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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
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 peer reiviewed journal

Data source

Reference
Reference Type:
publication
Title:
Teratogenicity and embryotoxicity study of green s in rats
Author:
S. A. CLODE
Year:
1987
Bibliographic source:
Food Chemical Toxicology. Vol. 25, No. 12, pp. 995-997, 1987

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
other: OECD 422
Principles of method if other than guideline:
Combined repeated dose repro-devp. Screen of Green S (Hydrogen [4-[4-(dimethylamino)α-(2-hydroxy-3,6-disulphonato-1-naphthyl)benzylidene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium, monosodium salt) in rats
GLP compliance:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
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
Specific details on test material used for the study:
- Name of test material (as cited in study report): Green S (Wool Green BS; CI (1971) No. 44090; EEC E.142, monosodium salt of 4,4'-bis(dimethylamino)-diphenylmethylene-2-naphthol-3,6-disulphonic acid, sodium 5-[4-dimethylamino-~-(4-dimethyliminocyclohexa - 2,5 - dienylidene)benzyl] - 6-hydroxy-7 sulphonatonaphthalene-2-sulphonate))
- Molecular formula (if other than submission substance): C27H26N2O7S2.Na
- Molecular weight (if other than submission substance): 576.6 g/mole
- Substance type: Organic
- Physical state: No data available
- Impurities (identity and concentrations): Dye 82% (80%); volatile matter, 3.37% (4.9%); water insoluble matter, 0.01% (0.07%); sodium chloride, 1.4% (2.6%); sodium sulphate, 8.5% (6.0%); pH (of a 1% solution in distilled water), 5.4 (2.9); lead, < 5 ppm; copper, 4 ppm (2 ppm); chromium, 6 ppm (8 ppm); zinc, 9 ppm (5 ppm); iron, 30 ppm (35 ppm); cadmium, < 1 ppm; mercury, 0.2 ppm (< I ppm); free aromatic amines (as aniline), 29 ppm (17 ppm); Michler's hydrol, <0.01% (<0.02%); Michler's ketone, < 0.01%; R-acid, 0.11% (0.05%); subsidiary dyes, < 1%; ether extractable material, 0.14%.

Test animals

Species:
rat
Strain:
Wistar
Details on test animals 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:
Body weigth and gross appearances were observed
Ovaries and uterine content:
Presence of cornified and epithelial cells of Estrous cyclicity, number of corpora lutea, implantation sites, Pre-implantation losses, Early resorptions, Late resorptions and Post-implantation losses was recorded.
Fetal examinations:
Weight of fetuses, Sex, Gross skeletal and soft-part abnormalities were observed.
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
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
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not specified
Other effects:
not specified

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 specified
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 specified
Changes in number of pregnant:
not specified
Details on maternal toxic effects:
Body weight:
No statistically significant differences were observed in treated female rats as compared to control.

Reproductive performance:
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.

Gross pathology:
Slightly green colouring in the gastro-intestinal tract and the placental tissue were observed in treated female rats.

No other abnormalities were found in the tissues of treated female rats.

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
gross pathology
maternal abnormalities
number of abortions
pre and post implantation loss
total litter losses by resorption
early or late resorptions
dead fetuses
necropsy findings
other: No effect

Maternal abnormalities

Abnormalities:
not specified
Localisation:
not specified

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:
not specified
Changes in litter size and weights:
not specified
Changes in postnatal survival:
not specified
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Visceral malformations:
not specified
Other effects:
not specified
Details on embryotoxic / teratogenic effects:
Mortality:
No effect were observed on Live foetuses f trated female rats as compared to contorl.

Body weight:
No significant effect was observed on fetuses of treated female rats as compared to control.

Gross pathology:
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.

Histopathology: 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.

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
other: No effect

Fetal abnormalities

Abnormalities:
not specified
Localisation:
other: not specified

Overall developmental toxicity

Developmental effects observed:
not specified
Treatment related:
not specified
Relation to maternal toxicity:
not specified
Dose response relationship:
not specified
Relevant for humans:
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 Green S/kg/day 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 Green S/kg/day in 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 Water female rats treated with Green S.
Executive summary:

In a Combined repeated dose repro-devp. Screen, Wistar male and female rats treated with Green S in the concentration of 0, 50, 500 and 1000 mg/kg body weight/day orally in diet. In Parental generation, No effect were observed on survival, body weight, Food consumption, compound intake and water consumption of treated male and female rats as compared to control. Signs of respiratory distress, faeces, fur and extremities of all treated animals became impregnated or coated with the colouring were observed in treated and control. In addition, Significant decrease were observed in absolute stomach and empty caecum weight and increased in relative kidney weight in male and increased in absolute and relative empty and full caecum, spleen, and gonads weight in female and full caecum weight in male were observed at 1000 mg/kg body weight/day treated rats. Occasional focal inflammatory lesions were observed in the liver, heart, kidney and stomach in 1000 mg/kg bw/day and presence of colouring in wall of the uterus as a distinct ring around the placenta, and appeared to be confined to the amniotic membrane were observed in 500 and 1000 mg/kg bw/day treated female rats as compared to control. Significant increased in isolated foci or as a more widespread multifocal lesion associated with slight parenchymal necrosis and a portal inflammatory infiltrate of liver in female rats and statistically significantly increase in number of submucosal granulocytes and a slight vacuolation of overlying epithelium of stomach in male rats were observed at 1000 mg/kg bw/day as compared to control. In F1, F2 and F3 generation, Significantly decrease in number of live foetuses were observed in P generation and Slightly earlier incisors were observed in F1, F2 and F3 generation at 500 and 1000 mg/kg bw/day and in F2 and F3 generation at 50 mg/kg bw/day. This was associated with a large number of late resorptions in four animals. Significantly increased pups weight was observed in P and F1 generation at 50 mg/kg bw/day as compared to control. Both changes in P and F1 stages were associated with slightly smaller litters. Similarly, Minor variations in the degree of skeletal ossification were observed in alizarin-stained preparations, there were no differences that could be associated with treatment. Therefore, NOAEL was considered to be 500 mg/kg body weight/day for P, F1, F2 and F3 generation when Wistar male and female rats were treated with Green S orally in diet for 229 days.