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Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
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 peer reiviewed journal

Data source

Reference
Reference Type:
publication
Title:
Teratogenicity and embryotoxicity study in rats
Author:
S. A. CLODE
Year:
1987
Bibliographic source:
Food Chemical Toxicology, 1987

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: as mentioned below
Principles of method if other than guideline:
Teratogenicity and embryotoxicity study of the test chemical was performed on rats
GLP compliance:
not specified
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Hydrogen [4-[4-(dimethylamino)-α-(2-hydroxy-3,6-disulphonato-1-naphthyl)benzylidene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium, monosodium salt
EC Number:
221-409-2
EC Name:
Hydrogen [4-[4-(dimethylamino)-α-(2-hydroxy-3,6-disulphonato-1-naphthyl)benzylidene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium, monosodium salt
Cas Number:
3087-16-9
Molecular formula:
C27H26N2O7S2.Na
IUPAC Name:
Hydrogen [4-[4-(dimethylamino)-α-(2-hydroxy-3,6-disulphonato-1-naphthyl)benzyli dene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium, monosodium salt
Test material form:
solid
Details on test material:
- Name of test material (as cited in study report): Hydrogen [4-[4-(dimethylamino)-α-(2-hydroxy-3,6-
disulphonato-1-naphthyl)benzylidene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium, monosodium
salt
- Molecular formula :C27H26N2O7S2.Na
- Molecular weight: 576.623 g/mol
- Substance type:Organic
- Physical state:Solid

Test animals

Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Specified-pathogen-free colony (Olac (1976) Ltd, Bicester, Oxon)
- Age at study initiation: No data available
- Weight at study initiation: No data available
- Fasting period before study: No data available
- Housing: Animals were housed in five per caged till 7 weeks and then individually.
- Diet (e.g. ad libitum): Spratt's Laboratory Diet No. 5, ad libitum
- Water (e.g. ad libitum): Tap-water, ad libitum
- Acclimation period: 7 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 2°C
- Humidity (%): 40-60%
- Air changes (per hr): No data available
- Photoperiod (hrs dark / hrs light): No data available

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 exposure:
PREPARATION OF DOSING SOLUTIONS: The dosing solutions were prepared at concentrations 0, 250, 500 and 1000 mg/kg such that a volume of 10 ml/kg was administered daily.

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

VEHICLE
- Justification for use and choice of vehicle (if other than water): Water
- Concentration in vehicle: 0, 250, 500 and 1000 mg/kg/day
- Amount of vehicle (if gavage): 10 ml/kg
- Lot/batch no. (if required): No data available
- Purity: No data available
Analytical verification of doses or concentrations:
not specified
Details on mating procedure:
- M/F ratio per cage: 1 : 1 ratio
- Length of cohabitation: Overnight
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 / day 1] of pregnancy Sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): Individually
- Any other deviations from standard protocol: This procedure was repeated over several consecutive days until at least 30 females had been allocated to each treatment group.
Duration of treatment / exposure:
19 days
Frequency of treatment:
Daily
Duration of test:
19 days
Doses / concentrations
Remarks:
0, 250, 500 and 1000 mg/kg bw/day
No. of animals per sex per dose:
Total: 120
0 mg/kg/day: 30 female
250 mg/kg/day: 30 female
500 mg/kg/day: 30 female
1000 mg/kg/day: 30 female
Control animals:
yes, concurrent vehicle
Details on study design:
No data available

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes / No / No data:
- Time schedule:
- Cage side observations checked in table [No.?] were included.

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

BODY WEIGHT: Yes / No / No data: yes
- Time schedule for examinations:Each animal was weighed on alternate days, commencing on day 0 of pregnancy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes / No / No data
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes / No / No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes / No / No data
- Time schedule for examinations:

POST-MORTEM EXAMINATIONS: Yes / No / No data: yes
- Sacrifice on gestation day # : On day 20 of pregnancy, 30 females from each group were killed by cervical dislocation and a postmortem examination was carried out
- Organs examined: a postmortem examination was carried out, during which the gross appearance of the maternal tissues was noted and the foetuses were removed. The numbers of corpora lutea and implantation sites and the numbers and positions of the sites with dead, live or resorbed foetuses were recorded.

OTHER: At 7 wk of age vaginal smears were prepared and examined for each female. Those females in pro-oestrus (indicated by the presence of mainly cornified and epithelial cells) were caged
individually overnight with a mature male.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes / No / No data: yes
Examinations included:
- Gravid uterus weight: Yes / No / No data: yes
- Number of corpora lutea: Yes / No / No data: yes
- Number of implantations: Yes / No / No data: yes
- Number of early resorptions: Yes / No / No data: yes
- Number of late resorptions: Yes / No / No data: yes
Fetal examinations:
- External examinations: Yes: [all per litter / half per litter / #? per litter ] / No / No data: yes
- Soft tissue examinations: Yes: [all per litter / half per litter / #? per litter ] / No / No data: tes
- Skeletal examinations: Yes: [all per litter / half per litter / #? per litter ] / No / No data: yes
- Head examinations: Yes: [all per litter / half per litter / #? per litter ] / No / No data: no data available
Statistics:
No data available
Indices:
Viability of fetuses were observed.
Historical control data:
No data available

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
not specified
Dermal irritation (if dermal study):
not specified
Mortality:
not specified
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no statistically significant differences in body weight between treated and control females
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
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
At autopsy, all the maternal animals given the test chemical were found to have colouring in the gastro-intestinal tract and the placental tissue also appeared to the slightly green. Apart from this,
no abnormalities were found in the tissues of the maternal animals.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no differences between the control and treated groups in the results of the examination of the reproductive system and the foetuses
Histopathological findings: neoplastic:
not specified
Other effects:
no effects observed
Description (incidence and severity):
No effect were observed on number of corpora lutea, implantation sites, Pre-implantation losses, early resorptions, late resorptions and post-implantation losses of treated female rats as compared to control.

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
No effect were observed on number of corpora lutea, implantation sites, Pre-implantation losses, early resorptions, late resorptions and post-implantation losses of treated female rats as compared to control.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
No effect were observed on the number of Pre-implantation and post-implantation losses of treated female rats as compared to control.
Total litter losses by resorption:
not specified
Description (incidence and severity):
no data available
Early or late resorptions:
no effects observed
Description (incidence and severity):
No effect were observed on number of early resorptions, late resorptions of treated female rats as compared to control.
Dead fetuses:
no effects observed
Description (incidence and severity):
No effect were observed on number of corpora lutea, implantation sites, Pre-implantation losses, early resorptions, late resorptions and post-implantation losses of treated female rats as compared to control.
Changes in pregnancy duration:
not specified
Changes in number of pregnant:
not specified
Other effects:
not specified

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
body weight and weight gain
dead fetuses
early or late resorptions
gross pathology
maternal abnormalities
necropsy findings
number of abortions
pre and post implantation loss
total litter losses by resorption

Maternal abnormalities

Abnormalities:
not specified
Localisation:
not specified

Results (fetuses)

Fetal body weight changes:
not specified
Reduction in number of live offspring:
not specified
Changes in sex ratio:
not specified
Changes in litter size and weights:
not specified
Changes in postnatal survival:
not specified
External malformations:
no effects observed
Description (incidence and severity):
a lack of teratogenic effect was indicated by the absence of any gross malformations of severe defects in the examination of the skeletal or soft parts.
Skeletal malformations:
no effects observed
Description (incidence and severity):
a lack of teratogenic effect was indicated by the absence of any gross malformations of severe defects in the examination of the skeletal parts.
Visceral malformations:
no effects observed
Other effects:
not specified
Description (incidence and severity):
Ossification of proximal phalanges and fourth metacarpals of Skeletal tissue were observed in 500 and 1000 mg/kg/day treated fetuses as compared to contorl. Despite this association with treatment, the nature of the finding does not indicate an adverse effect.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
fetal/pup body weight changes
external malformations
skeletal malformations

Fetal abnormalities

Abnormalities:
not specified
Localisation:
other: not specified

Overall developmental toxicity

Developmental effects observed:
no
Treatment related:
no

Any other information on results incl. tables

Table 1.Results of the examination of the reproductive system and foetuses of female rats given 0-1000mg/kg/day test chemical in aqueous solution, by oral intubation, throughout pregnancy

 

Mean no. (value)/pregnant female given doses (mg/kg/day) of:

Observation

0

250

500

1000

Corpora lutea

12.13

11.57

11.67

12.03

Implantations

10.60

10.93

10.13

10.83

Pre-implantation losses

1.57

0.64

1.54

1.20

Early resorptions

0.50

0.60

0.40

0.57

Late resorptions

0.03

0.13

0.17

0.13

Post-implantation losses

0.53

0.73

0.57

0.70

Live foetuses

10.07

10.20

9.53

10.13

Litter weight (g)

34.2

34.7

33.6

35.9

Mean foetal weight (g)

3.40

3.42

3.49

3.53

The results are means for groups of 30 females. There were no significant differences between the control and test groups (the exact test of Fisher, 1934): P > 0.05.

Table 2. Incidence of statistically significant findings in rat pups from females given 0-1000mg/kg/day test chemicalin aqueous solution, by oral intubation, throughoutpregnancy

Finding

Dose level (mg/kg/day)...

No. of pups affected

 

 

0

250

500

1000

Visceral findings

 

 

 

 

 

 

No. of pups examined...

 

138

137

133

136

Mucus in tracea

 

3

11*

13**

13**

Skeletal findings

 

 

 

 

 

No. of pups examined...

 

131

134

129

135

Proximal phalanges ossified

 

23

19

35

38*

Metacarpals ossified

 

53

56

71*

82***

Values marked with asterisks differ significantly (chi-square test) from those of the control: *P <0.05; **P < 0.01; ***P < 0.001.

Applicant's summary and conclusion

Conclusions:
NOAEL was considered to be 1000 mg/kg/day for P and F1 generation when Wistar female rats treated with the test chemical
Executive summary:

Teratogenicity and embryotoxicity study of the test chemical was performed on rats. Daily oral doses of 0 (control), 250, 500 or 1000 mg/kg body weight were given to groups of 30 pregnant Wistar rats on days 0-19 of pregnancy.The dosing solutions were prepared at concentrations such that a volume of 10ml/kg was administered daily. The controls received water. Each animal was weighed on alternate days, commencing on day 0 of pregnancy. On day 20 of pregnancy, 30 females from each group were killed by cervical dislocation and a postmortem examination was carried out, during which the gross appearance of the maternal tissues was noted and the foetuses were removed. The numbers of corpora lutea and implantation sites and the numbers and positions of the sites with dead, live or resorbed foetuses were recorded. The live foetuses were weighed and examined for gross abnormalities.This treatment did not adversely influence maternal body weight, the numbers of implanations, pre- or post-implantation losses or of live foetuses, the sex ratio or the weight of the litters or foetuses. No definite abnormalities were seen and the only finding in the examination of stained skeletons was a slightly more advanced ossification of the forelimbs of the offspring from females given 500 or 1000 mg /kg/day. More foetuses with mucus in the trachea were found in the treated groups than in the controls but this was not considered to be a teratogenic effect. Thus no embryotoxic or teratogenic effects were detected with doses of up to 1000 mg/kg/day throughout pregnancy. NOAEL was considered to be 1000 mg/kg/day for P and F1 generation when Wistar female rats treated with the test chemical