sexual maturity.


ORGAN WEIGHTS (OFFSPRING)
Intergroup differences in organ weights, adjusted for final bodyweight as appropriate, were only slight (P>0.05) and revealed no clear or consistent changes in either sex which could be attributed to treatment.

GROSS PATHOLOGY (OFFSPRING)
The macroscopic examination performed at terminal autopsy of surviving adults and offspring revealed no treatment-related changes.

HISTOPATHOLOGY (OFFSPRING)
No treatment-related findings were detected in the tissues examined.

No microscopic findings were detected which might have been associated with the failure of a small number of male/female pairings amongst rats receiving 62.5 or 250 mg/kg/day to produce offspring.

The few microscopic findings seen in the adult rats examined were considered to be spontaneous in origin and of no toxicological importance.

OTHER FINDINGS (OFFSPRING)

Key result

Dose descriptor:

NOEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: There were no adverse effects on the growth and reproductive capacity of male and female rats or the development of their offspring.

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

The dosage at which no signs of toxicity were recorded is therefore considered to be NOEL 1000 mg/kg/day, the highest dose tested.

Reproductive effects observed:

not specified

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.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Klimisch 1

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Justification for selection of Effect on fertility via oral route:

Only study available.

Effects on developmental toxicity

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

Reproduction

All dosages were well tolerated throughout. Adult bodyweight change, food and water intake, mating performance and pup survival, growth and development to weaning were similar for all groups and no changes were noted that were considered to be treatment related. The NOEL was determined to be 1000 mg / kg bw /day.

Based on these findings the substance ODB-2 will not be classified as a reproductive toxicant.

Developmental

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.

Based on these findings the substance ODB-2 will not be classified as a developmental reproductive toxicant.