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Toxicological information

Toxicity to reproduction

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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 peer reviewed 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
Sex:
female
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: (P) x wks; (F1) x wks: No data available
- Weight at study initiation: (P) Males: x-x g; Females: x-x g; (F1) Males: x-x g; Females: x-x g: 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

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
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.
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
19 days
Frequency of treatment:
Daily
Details on study schedule:
- Dose selection rationale: No data available
- Rationale for animal assignment (if not random): The day of sperm detection was designated day 0 of pregnancy and the mated females were randomly allocated to one of four treatment groups. This procedure was repeated over several consecutive days until at least 30 females had been allocated to each treatment group.
- Rationale for selecting satellite groups: No data available
- Post-exposure recovery period in satellite groups: No data available
- Section schedule rationale (if not random): No data available
Doses / concentrations
Remarks:
0, 250, 500 and 1000 mg/kg/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
Positive control:
No data available

Examinations

Parental animals: Observations and examinations:
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.
Oestrous cyclicity (parental animals):
The numbers of corpora lutea were recorded
Sperm parameters (parental animals):
No data available
Litter observations:
no data available
Postmortem examinations (parental animals):
Gross appearances were observed.

Postmortem examinations (offspring):
- 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

SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at [#?] days of age. : 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
Statistics:
No data available

Reproductive indices:
Number of corpora lutea, implantation sites, Pre-implantation losses, Early resorptions, Late resorptions and Post-implantation losses was recorded.
Offspring viability indices:
Viability of fetuses were observed.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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):
No statistically significant differences were observed in treated female 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:
not specified
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 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.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
body weight and weight gain
gross pathology
histopathology: non-neoplastic
reproductive performance

Target system / organ toxicity (P0)

Critical effects observed:
not specified
System:
other: not specified
Organ:
not specified

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:
not specified
Dermal irritation (if dermal study):
not specified
Mortality:
not specified
Body weight and weight changes:
not specified
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:
not specified
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not specified
Other effects:
not specified

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:
not specified
Reproductive function: sperm measures:
not specified
Reproductive performance:
not specified

Effect levels (P1)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive function (oestrous cycle)
reproductive performance

Target system / organ toxicity (P1)

Critical effects observed:
not specified
System:
other: not specified
Organ:
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 significant effect was observed on fetuses of treated female rats as compared to control.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No significant effect was observed on fetuses of treated female 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:
not specified
Sexual maturation:
not specified
Anogenital distance (AGD):
not specified
Nipple retention in male pups:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Statistically significant increase in fetuses with mucus in tracea were observed in 250, 500 and 1000 mg/kg/day treated female rats as compared to control.
This findings were observed at all dose levels and the incidence was not dose related.Ossification of proximal phalanges and fourth metacarpals of Skeletal tissue were observed in 500 and 1000 mg/kg/day treated fetoses as compared to contorl. Despite this association with treatment, the nature of the finding does not indicate an adverse effect.
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:
1 000 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

Target system / organ toxicity (F1)

Critical effects observed:
not specified
System:
other: not specified
Organ:
not specified

Results: F2 generation

General toxicity (F2)

Clinical signs:
not specified
Dermal irritation (if dermal study):
not specified
Mortality / viability:
not specified
Body weight and weight changes:
not specified
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:
not specified
Histopathological findings:
not specified
Other effects:
not specified

Developmental neurotoxicity (F2)

Behaviour (functional findings):
not specified

Developmental immunotoxicity (F2)

Developmental immunotoxicity:
not specified

Effect levels (F2)

Dose descriptor:
other: not specified
Generation:
other: not specified
Based on:
not specified
Sex:
not specified
Basis for effect level:
other: not specified
Remarks on result:
other: not specified

Target system / organ toxicity (F2)

Critical effects observed:
not specified
System:
other: not specified
Organ:
not specified

Overall reproductive toxicity

Reproductive effects observed:
no
Treatment related:
not specified

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 chemical in aqueous solution, by oral intubation, throughout pregnancy

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 fetuses 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.