Xanthylium, 9-[2-(ethoxycarbonyl)phenyl]-3,6-bis(ethylamino)-2,7-dimethyl-, molybdatetungstatephosphate

Toxicity to reproduction

Administrative data

Endpoint:

reproductive toxicity, other

Remarks:

Teratogenicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

Data is from publication

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:

according to guideline

Guideline:

other: as below

Principles of method if other than guideline:

Teratogenic Potential study of D & C Yellow No. 8, sodium fluoresce in by Oral Administration in Rats

GLP compliance:

not specified

Limit test:

no

Justification for study design:

No data available

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

Details on test material:

- Name of test material : D & C Yellow No. 8, sodium fluorescein
- Molecular formula : C20H10Na2O5
- Molecular weight : 376.274 g/mol
- Substance type: Organic
- Physical state: No data

Specific details on test material used for the study:

- Name of test material : D & C Yellow No. 8, sodium fluorescein
- Molecular formula : C20H10Na2O5
- Molecular weight : 376.274 g/mol
- Substance type: Organic
- Physical state: No data

Test animals

Species:

rat

Strain:

Sprague-Dawley

Remarks:

CD

Details on species / strain selection:

No data available

Sex:

female

Details on test animals or test system and environmental conditions:

- Source:
Female rats: Charles River Breeding Laboratories,Portage, Michigan.
Male rats: Langshaw Farms, Augusta, Michigan
- Age at study initiation:18 weeks
- Weight at study initiation:No data
- Fasting period before study:No data
- Housing:The animals were housed in wire bottomed cages
- Diet (e.g. ad libitum):Purina Certified Rodent Chow No. 5002 ad libitum
- Water (e.g. ad libitum):Water ad libitum
- Acclimation period:4 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C):22.2±-15.5 ˚C
- Humidity (%):50± 15%
- Air changes (per hr):No data
- Photoperiod (hrs dark / hrs light):12 hrs light/dark cycle

IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:

oral: gavage

Type of inhalation exposure (if applicable):

not specified

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test chemical was mixed with vehicle at dose levels of 0, 100, 500 or 1500 mg/kg body weight and prepared daily

DIET PREPARATION
- Rate of preparation of diet (frequency):No data
- Mixing appropriate amounts with (Type of food):No data
- Storage temperature of food:No data

VEHICLE
- Justification for use and choice of vehicle (if other than water):No data
- Concentration in vehicle:0, 100, 500 or 1500 mg/kgbw
- Amount of vehicle (if gavage):10 mL/Kg
- Lot/batch no. (if required):No data
- Purity:No data

Details on mating procedure:

- M/F ratio per cage: 1: 1.
- 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 was designated as day 0 of gestation

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

14 days (days 6-19 of gestation)

Frequency of treatment:

Daily

Details on study schedule:

not specified

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

500 mg/kg bw/day

Doses / concentrations 4

Dose / conc.:

1 500 mg/kg bw/day

No. of animals per sex per dose:

Total: 100 females
0 mg/Kg bw: 25 females
100 mg/Kg bw: 25 females
500 mg/Kg bw: 25 females
1500 mg/Kg bw: 25 females

Control animals:

yes, concurrent vehicle

Details on study design:

not specified

Positive control:

not specified

Examinations

Parental animals: Observations and examinations:

Survival, clinical sign and body weights were examined.

Oestrous cyclicity (parental animals):

not specified

Sperm parameters (parental animals):

not specified

Litter observations:

sex and body weight were examined.

Postmortem examinations (parental animals):

Gross pathology were examined.

Postmortem examinations (offspring):

Gross external malformations and variations were examined.

Statistics:

Differences in the fetal sex distribution and the number of litters with malformations between control and treated groups wcre compared using the 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). 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 t\)etal weights were compared between groups by analysis of variance (oneway classification), Bartlett's test l\~r 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:

Viable and non-viable foetuses, early and late resorptions, total implantations and corpora lutea were examined.

Offspring viability indices:

not specified

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, Six rats died during the dosing period as compared to control.

Survival was 100% in the controls and the groups receiving 100 and 500 mg/kg of dye.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

When treated with 1500 mg/kg bw, slight reductions in body-weight gains as compared to controls, throughout the dosing period

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 noted in all treated rats during the treatment period.

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:

not specified

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

Description (incidence and severity):

No effect on Viable and non-viable foetuses, early and late resorptions, total implantations and corpora lutea and sex ratio of fetuses were observed as compared to control.

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

1 500 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

mortality

body weight and weight gain

urinalysis

gross pathology

reproductive performance

other: No effect observed

Remarks on result:

other: No effects on reproductive performance

Target system / organ toxicity (P0)

Critical effects observed:

not specified

System:

other: not specified

Organ:

not specified

Treatment related:

not specified

Dose response relationship:

not specified

Relevant for humans:

not specified

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 Viable and non-viable fetuses were observed as compared to control.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No effect on Body weight of fetuses were observed 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:

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):

When treated with 1500 mg/kg bw, A slight increase in the number of litters with unossified sternebrae (sternebrae nos 1-6) and rudimentary 14th rib(s) was observed however, these values fell within the ranges of historical control data.

No biologically meaningful or statistically significant differences in the number of litters and number of fetuses with malformations and number of fetuses or litters with developmental variations were observed in treated rats as compared to control.

Histopathological findings:

not specified

Other effects:

not specified

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not specified

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not specified

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 500 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

body weight and weight gain

gross pathology

other: No effect observed

Target system / organ toxicity (F1)

Critical effects observed:

not specified

System:

other: not specified

Organ:

not specified

Treatment related:

not specified

Dose response relationship:

not specified

Relevant for humans:

not specified

Overall reproductive toxicity

Reproductive effects observed:

not specified

Treatment related:

not specified

Relation to other toxic effects:

not specified

Dose response relationship:

not specified

Relevant for humans:

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

Parameter	Observation
	0(control)						100						500						1500
	No.		%		SD		No.		%		SD		No.		%		SD		No.		%		SD
Animals on study		25		-		-		25		84.0		-		25		-		25		25		-		-
Animals that were gravid		25		100.0		-		21		0.0		-		21		84.0		24		24		96.0		-
Animals that died gravid		0		0.0		-		0		100		-		0		0.0		6		6		24.0		-
Animals examined at caesarean section		25		100.0		-		25		16.0		-		25		100		19		19		76.0		-
Non-gravid		0		0.0		-		4		84.0		-		4		16		1		1		5.3		-
Gravid		25		100.0		-		21		-		2.55		21		84		18		18		94.7		-
Dams with viable foetuses		25		100.0		-		21		-		1.06		21		84		18		18		94.7		-
Viable foetuses/dam		13.3		-		2.92		13.1		-		2.16		12.6		-		13.9		13.9		-		1.30
Post-implantation loss/dam		0.9		-[		1.78		0.9		-		1.88		0.6		-		2.89		0.5		-		0.79
Total implantations/dam		14.2		-		2.24		14.0		-		-		13.1		-		2.13		14.4		-		1.10
Corpora lutea/dam		15.6		-		2.04		15.1		-		-		15.3		-		-		15.8		-		1.56
Foetal sex: male                     Female		178 154		53.6 46.4		-		148 132		52.9 47.1		-		130 134		49.2 50.8		-		118 133		47.0 53.0		-
Mean foetal body weight (g)		3.4		-		0.29		3.5		-		0.36		3.5		-		0.36		3.5		-		0.45

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

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

Applicant's summary and conclusion

Conclusions:

NOAEL was considered to be 1500 mg/kg/day for P and F1 generation when CD Sprague-Dawley female rats treated with D & C Yellow No. 8, sodium fluorescein orally by gavage for 14 days.

Executive summary:

In a Teratogenic Potential Test, CD Sprague-Dawley female rats treated with D & C Yellow No. 8, sodium fluorescein in the concentration of 0, 100, 500 or 1500 mg/kg bw orally by gavage in water for 14 days (days 6-19 of gestation). Six rats died during the dosing period at 1500 mg/kg bw as compared to control. Survival was 100% in the controls and the groups receiving 100 and 500 mg/kg of dye. Slight reductions in body-weight gains at 1500 mg/kg bw and Orange discoloration of the urine was noted in all treated rats during the treatment period. No effect on reproductive parameters such as Viable and non-viable fetuses, early and late resorptions, total implantations and corpora lutea and sex ratio of fetuses were observed in treated female rats as compared to control. Green discoloration of the amniotic fluid was observed in 1, 10 and 16 rats at 100, 500 and 1500 mg/kg/day groups, respectively, and the small intestines were green in colour in many rats at 500 mg/Kg group. In addition, No effect on Body weight of fetuses were observed as compared to control. A slight increase in the number of litters with unossified sternebrae (sternebrae nos 1-6) and rudimentary 14th rib(s) was observed at 1500 mg/kg bw however, these values fell within the ranges of historical control data. No biologically meaningful or statistically significant differences in the number of litters and number of fetuses with malformations and number of fetuses or litters with developmental variations were observed in treated rats as compared to control. Therefore, NOAEL was considered to be 1500 mg/kg/day for P and F1 generation when CD Sprague-Dawley female rats treated with D & C Yellow No. 8, sodium fluorescein orally by gavage for 14 days.