Dihydrogen (ethyl)[4-[4-[ethyl(3-sulphonatobenzyl)]amino]-2'-sulphonatobenzhydrylidene]cyclohexa-2,5-dien-1-ylidene](3-sulphonatobenzyl)ammonium, disodium salt

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1972

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1972

Report date:

1972

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

treatment from day 6 until day 15 post mating (caesarean section on day 20); no clinical observations recorded, no examination of food consumption; no examination of maternal thyroid glands; no measurement of anogenital distance (AGD) in live fetuses

Principles of method if other than guideline:

Teratology study in rats. Rats were treated from day 6 through day 15 of gestation (day 0 = day of mating). The surviving rats were sacrificed on day 20 and the corpora lutea of pregnancy and uterine contents were carefully recorded. All fetuses were carefully examined for malformations. Approximately two-thirds of the fetuses were cleared and their skeletons stained with Alizarin Red for visuallzation of skeletal ossification variations and anomalies. The remaining one-third were fixed in Bouin's solution and examined by the slicing method of Wilson.

GLP compliance:

no

Remarks:

pre-GLP study

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Dihydrogen (ethyl)[4-[4-[ethyl(3-sulphonatobenzyl)]amino]-2'-sulphonatobenzhydrylidene]cyclohexa-2,5-dien-1-ylidene](3-sulphonatobenzyl)ammonium, disodium salt

EC Number:

223-339-8

EC Name:

Dihydrogen (ethyl)[4-[4-[ethyl(3-sulphonatobenzyl)]amino]-2'-sulphonatobenzhydrylidene]cyclohexa-2,5-dien-1-ylidene](3-sulphonatobenzyl)ammonium, disodium salt

Cas Number:

3844-45-9

Molecular formula:

C37 H34 N2 Na2 O9 S3

IUPAC Name:

disodium 2-({4-[ethyl(3-sulfonatobenzyl)amino]phenyl}{4-[ethyl(3-sulfonatobenzyl)iminio]cyclohexa-2,5-dien-1-ylidene}methyl)benzenesulfonate

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Supplier: Warner-Jenkinson Manufacturing Company
- Lot number: CC1C-8

Test animals

Species:

rat

Strain:

Long-Evans

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Diet: commercial rat food ad libitum
- Water: ad libitum
- Acclimation period: suitable equilibration period

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 0.5% methylcellulose

Details on exposure:

- administered volume: 10 mL/kg bw

Details on mating procedure:

- M/F ratio per cage: 2 males / 2 females
- Length of cohabitation: nightly
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0

Duration of treatment / exposure:

day 6 - day 15 of gestation

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

200 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

600 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

2 000 mg/kg bw/day

No. of animals per sex per dose:

66 dams in control group (3 groups á 22 dams)
22 dams (positive control)
24 dams (200 mg/kg bw)
22 dams (600 mg/kg bw)
22 dams (2000 mg/kg bw)

Control animals:

yes

Examinations

Maternal examinations:

BODY WEIGHT:
- Time schedule for examinations: days 0, 6, 15 and 20

SACRIFICE
- All females were sacrificed on day 20 by chloroform inhalation.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: not specified
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes (all fetuses)
- Soft tissue examinations: Yes (approx. one-third of the fetuses)
- Skeletal examinations: Yes (approx. two-thirds of the fetuses)
- Body weight: Yes (all fetuses)
- Sex: Yes (all fetuses)

Statistics:

Statistical comparisons between control and test groups were made where applicable by the chi-square method or the t-test, including appropriate adjudgement if the variances were significantly different (F-test).

Indices:

Pregnancy rate and implantation efficiency was calculated according to the formulas in section "Any other information on materials and methods incl. tables"

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

not specified

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

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:

not specified

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not examined

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

As treatment was not initiated until day 6, no treatment effect was expected or observed upon pregnancy rate.

Other effects:

no effects observed

Details on maternal toxic effects:

The positive control group had a statistically significant increase in resorptions when compared to the negative control groups.

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

2 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: No adverse effect up to and including the highest dose.

Maternal abnormalities

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The incidences of fetal malformations and number of litters with malformations in the mid dose level (600 mg/kg bw/d) was statistically significantly greater than the combined control. These differences were not considered compound-related, as most of the malformations were cases of hydronephrosis, a not incommon finding in the testing laboratory.

Other effects:

no effects observed

Details on embryotoxic / teratogenic effects:

The positive control group had a statistically significant increase in malformed fetuses when compared to all of the negative control groups. In addition, the number of Trypan Blue treated dams with malformed fetuses was statistically significantly greater. The anomalies observed in the Trypan Blue treated group are typical of those reported to be induced by Trypan Blue administration which demonstrates the susceptibility of the strain of rats used to a known teratogen.

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

2 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: No adverse effects observed up to and including the highest dose

Fetal abnormalities

Abnormalities:

no effects observed

Overall developmental toxicity

Developmental effects observed:

no

Any other information on results incl. tables

Table 1: Reproduction and fetal data

Group	No. pregnant females	Mean no. corpora lutea	Mean no. implantations	Implantation efficiencies (%)	Mean no. viable fetuses	Mean no. resorptions	Females with one or more resorptions		Mean fetal weight (g)	Pregnancy rate (%)	Fetal sex ratio (M/F)
							Total	%
C-I-III	63	13.3	12.2	91.4	11.4	0.73	21	33.3	3.584	95.5	0.93
PC-I93	22	12.9	12.1	94.2	6.8	5.27**	7	100	3.579	100	0.74
T-I	22	12.9	11.8	91.5	11.3	0.5	7	31.8	3.579	91.7	0.96
T-II	21	13.2	12.2	97.3	11.6	0.67	11	52.4	3.713	95.5	0.85
T-III	22	12.9	12	93.3	11.5	0.5	7	31.8	3.611	100	0.78

* statistically significantly different from C-I-III at p = 0.01

C = control

PC = positive control

T = treatment (T-I: 200 mg/kg; T-II: 600 mg/kg; T-III: 2000 mg/kg)

Table 2:

		Fetuses with ossification variations
	Number of full-term fetuses examined skeletally	number	%	Number of fetuses with gross malformations
Control I	159	121	76	16
Control II	158	99	63	17
Control III	163	111	68	12
C I+II+III	480	331	69	45
Positive control I	102	83	81	32**
T-I (200 mg/kg bw)	166	116	70	16
T-II (600 mg/kg bw)	163	98	60	30**
T-III (2000 mg/kg bw)	171	102	60	19

Applicant's summary and conclusion

Executive summary:

The test substance was administered orally at doses of 200, 600 and 2000 mg/kg bw/day to mated, sexually mature, female Long-Evans rats from day 6 through day 15 of gestation (day 0 = day of mating). Three groups of vehicle controls as well as a positive control group recieving an aqueous solution of Trypan Blue (30 mg/kg bw/day) (subcutaneously) were included in the study.

Rats which survived the duration of the experiment were sacrificed on day 20 and the corpora lutea of pregnancy and uterine contents were carefully recorded. All fetuses were carefully examined for malformations. Approximately two-thirds of the fetuses were cleared and their skeletons stained with Alizarin Red for visualization of skeletal ossification variations and anomalies. The remaining one-third were fixed in Bouin's solution and examined by the slicing method of Wilson.

No signs of fetal toxicity or anomalies were observed within the test substance treated groups. Thus, the test substance is adjudged to be non-fetal-toxic and non-teratogenic under the conditions of this experiment.