3',6'-bis(diethylamino)spiro[isobenzofuran-1(3H),9'-[9H]xanthene]-3-one

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Data is from per reviewed journal

Data source

Reference

Reference Type:

publication

Title:

THE TERATOGENIC POTENTIAL IN RATS AND RABBITS OF D&C YELLOW NO. 8

Author:

C. M. BURNETI, E. I. GOLDENTHAL

Year:

1986

Bibliographic source:

Fd Chem. Toxic, Vol. 24, No. 8, pp. 819 823, 1986

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

other: as mentioned below

Principles of method if other than guideline:

Equivalent or similar to Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test in rats.

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Disodium 2-(3-oxo-6-oxidoxanthen-9-yl)benzoate

EC Number:

208-253-0

EC Name:

Disodium 2-(3-oxo-6-oxidoxanthen-9-yl)benzoate

Cas Number:

518-47-8

Molecular formula:

C20H12O5.2Na

IUPAC Name:

disodium 3-oxo-3H-spiro[2-benzofuran-1,9'-xanthene]-3',6'-diolate

Constituent 2

Reference substance name:

D&C YELLOW NO. 8

IUPAC Name:

D&C YELLOW NO. 8

Details on test material:

- Name of test material (as cited in study report): D & C Yellow No. 8, sodium fluorescein (C.I. No. 45350)
- Molecular formula (if other than submission substance): C20H12O5.2Na
- Molecular weight (if other than submission substance): 376.274 g/mole
- Substance type: Organic
- Physical state: No data available
- Impurities (identity and concentrations): 13 %

Test animals

Species:

rat

Strain:

other: CD Sprague-Dawley

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories
- Age at study initiation: (P) x wks; (F1) x wks: P- 18 weeks
- Weight at study initiation: (P) Males: x-x g; Females: x-x g; (F1) Males: x-x g; Females: x-x g: P-246 to 379 g
- Fasting period before study: No data available
- Housing: Animals were housed individually and identified by ear tag.
- Diet (e.g. ad libitum): Animals were housed individually
- Water (e.g. ad libitum): water, ad lib.
- Acclimation period: 4 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.22 ± 15
- Humidity (%):50 ± 15%
- Air changes (per hr): No data available
- Photoperiod (hrs dark / hrs light): 12-hr light/dark cycle.

IN-LIFE DATES: From: To: No data available

Administration / exposure

Route of administration:

oral: gavage

Type of inhalation exposure (if applicable):

not specified

Vehicle:

water

Details on mating procedure:

- M/F ratio per cage: 1:1 ratio
- Length of cohabitation: No data available
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 / day 1] of pregnancy Copulatory plug or presence of sperm in vaginal washings day 0 of pregnancy
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility. No data available
- Further matings after two unsuccessful attempts: [no / yes (explain)] No data available
- After successful mating each pregnant female was caged (how): Individually
- Any other deviations from standard protocol: Mated females were assigned to treatment and control group.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

14 days

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Basis: nominal in water

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Basis: nominal in water

Doses / concentrations 3

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

Basis: nominal in water

Doses / concentrations 4

Dose / conc.:

1 500 mg/kg bw/day (nominal)

Remarks:

Basis: nominal in water

No. of animals per sex per dose:

Total: 100
0 mg/kgbw/day: 25 female
100 mg/kgbw/day: 25 female
500 mg/kgbw/day: 25 female
1500 mg/kgbw/day: 25 female

Control animals:

yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:

Survival, clinical sign and body weight were examined

Oestrous cyclicity (parental animals):

The number and location of viable and non-viable foetuses, Early and late resorptions, total implantations and corpora lutea were recorded.

Sperm parameters (parental animals):

No data available

Litter observations:

Feotus weight and sex were observed.

Postmortem examinations (parental animals):

The thoracic and abdominal cavities and the internal organs of the dams were examined for gross morphological changes.

Postmortem examinations (offspring):

Visceral and skeletal malformations were examination.

Statistics:

Statistical analysis were performed by using Chi-square test criterion with Yates" correction for 2 x 2 contingency lables and, or Fisher's exact probability test as described by Sicgel (1956) to Differences in the foetal sex distribution and the number of litters with malformations between control and treated groups. The numbers of early and late resorptions, dead foetuses and post-implantation losses were compared between groups by the Mann Whitney U test as described by Siegel (1956) and Well (1970). The mean numbers of viable foetuses, total implantations, corpora lutca and mean foetal weights were compared between groups by analysis of variance (one way classification), Bartlett's test for homogeneity of variances, and the appropriate t- test (for equal or unequal variances) as described by Steel & Torric (1960) using Dunnctt's multiple comparison tables (1964).

Reproductive indices:

Dams with viable foetuses were examined.

Offspring viability indices:

Viable foetuses/dam were examined.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

not specified

Dermal irritation (if dermal study):

not specified

Mortality:

mortality observed, treatment-related

Description (incidence):

When treated with 1500 mg/kg bw/day, Six rats died during the dosing period as compared to control.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

When treated with 1500 mg/kg bw/day, decrease in body weight gain was observed in treated rats as compared to control.

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Orange discoloration of the urine was observed in all the treated rats as compared to control.

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

No effect on Post-implantation loss/dam, Total implantations/dam and Corpora lutea/dam were observed in treated rats as compared to control.

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

Description (incidence and severity):

There were no biologically meaningful or statistically significant differences in the number of litters and Foetal sex of treated rats as compared to control.

Details on results (P0)

Mortality:
When treated with 1500 mg/kg bw/day, Six rats died during the dosing period as compared to control.

Clinical signs: Not specified

Body weight :
When treated with 1500 mg/kg bw/day, decrease in body weight gain was observed in treated rats as compared to control.

food consumption: No data available

Test substance intake: No data available

Reproductive function: estrous cycle: No effect on Post-implantation loss/dam, Total implantations/dam and Corpora lutea/dam were observed in treated rats as compared to control.

Reproductive function: sperm measures: No data available

Reproductive performance: There were no biologically meaningful or statistically significant differences in the number of litters and Foetal sex of treated rats as compared to control.

Organ weights No data available

Gross pathology: When treated with 1500 mg/kg bw/day, green discoloration of the amniotic fluid and green colored small intestines were observed in treated rats as compared to control.

When treated with 100 and 500 mg/kg bw/day, green discoloration of the amniotic fluid was observed in treated rats as compared to control.

Histopathology: No data available

other findings No data available

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

1 500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

reproductive function (oestrous cycle)

reproductive performance

Target system / organ toxicity (P0)

Critical effects observed:

not specified

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:

not specified

Dermal irritation (if dermal study):

not specified

Mortality:

no mortality observed

Description (incidence):

No effect on viability of foetuse were observed in treated rats as compared to control.

Results: F1 generation

General toxicity (F1)

Clinical signs:

not specified

Dermal irritation (if dermal study):

not specified

Mortality / viability:

no mortality observed

Description (incidence and severity):

No effect on viability of foetuse were observed in treated rats as compared to control.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No effect on foetal body weight was observed in treated rats as compareThere were no biologically meaningful or statistically significant differences in number of fetuses with malformations, number of foetuses or litters with developmental variations in any of the treated groups.d to control.

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Sexual maturation:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no biologically meaningful or statistically significant differences in number of fetuses with malformations, number of foetuses or litters with developmental variations in any of the treated groups.

Histopathological findings:

no effects observed

Description (incidence and severity):

When treated wtih 1500 mg/kg bw/day, slight increase in the number of litters with unossified sternebrae (sternebrae nos 1-6) and rudimentary 14th rib(s) was observed in feotus of treated rats as compared to control, but the observed effect fell within the ranges of historical control data.

Other effects:

not specified

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not specified

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not specified

Details on results (F1)

Mortality:
No effect on viability of foetuse were observed in treated rats as compared to control.

Clinical signs:
No data available

Body weight:
No effect on foetal body weight was observed in treated rats as compareThere were no biologically meaningful or statistically significant differences in number of fetuses with malformations, number of foetuses or litters with developmental variations in any of the treated groups.d to control.

Food consumption:
No data available

Test substance intake: No data available

Reproductive function: estrous cycle: No data available

Reproductive function: sperm measures: No data available

Reproductive performance: No data available

Organ weights: No data available

Gross pathology: There were no biologically meaningful or statistically significant differences in number of fetuses with malformations, number of foetuses or litters with developmental variations in any of the treated groups.

Histopathology: When treated wtih 1500 mg/kg bw/day, slight increase in the number of litters with unossified sternebrae (sternebrae nos 1-6) and rudimentary 14th rib(s) was observed in feotus of treated rats as compared to control, but the observed effect fell within the ranges of historical control data.

other findings: No data available

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

mortality

body weight and weight gain

gross pathology

histopathology: non-neoplastic

Target system / organ toxicity (F1)

Critical effects observed:

not specified

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Summary of maternal and foetal observations in study of rats given D&C Yellow No. 8 by gavage on days 6-19 of gestation

		Observations
	Dose (mg,'kg/day)	0 (control)			100			500			1500
Parameter		No.	%	SD	No.	%	SD	No.	%	SD	No.	%	SD
Animals on study		25	--	--	25	--	--	25	--	--	25	--	--
Animals that were gravid		25	100.0	--	21	84.0	--	21	84.0	--	24	96.0	--
Animals that died; gravid		0	0.0	--	0	0.0	--	0	0.0	--	6	24.0	--
Animals examined at caesarean section		25	100.0	--	25	100.0	--	25	100.0	--	19	76.0	--
Nongravid		0	0.0	--	4	16.0	--	4	16.0	--	1	5.3	--
Gravid
Dams with viable fetuses		25	100.0	--	21	84.0	---	21	84.0	---	18	94.7	--
Viable foetuses/dam		13.3	--	2.92	13.1	--	2.55	12.6	--	2.62	13.9	--	1.30
Post-implantation loss/dam		0.9	--	1.78	0.9	--	1.06	0.6	--	0.87	0.5	--	0.79
Total implantations/dam		14.2	--	2.24	14.0	--	2.16	13.1	--	2.89	14.4	--	1.10
Corpora lutea/dam		15.6	--	2.04	15.1	--	1.88	15.3	--	2.13	15.8	--	1.56
Foetal sex: males		178	53.6	--	148	52.9	--	130	49.2	--	118	47.0	--
Females		154	46.4	--	132	47.1	--	134	50.8	--	133	53.0	--
Mean foetal body weight (g)		3.5	--	0.29	3.5	--	0.36	3.5	--	0.36	3.5	--	0.45

SD = Standard deviation

None of the values differed significantly at P ≤ 0.05.

Summary of the incidence of malformations and developmental and genetic variations observed among foetuses from rats given D & C Yellow No. 8 by gavage on days 6-19 of gestation

		No. of foetuses (no. of litters)
Observations	Dose (mg,'kg/day)	0 (control)	100	500	1500
No. of litters examined		25	21	21	18
No. of foetuses examined externally		332	275	264	251
No. of foetuses examined viscerally		99	81	93	70
No. of foetuses examined skeletany		219	179	167	164
Malformations observed
Skull anomaly		2 (1)
Thoracoschisis		1 (1)
Gastroschisis		(1)
Sternoschisis		( 1 )
Bent ribs		1(1)	6 (3)
Radius bent				1 (1)
Carpal flexure		1 (1)
Foetal anasarca		2 (1)
Total foetuses (litters) with malformations:		3 (2)	6 (3)	1 (1)	0 (0)
Variations observed
27 presacral vertebrae		3 (2)		1(1)	2 (1)
14^thrudimentary rib(s)		27 (11)	23 (9)	17 (8)	49 (14)
14^thfull rib(s)		2 (1)			2 (1)
12 full pairs of ribs with bilateral 13^thrudimentary ribs		1 (1)
Skull reduced in ossification		4 (3)	4 (2)	5 (3)	4 (2)
Hyoid unossified		7 (5)	4 (3)	2 (1)	9 (3)
Vertebrae reduced in ossification		2 (I)
Femur reduced in ossification		1 (1)
Pubis unossified		2 (1)
lschium unossified		2 (1)
Femur unossified		1(1)
Metatarsals unossified		1(1)
Sternebrae no. 5 and/or no. 6 unossified		33 (9)	18 (8)	33 (12)	29 (11)
Other sternebrae unossified				1 (1)	9 (4)
Sternebrae misaligned				1 (1)	1 (1)
Major vessel variation				1 (1)
Renal papillae not developed and/or distended ureter		8(6)	2 (2)	1 (1)	2 (2)

 

Applicant's summary and conclusion

Conclusions:

Reproductive NOAEL was considered to be 1500 mg/kg body weight /day for F0 and F1 generation when CD Sprague-Dawley female rats were treated with D&C Yellow no. 8.

Executive summary:

In a Reproductive/Teratogenic toxicity study, CD Sprague-Dawley female rats were treated with D&C Yellow no. 8 in the concentration of 0, 100, 500 and 1500 mg/kg body weight/day by oral gavage.Six rats died during the dosing period and at 1500 mg/kg bw/day and Orange discoloration of the urine was observed in all the treated rats as compared to control. Similarly, green discoloration of the amniotic fluid and green colored small intestines were observed at 1500 mg/kg bw/day and green discoloration of the amniotic fluid was observed at 100 and 500 mg/kg bw/day treated rats as compared to control.No effect on Post-implantation loss/dam, Total implantations/dam, Corpora lutea/dam and There were no biologically meaningful or statistically significant differences in the number of litters and Foetal sex of treated rats were observed as compared to control. In addition, No effect on viability of foetuse, number of fetuses with malformations, number of foetuses or litters with developmental variations were observed in any of the treated groups.Slight increase in the number of litters with unossified sternebrae (sternebrae nos 1-6) and rudimentary 14^thrib(s) was observed in feotus of 1500 mg/kg bw/day treated rats as compared to control, but the observed effect fell within the ranges of historical control data. Therefore, Reproductive NOAEL was considered to be 1500 mg/kg body weight /day for F0 and F1 generation when CD Sprague-Dawley female rats were treated with D&C Yellow no. 8.