2'-anilino-6'-[ethyl(3-methylbutyl)amino]-3'-methylspiro[isobenzofuran-1(3H),9'-[9H]xanthene]-3-one

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

one-generation reproductive toxicity

Remarks:

based on test guideline (migrated information)

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

30 November 1992 to 03 March 1993

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study undertaken at GLP accredited laboratory to internationally accepted guidelines. The restriction is due to the use of the read across approach: the test was performed not with S-205 but with Black 400, a substance which has been demonstrated to be very similar in structure, physical/chemical properties and the toxicological profile .

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EEC 87/302/EEC, 30 May 1988.

Deviations:

no

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
- 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: during the pre-mating period, males and females were housed separately, four to a cage. Cages of males were interspersed amongst those holding females to promote development of regular oestrous cycles.During the mating period, animals were housed on the basis of one male to one female in plastic breeding cages (RM-2 type).At the end of the mating period the males were rehoused with their former cage mates in metal cages
and the females remained in individual breeding cages for the birth and rearing of young.Following the sacrifice of weanIings, females were rehoused with their former cage mates in metal cages.
- Diet: ad libitum
- 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:

other: 1% methylcellulose

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.
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).

VEHICLE
- Concentration in vehicle: 0.625, 2.5 and 10.0% w/v
- Amount of vehicle (if gavage): a constant dose volume of 1 ml / 100 g bodyweight was assumed throughout.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: 20 days
- Proof of pregnancy: vaginal plug or smear referred to as day 0 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. the treatment lasted 17 weeks for the males and 13 weeks for the females.

Frequency of treatment:

Daily

Details on study schedule:

- Age at mating of the mated animals in the study: 16 weeks for the males and 19-20 weeks for the females

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 vehicle

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.

Positive control:

None

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule:

DETAILED CLINICAL OBSERVATIONS: Yes / No / No data
- Time schedule:

BODY WEIGHT: Yes
- Time schedule for examinations: all animals were weighed at the start of treatment and subsequently at weekly intervals.During the mating period, all females were weighed daily throughout and daily weighing continued until parturition. Dams that littered were weighed on Days 0, 7, 14 and 21 post partum.

OPHTHALMOSCOPY
Prior to treatment and scheduled terminal sacrifice, all surviving animals were subjected to an
ophthalmoscopic examination.

REPRODUCTIVE PARAMETERS
Mating performance was assessed by the length of an oestrous cycle, the median pre-coital time, the type of smear recorded on the day of conception, the incidence and distribution of successful matings, the pregnancy rate and the duration of pregnancy.

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 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities,
other:surface righting, startle, air righting and pupil reflexes

GROSS EXAMINATION OF DEAD PUPS: yes

Postmortem examinations (parental animals):

SACRIFICE
-Shortly after the pups had weaned, adult males and adult females were sacrificed.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations.

HISTOPATHOLOGY / ORGAN WEIGHTS
- From all adults, the organs listed below were weighed and a full range of tissues preserved for histopathological examination. In addition, any tissue showing macroscopic abnormality was similarly preserved.
- Histology was performed on reproductive tract associated tissues of:
* all adults of the control group and at the highest dose;
* all apparently infertile males and females from the lowest and intermediate dose;
* any animals which died.
The routine stains used were haematoxylin and eosin.
The uteri of apparently non-preguant females were examined by a modified Salewski technique (Salewski, 1964) prior to storage in fixative. The uterus of every female which gave birth was visually inspected for implantation sites and the number of sites recorded.

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

Postmortem examinations (offspring):

SACRIFICE
- On or shortly after Day 21 all pups were sacrificed
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations for abnormalities. Sex of the pups was confirmed by gonadal inspection.

HISTOPATHOLOGY
Any tissues showing macroscopic abnormality were preserved in neutral buffered 10% formalin.

Statistics:

All statistical analyses were carried out separately for males and females. Significance tests, employing analysis of variance following by an intergroup comparison with the control, were performed on the following parameters:
- weekly bodyweight and female bodyweight change during pregnancy and lactation, food and water consumption, litter data and organ weights.
Dependent on the heterogeneity of variance between treatment groups, parametric tests (analysis of variance (Snedecor and Cochran, 1967) followed by Williams' test (Williams, 1971/2)) or nonparametric tests (Kruskal-Wallis (Hollander and Wolfe, 1968) followed by Shirley's test (Shirley, 1977)) were used to analyse these data, as appropriate.
For bodyweight and bodyweight change, the analyses were carried out using the individual animal as the experimental unit. Data relating to food and water consumption were analysed on a cage basis. For litter data, the basic sample unit was the litter and, due to the preponderance of non-normal
distributions, non-parametric analyses were routinely used. Organ weight data were analysed using bodyweight at post mortem as covariate, to allow for differences in bodyweight which may have influenced organ weight values.
Where 75% or more of the values for a given variable were the same, a Fisher's exact test (Fisher, 1950) was used, when considered necessary.
All significant (ie P<0.05) intergroup differences from the control are reported and were supported by a significant analysis of variance (P< 0.05).

Offspring viability indices:

- Pup loss at birth was calculated as a percentage from the formula:
[(total no. of young at birth - no. of live young)/ Total no. of young at birth]x 100
- Cumulative pup loss was similarly calculated from the formula:
[(total no. of young at birth - no. of live youngat Day X)/ Total no. of young at birth]x 100 where 'x' is the day of weighing the litter
- Sex ratio at birth and weaning was calculated from the formula:
(Total no. of males/ Total no. of young) x 100

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:

no effects observed

Reproductive performance:

no effects observed

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 [mdings 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.

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

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)
Two females (one each at 62.5 and 250 mg/kg/day) resorbed their single implant. A single implantation is atypical and is generally considered insufficient to maintain pregnancy thus, in the absence of similar occurrences at the high dose level, the 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 (P>0.05).

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)
Bodyweight for pups was generally similar for all groups at birth, and Days 4, 8, 12, 16 and 21 .

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.

Dose descriptor:

NOEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

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 1000 mg/kg/day.

Executive summary:

S-205 and ODB-2 (Black 400), which is tested for its genotoxicity in a mouse micronucleus test, are both colour precursor for heat sensitive record sheets. These substances have been demonstrated to be very similar in structure, physical/chemical properties and the toxicological profile. Due to the fact that S-205 and ODB-2 have nearly the same chemical structure. The same mode of interaction with bio-macromolecules, living cells and tissue and metabolic pathway is expected. Therefore, a read-across from S-205 to data obtained with ODB-2 is scientifically justified.

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:

The study was undertaken to GLP by an accredited laboratory. Therefore the study would be assigned a Klimisch 1. However as it has been used as a read-across, it has been assigned Klimisch 2.

Additional information

Justification for selection of Effect on fertility via oral route:
This was the only study available.

Effects on developmental toxicity

Additional information

The substance shows no evidence of toxicity and no evidence of toxicity to reproduction, from the one generation study, therefore, on these grounds it cannot be justified to undertake an additional vertebrate study.

Justification for classification or non-classification

The substance has not been classified as toxic to reproduction at this level. The one generation study, a read-across from Black 400, showed no indication of toxicity, resulting in a NOEL 1000 mg/kg bw/day.