Sodium 4-[(9,10-dihydro-4-hydroxy-9,10-dioxo-1-anthryl)amino]toluene-3-sulphonate

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 13 August 1999 to 24 September 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

Adopted 12 May 1981

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Sodium 4-[(9,10-dihydro-4-hydroxy-9,10-dioxo-1-anthryl)amino]toluene-3-sulphonate

EC Number:

224-618-7

EC Name:

Sodium 4-[(9,10-dihydro-4-hydroxy-9,10-dioxo-1-anthryl)amino]toluene-3-sulphonate

Cas Number:

4430-18-6

Molecular formula:

C21H15NO6S.Na

IUPAC Name:

sodium 2-[(4-hydroxy-9,10-dioxo-9,10-dihydroanthracen-1-yl)amino]-5-methylbenzenesulfonate

Test material form:

solid: particulate/powder

Remarks:

Dark Red Violet

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Batch No. 0609RA Lot no. AK0709
- Expiration date of the lot/batch: May 2001
- Purity test date: 18 March 1999

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:at room temperature, light and humidity protected
- Stability under test conditions: Stable for 4 hours in vehicle at room temperature
- Solubility and stability of the test substance in the solvent/vehicle: at maximum dose used : 100 mg/ml

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
The mixtures of the test article and vehicle were prepared daily before administration. The test article was weighed into a glass beaker on a tared precision balance and the vehicle added (w/v).The mixtures were prepared using a homogenizer. During the daily administration period, the homogeneity was maintened using a magnetic stirrer

Test animals

Species:

rat

Strain:

Wistar

Remarks:

WIST HanIbm

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: RCC Ltd. Biotechnology and Animal Breeding Division
- Age at study initiation: 11 weeks minimum
- Weight at study initiation: 191 to 243 grams
- Fasting period before study: not specified
- Housing: Animals were housed individually in Makrolon cages (type-3) with wire mesh tops and standardized granulated softwood beddings
- Diet (e.g. ad libitum): Pelleted standard Kliba 3433 rat/mouse maintenance diet (Provimi Kliba AG, Switzerland), ad libitum
- Water (e.g. ad libitum):tap water in bottles ad libitum
- Acclimation period: at least ten days prior to pairing under test conditions

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3 deg Celsius
- Humidity (%): 40+-70% relative humidity
- Air changes (per hr): 10-15 air changes per hour
- Photoperiod (hrs dark / hrs light):12h/12h with background music played at a centrally defined low volume for at least 8 hours during the light period

IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

1% diluted in bi-distilled water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

The mixtures of the test article and vehicle were prepared daily before administration. The test article was weighed into a glass beaker on a tared precision balance and the vehicle added (w/v).The mixtures were prepared using a homogenizer. During the daily administration period, the homogeneity was maintened using a magnetic stirrer

VEHICLE
- Justification for use and choice of vehicle (if other than water): no information
- Concentration in vehicle: 10, 30 and 100 mg/ml of test item in vehicle
- Amount of vehicle (if gavage): 10ml/kg
- Lot/batch no. (if required): no information
- Purity: no information

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

During the course of the study, samples for confirmation of concentration, homogeneity and stability were taken during the forst and the last week of the treatment period. Samples were taken immediately after preparation and again 4 hours later. Analysis were performed using a High Performance Liquid Chromatography HPLC method.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1
- Length of cohabitation: overnight
- Further matings after two unsuccessful attempts: no
- Verification of same strain and source of both sexes: not specified, supplied by RCC, no more details provided
- Proof of pregnancy: vaginal plug and copulation plug referred to as day 0 of pregnancy

Duration of treatment / exposure:

12 days ( day 6 to day 17 of pregnancy)

Frequency of treatment:

Daily

Duration of test:

17 days

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

22 females per dose were used

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: no information
- Rationale for animal assignment (if not random):computer-generated random algorithm

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily for signs of reaction to treatment and/or symptoms off ill heath

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: Body weight were recorded daily from day 0 until day 21 post coitum

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
Food consumption was recorded for the following periods : days 0-6, 6-12, 12-18, 18-21 post coitum
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No : calculated for grams/animal/day
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #21
- Organs examined: all internal organs, with emphasis on the uterus, uterine contents, position of the fetuses in the uterus and number of corpora lutea

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No

Fetal examinations:

- External examinations: Yes :all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes :half per litter
- Head examinations: No data

Statistics:

The following statistical methods were used to analyse the body weights, food consumption, reproduction and skeletal examination data:
-Means and standard deviations of various data were calculated and included in the report
-If the variables could be assumed to follow a normal distribution, The Dunnett many-one t-test, based on a pooled variance estimate, was used for intergroup commparisons (i.e. single treatment groups against the control group)
-The Steel test (many-one rank test) was applied when the data could not be assumed to follow a normal distribution
-Fisher's Exact test for 2x2 tables was applied if the variables could be dichotomized without loss of information.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

no effects observed

Description (incidence and severity):

Dark discolored feces was noted from day 7 post coitum (second day of the treatment) until day 18 post coitum il all females of the high dose level group. No further signs or reactions to treatment were observed in any female of any group.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No female died during the course of this study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The mean bodyweight gain in all groups was similar during the entire study. Further, the mean corrected body weight gain (corrected for uterus weight) gave no indication for test article-related effects.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no test article-related differences amongst the mean food consumption of group 1, and all the treated groups.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

In two females which had no fetuses in the high dose level group, the uterine horns contained fluid (a common finding in female rats of the strain and age). No further abnormal macroscopical findings were noted in group 4 and ni macroscopical changes were noted in the females of group 1, 2 or 3.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

One female of group 2 (100 mg/kg/day) had only empty implantation sites. These finding was considered to be incidental.

Description (incidence and severity):

a further female in group of 100 mg/kg/day only embryonic resoptions at caesarian section on day 21 post coitum. These finding was considered to be incidental.

Early or late resorptions:

not examined

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed

Changes in number of pregnant:

no effects observed

Other effects:

not examined

Details on maternal toxic effects:

There were 20 to 22 pregnant females per group. In the group who received 100 mg/kg/day one female had only embryonic resorptions and in the highest dose group two females were not pregnant, one female had only empty implantation sites and a further one only embryonic resorptions at Caesarean section. These findings were considered to be incidental as a dose relation was missing. No deaths were reported, and clinical signs were limited to discoloured faeces at 1000 mg/kg/day.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

mortality

pre and post implantation loss

total litter losses by resorption

Results (fetuses)

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

Effect observed but no significant.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not examined

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Description (incidence and severity):

In group 1 (control), 2 (100 mg/kg/day), 4 (1000 mg/kg/day) the sex ratio of fetuses was similar. In group 3 (300mg/kg/day), the number of males was increased and consequently the number of female reduced. The differences were within the range of th historical control data and considered to be incidental.

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

no effects observed

External malformations:

no effects observed

Description (incidence and severity):

In control gorup, one out 275 fetuses was noted with malformations as anal atresia and dysplastic tail.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

During skeletal examination of the fetuses, abnormal findings were noted in all groups (including control and treated group). The type and frequencies of the common abnormal findings (wavy ribs, rudimentary cervical ribs, dumbbell shaped thoracic vertebral body, abnormally shaped sternebrae) gave no indication of test article-related effects.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Only abnormal findings were observed in control group, group of 100 mg/kg/day and 1000 mg/kg/day as coagula in the head or thorax, and did not indicate test article-related effect and were considered to be incidental.

Other effects:

not examined

Details on embryotoxic / teratogenic effects:

No effects on litter parameters or foetal weight were observed. Foetal and litter incidences of external, soft tissue and skeletal anomalies were similar for control and treated groups.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

changes in sex ratio

fetal/pup body weight changes

external malformations

skeletal malformations

visceral malformations

Remarks on result:

not determinable due to absence of adverse toxic effects

Fetal abnormalities

Abnormalities:

no effects observed

Overall developmental toxicity

Developmental effects observed:

no

Any other information on results incl. tables

Table 1 :Summaryof reproductive data

			Group 1 0 mg/kg	Group 2 100 mg/kg	Group 3 300 mg/kg	Group 4 1000 mg/kg
	Number of dams	Mean	22	21	22	18
	Corpora Lutea	N	312	293	298	257
		Mean	14,2	14	13,5	257
		Standard deviation	1,8	1,8	1,8	1,5
	Pre-implantationloss	N	15	22	15	20
		% of corp.Lutea	4,8	7,5	5	7,8
		Mean	0,7	1	0,7	1,1
		Standard deviation	1,1	1,3	0,9	1,3
		Number of dams affected	8	10	9	10
	Implantation site	N	297	271	283	237
		% of corp.Lutea	95,2	92,5	95	92,2
		Mean	13,5	12,9	12,9	13,2
		Standard deviation	1,7	2	1,7	1,6
	Post-implantationloss	N	22	14	22	13
		% of implantation sites	7,4	5,2	7,8	5,5
		Mean	1	0,7	1	0,7
		Standard deviation	1,5	0,8	1,4	1,1
		Number of dams affected	11	10	11	7
	Implantation sitescars	N	0	0	0	0
	Embryonic/fetal deaths total	N	22	14	22	13
	Embryonic resorptions	N	20	14	21	11
		% of implantation sites	6,7	5,2	7,4	4,6
		Mean	0,9	0,7	1	0,6
		Standard deviation	1,5	0,8	1,5	1,1
		Number of dams affected	10	10	10	5
	Fetalresorptions		2	0	1	2
Fetuses	Totalfetuses	N	275	257	261	224
		% of implantation sites	92,6	94,8	92,2	94,5
		Mean	12,5	12,2	11,9	12,4
		Standard deviation	1,7	1,9	2,1	2,1
	Live fetuses	N	275	257	261	224
	Abnormal fetuses		1	0	0	0
		% offetuses	0,4
		Mean	0
		Standard deviation	0,2
		Numberofdamsaffected	1
		Abnormal live fetuses at external examination	1	0	0	0
		Abnormal dead fetuses at external examination	0	0	0	0
	Number of dams	N	22	21	22	18
	Sex of fetuses	Total male	129	124	143	100
		% offetuses	46,9	48,2	54,8#	44,6
		Mean	5,9	5,9	6,5	5,6
		Standard deviation	1,5	2,1	2,1	1,9
		totalfemale	146	133	118	124
		% offetuses	53,1	51,8	45,2#	55,4
		Mean	6,6	6,3	5,4	6,9
		Standard deviation	1,7	1,8	1,6	2,2
		Live males	129	124	143	100
		Livefemales	146	133	118	124
Weights of fetuses (litter basis)	Total fetuses	N (liters)	22	21	22	18
		Mean	4,6	4,7	4,8	4,7
		Standard deviation	0,3	0,3	0,3	0,3
	males	N (litters)	22	21	22	18
		Mean	4,7	4,8	4,9*	4,9
		Standard deviation	0,3	0,3	0,3	0,3
	females	N (litters)	22	21	22	18
		Mean	4,5	4,6	4,6	4,6
		Standard deviation	0,3	0,3	0,3	0,3
Weights of fetuses (fetuses basis)	total fetuses	n (fetuses)	275	257	261	224
		Mean	4,6	4,7**	4,8**	4,7**
		Standard deviation	0,4	0,4	0,4	0,4
	males	n (fetuses)	129	124	143	100
		Mean	4.7	4,9**	4,9**	4,9**
		Standard deviation	0,4	0,4	0,4	0,3
	females	n (fetuses)	146	133	118	124
		Mean	4,5	4,6**	4,6*	4,6
		Standard deviation	0,3	0,4	0,4	0,4

 */** Dunett's based on pooled variance significant at a level 5%* or 1%**

# Fisher's exact test significant at level 5% # or 1% ##

Applicant's summary and conclusion

Conclusions:

Under experimental condtion of this study, the registered substance External D&C Violet No. 2 did not induce toxic effect on rats and on embryo which were exposed during organogenesis. Hence, The No Observed Adverse Effect Level (NOAEL) for teratogenicity and maternal toxicity was defined as 1000mg/kg/day.

Executive summary:

The purpose of the GLP compliant study was to detect effects on the pregnant female rats and development of the embryo and fetus consequent to exposure of the female rats to the test item according OECD Guideline 414 method.

The test substance (in 1% carboxymethylcellulose in water) was given daily at dose volumes of 10 ml/kg bw by oral gavage. The doses were selected on the basis of the results of a preliminary study in rats. Maternal evaluations and measurements included daily clinical signs and body weight/food intake recorded at designated intervals. The females were killed on gestation day 21, subjected to macroscopic examination, and foetuses were removed by Caesarean section. Common litter parameters were recorded and foetuses were sexed, weighed and submitted to external examination. About one half

of the fetuses were also examined for soft tissue anomalies whereas remaining fetuses were examined for skeletal anomalies following alizarin red staining.

There were 20 to 22 pregnant females per group. In the group who received 100 mg/kg/day one female had only embryonic resorptions and in the highest dose group two females were not pregnant, one female had only empty implantation sites and a further one only embryonic resorptions at Caesarean section. These findings were considered to be incidental as a dose relation was missing. No deaths were reported, and clinical signs were limited to discoloured faeces at 1000mg/kg/day. No effects on litter parameters or foetal weight were observed. Foetal and litter incidences of external, soft, tissue and skeletal anomalies were similar for control and treated groups.

Under experimental condtion of this study, the registered substance External D&C Violet No. 2 did not induce toxic effect on rats and on embryo which were exposed during organogenesis. Hence, The No Observed Adverse Effect Level (NOAEL) for teratogenicity and maternal toxicity was defined as 1000mg/kg/day.