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EC number: 221-409-2 | CAS number: 3087-16-9
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
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- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Endpoint summary
Administrative data
Key value for chemical safety assessment
Effects on fertility
Description of key information
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.
Link to relevant study records
- 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
- Qualifier:
- equivalent or similar to guideline
- 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
- 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%. - 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 - 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 - 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
- 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.
- 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: oestrous cycle:
- not specified
- Reproductive function: sperm measures:
- not specified
- Reproductive performance:
- no effects observed
- 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
- 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
- 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: oestrous cycle:
- not specified
- Reproductive function: sperm measures:
- not specified
- Reproductive performance:
- not specified
- 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
- 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
- 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
- Behaviour (functional findings):
- not specified
- Developmental immunotoxicity:
- not specified
- 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
- 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
- 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
- Behaviour (functional findings):
- not specified
- Developmental immunotoxicity:
- not specified
- 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
- 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
- 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
- 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.
Reference
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.
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.
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.
Effect on fertility: via oral route
- Endpoint conclusion:
- no adverse effect observed
- Dose descriptor:
- NOAEL
- 1 000 mg/kg bw/day
- Study duration:
- subacute
- Species:
- rat
- Quality of whole database:
- Data is Klimisch 2 and from peer reviewed journal
Effect on fertility: via inhalation route
- Endpoint conclusion:
- no study available
Effect on fertility: via dermal route
- Endpoint conclusion:
- no study available
Additional information
Reproductive toxicity: Oral
In different studies, Hydrogen [4-[4-(dimethylamino)-α-(2-hydroxy-3,6-disulphonato-1-naphthyl)benzylidene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium, monosodium salt (Green S) has been investigated for reproductive toxicity based on in vivo experiments in rodents, i.e. most commonly in mice and rats for Hydrogen [4-[4-(dimethylamino)-α-(2-hydroxy-3,6-disulphonato-1-naphthyl)benzylidene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium, monosodium salt.
In a Combined repeated dose repro-devp. Screen by Clodeet al(Food Chemical Toxicology. Vol. 25, No. 12, pp. 995-997, 1987), Water female rats treated with Green S in the concentration of 0, 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 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. 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. These 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.
In another Three Generation reproduction toxicity study by Moorhouseet al(Food Chem. Toxic. Vol. 25, No. 12, pp. 985-993, 1987), 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 F0 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.
Also it is further supported by combined repeated dose carcinogenicity study by Brantomet al(Food Chem. Toxic. Vol. 25, No. 12, pp. 977-983, 1987), CD-1 male and female mice treated with Green S in the concentration of 0, 49.5, 495 and 530 or 660 mg/kg bw/day orally in diet. No effects were observed on survival, clinical sign and body weight of F0 and F1 treated mice as compared to control. Very few statistically significant differences were observed in hematology on 14, 28, 51 and 106 week in P and F1 mice, but the differences present did not occur consistently at all times of examination or in both sexes. Increase in absolute stomach weight was observed in female mice at 530 mg/kg bw dose but was not present when the weights were expressed relative to body weight. Similarly, Green coloration of the gastro-intestinal tract was observed in all the treated mice. In addition, Non – neoplastic hyperplasia in Caecum, Luminal casts in Epididymis, Focal epithelial hyperplasia of nephron in Kidney, Necrotic/inflammatory foci in Liver and oedema in rectum of P and F1 male mice and Pneumonia in Lung at 530 mg/kg bw and Hepatitis of Liver at 495 mg/kg bw in F1 female mice were observed. Neoplastic cyst adenoma in Harderian Gland, Interstitial-cell tumors in Testes were observed in F1 male mice as compared to control. The total numbers of tumours and animals with tumours, calculated for both malignant and benign tumours show no positive dose-related trends. Therefore, NOAEL was considered to be 530 mg/kg bw for femlae and 660 mg/kg body weight/day in males for P and F1 generation when CD-1 male and female mice treated with Green S.
Thus, based on the above studies on Hydrogen [4-[4-(dimethylamino)-α-(2-hydroxy-3,6-disulphonato-1-naphthyl)benzylidene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium, monosodium salt, it can be concluded that NOAEL value is 1000 mg/kg bw. Thus, comparing this value with the criteria of CLP regulation, Hydrogen [4-[4-(dimethylamino)-α-(2-hydroxy-3,6-disulphonato-1-naphthyl)benzylidene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium, monosodium salt can be can be considered as Not Classified for reproductive toxicity.
Effects on developmental toxicity
Description of key information
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.
Link to relevant study records
- 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
- Qualifier:
- equivalent or similar to guideline
- 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
- 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%. - 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 - 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
- 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
- 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
- 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
- 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. - 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
- other: No effect
- Abnormalities:
- not specified
- Localisation:
- not specified
- 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. - 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
- Abnormalities:
- not specified
- Localisation:
- other: not specified
- 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
- 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.
Reference
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.
Effect on developmental toxicity: via oral route
- Endpoint conclusion:
- no adverse effect observed
- Dose descriptor:
- NOAEL
- 1 000 mg/kg bw/day
- Study duration:
- subacute
- Species:
- rat
- Quality of whole database:
- Data is Klimisch 2 and from peer reviewed journal
Effect on developmental toxicity: via inhalation route
- Endpoint conclusion:
- no study available
Effect on developmental toxicity: via dermal route
- Endpoint conclusion:
- no study available
Additional information
Developmental toxicity: Oral
In different studies, Hydrogen [4-[4-(dimethylamino)-α-(2-hydroxy-3,6-disulphonato-1-naphthyl)benzylidene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium, monosodium salt (Green S) has been investigated for developmental toxicity based on in vivo experiments in rodents, i.e. most commonly in mice and rats for Hydrogen [4-[4-(dimethylamino)-α-(2-hydroxy-3,6-disulphonato-1-naphthyl)benzylidene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium, monosodium salt.
In a Combined repeated dose repro-devp. Screen by Clodeet al(Food Chemical Toxicology. Vol. 25, No. 12, pp. 995-997, 1987), Water female rats treated with Green S in the concentration of 0, 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 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. 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. These 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.
In another Three Generation reproduction toxicity study by Moorhouseet al(Food Chem. Toxic. Vol. 25, No. 12, pp. 985-993, 1987), 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.
Also it is further supported by combined repeated dose carcinogenicity study by Brantomet al(Food Chem. Toxic. Vol. 25, No. 12, pp. 977-983, 1987), CD-1 male and female mice treated with Green S in the concentration of 0, 49.5, 495 and 530-660 mg/kg bw/day orally in diet. No effects were observed on survival, clinical sign and body weight of F0 and F1 treated mice as compared to control. Very few statistically significant differences were observed in hematology on 14, 28, 51 and 106 week in P and F1 mice, but the differences present did not occur consistently at all times of examination or in both sexes. Increase in absolute stomach weight was observed in female mice at 530 mg/kg bw dose but was not present when the weights were expressed relative to body weight. Similarly, Green coloration of the gastro-intestinal tract was observed in all the treated mice. In addition, Non – neoplastic hyperplasia in Caecum, Luminal casts in Epididymis, Focal epithelial hyperplasia of nephron in Kidney, Necrotic/inflammatory foci inLiver and oedema in rectum of F0 and F1 male mice and Pneumonia in Lung at 530 mg/kg bw and Hepatitis of Liver at 495 mg/kg bw in F1 female mice were observed. Neoplastic cyst adenoma in Harderian Gland, Interstitial-cell tumors in Testes were observed in F1 male mice as compared to control. The total numbers of tumours and animals with tumours, calculated for both malignant and benign tumours show no positive dose-related trends. Therefore, NOAEL was considered to be 530 mg/kg bw for female and 660 mg/kg body weight/day in males for P and F1 generation when CD-1 male and female mice treated with Green S. orally in diet for 2 years
Thus, based on the above studies on Hydrogen [4-[4-(dimethylamino)-α-(2-hydroxy-3,6-disulphonato-1-naphthyl)benzylidene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium, monosodium salt, it can be concluded that NOAEL value is 1000 mg/kg bw for developmental toxicity. Thus, comparing this value with the criteria of CLP regulation, Hydrogen [4-[4-(dimethylamino)-α-(2-hydroxy-3,6-disulphonato-1-naphthyl)benzylidene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium, monosodium salt can be can be considered as Not Classified for developmental toxicity.
Justification for classification or non-classification
Based on the above studies on Hydrogen [4-[4-(dimethylamino)-α-(2-hydroxy-3,6-disulphonato-1-naphthyl)benzylidene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium, monosodium salt, it can be concluded that NOAEL value is 1000 mg/kg bw for reproduction and developmental toxicity. Thus, comparing this value with the criteria of CLP regulation, Hydrogen [4-[4-(dimethylamino)-α-(2-hydroxy-3,6-disulphonato-1-naphthyl)benzylidene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium, monosodium salt can be can be considered as Not Classified for reproduction and developmenal toxicity.
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