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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:
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:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
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
Sex:
female
Details on test animals 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 no treatment
yes, concurrent vehicle
Details on study design:
No data available
Positive control:
No data available

Examinations

Parental animals: Observations and examinations:
Body weigth were observed.
Oestrous cyclicity (parental animals):
Presence of cornified and epithelial cells of Estrous cyclicity were observed.
Sperm parameters (parental animals):
No data available
Litter observations:
Weight of fetuses, Dead, live or resorbed fetuses were recorded.
Postmortem examinations (parental animals):
Gross appearances were observed.

Postmortem examinations (offspring):
Sex, Gross skeletal and soft-part abnormalities were observed
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
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

Details on results (P0)

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 (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
other: No effect

Target system / organ toxicity (P0)

Critical effects observed:
not specified
System:
other: not specified
Organ:
not specified
Treatment related:
not specified
Dose response relationship:
not specified
Relevant for humans:
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:
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 (P1)

Critical effects observed:
not specified
System:
other: not specified
Organ:
not specified
Treatment related:
not specified
Dose response relationship:
not specified
Relevant for humans:
not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:
not specified
Dermal irritation (if dermal study):
not specified
Mortality / viability:
no mortality observed
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
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
effects observed, non-treatment-related
Histopathological findings:
not specified
Other effects:
not specified

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not specified

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not specified

Details on results (F1)

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.
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 (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
Treatment related:
not specified
Dose response relationship:
not specified
Relevant for humans:
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
Treatment related:
not specified
Dose response relationship:
not specified
Relevant for humans:
not specified

Overall reproductive toxicity

Reproductive effects observed:
not specified
Treatment related:
not specified
Relation to other toxic effects:
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,Water female rats treated with Green S in the concentration of0, 250, 500 and 1000 mg/kg/day orally by gavage. No effect was observed on body weight of treated rats as compared to control. Similarly,No effect were observed onnumber of corpora lutea, implantation sites, Pre-implantation losses, early resorptions, late resorptions and post-implantation losses of treated female rats as compared to control. No effect was observed on fetal weight as compared to control. In addition,Slightly green colouring in the gastro-intestinal tract and the placental tissue were observed in treated female rats. Green colouring of the gastro-intestinal tract and placenta were observed because Green S is largely unabsorbed.Statistically significant increase in fetuses with mucus in tracea were observed in 250, 500 and 1000 mg/kg/day and Ossification of proximal phalanges and fourth metacarpals of Skeletal tissue were observed in 500 and 1000 mg/kg/day treated fetoses as compared to control. This findings were observed at all dose levels and the incidence was not dose related. Despite this association with treatment, the nature of the finding does not indicate an adverse effect. Therefore,NOAEL was considered to be 1000 mg/kg/day for P and F1 generation when Water female rats treated with Green S orally by gavage for 19 days.