Hydrogen [4-[4-(dimethylamino)-α-(2-hydroxy-3,6-disulphonato-1-naphthyl)benzylidene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium, monosodium salt

Endpoint summary

Description of key information

Combined repeated dose repro-devp. screen was performed for Green S orally in female rats. Wistar female rats were treated with Green S in the concentration of 0, 250, 500 and 1000 mg/kg/day orally by gavage for 19 days. The animals were observed for changes in body weight and gross pathology. No effect was observed on body weight of treated rats as compared to control. Similarly, no effects were observed on reproductive system and the foetuses of treated female rats. 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. Therefore, No Observed Adverse Effect level (NOAEL) was considered to be 1000 mg/kg/day when Wistar female rats were treated with Green S orally by gavage for 19 days.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

repeated dose toxicity: oral, other

Remarks:

Combined repeated dose repro-devp. Screen

Type of information:

experimental study

Adequacy of study:

key study

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 publication

Qualifier:

according to guideline

Guideline:

other: Refre below principle

Principles of method if other than guideline:

Combined repeated dose repro-devp. Screen was performed for Green S orally in female rats

GLP compliance:

not specified

Limit test:

no

Specific details on test material used for the study:

- Name of the test material: Green S
- EC name: 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
- Smiles: c12c(\C(c3ccc(N(C)C)cc3)=C3/C=C\C(=[N+](/C)C)C=C3)c(O)c(S(=O)(=O)[O-])cc1cc(S(=O)(=O)[O-])cc2.[Na+]
- Purity: 82%
- Impurity: volatile matter at 135°C, 3.37%; water-insoluble matter, 0.01%; sodium chloride, 1.4%; sodium sulphate, 8.5%; pH of a 1%o solution in distilled water, 5.4; inorganic impurities (ppm)--lead <5, copper 4, chromium 6, zinc 9, iron 30, cadmium <1 and mercury <0.2; organic impurities--free aromatic amines (as aniline) 29 ppm, Michler's hydrol <0.01%o, Michler's ketone <0.01% and R-acid 0.11%.

Species:

rat

Strain:

Wistar

Details on species / strain selection:

No data

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: Weanling
- 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

Details on route of administration:

No data

Vehicle:

water

Details on oral 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 analytical verification of doses or concentrations:

No data

Duration of treatment / exposure:

19 days

Frequency of treatment:

Daily

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:

- 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

Positive control:

No data

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: No data available
- Cage side observations checked in table [No.?] were included. No data available

DETAILED CLINICAL OBSERVATIONS: No data available
- Time schedule: No data available

BODY WEIGHT: Yes
- Time schedule for examinations: Alternate
Days

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

FOOD EFFICIENCY: No data available
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data available

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

OPHTHALMOSCOPIC EXAMINATION: No data available
- Time schedule for examinations: No data available
- Dose groups that were examined: No data available

HAEMATOLOGY: No data available
- Time schedule for collection of blood: No data available
- Anaesthetic used for blood collection: No data available
- Animals fasted: No data available
- How many animals: No data available
- Parameters checked in table [No.?] were examined. No data available

CLINICAL CHEMISTRY: No data available
- Time schedule for collection of blood:
- Animals fasted: No data available
- How many animals: No data available
- Parameters checked in table [No.?] were examined. No data available

URINALYSIS: No data available
- Time schedule for collection of urine: No data available
- Metabolism cages used for collection of urine: No data available
- Animals fasted: No data available
- Parameters checked in table [No.?] were examined. No data available

NEUROBEHAVIOURAL EXAMINATION: No data available
- Time schedule for examinations: No data available
- Dose groups that were examined: No data available
- Battery of functions tested: sensory activity / grip strength / motor activity / other:v No data available

OTHER:
No data available

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Gross appearances were observed.

HISTOPATHOLOGY: No data available

Other examinations:

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 fetuses were weighed and examined for gross abnormalities.

Statistics:

Statistical analysis were performed by using exact test of Fisher and chi-square test

Clinical signs:

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, treatment-related

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Details on results:

Clinical signs and mortality
Mortality: No data available

Clinical signs: No data available

Body weight and weight gain: No statistically significant differences were observed in treated female rats as compared to control.

Food consumption and compound intake: No data available

Food efficiency: No data available

Water consumption and compound intake: No data available

Opthalmoscopic examination: No data available

Haematology: No data available

Clinical chemistry: No data available

Urinanalysis: No data available

Neurobehaviour: No data available

Organ weights: No data available

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.

Histopathology: No data available

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No adverse effect on body weight and gross pathology

Critical effects observed:

not specified

Conclusions:

The No Observed Adverse Effect level (NOAEL) was considered to be 1000 mg/kg/day when female Wistar rats were treated with Green S by the gavage route of administration for 19 days.

Executive summary:

Combined repeated dose repro-devp. screen was performed for Green S orally in female rats. Wistar female rats were treated with Green S in the concentration of 0, 250, 500 and 1000 mg/kg/day orally by gavage for 19 days. The animals were observed for changes in body weight and gross pathology. No effect was observed on body weight of treated rats as compared to control. Similarly, no effects were observed on reproductive system and the foetuses of treated female rats. 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. Therefore, No Observed Adverse Effect level (NOAEL) was considered to be 1000 mg/kg/day when Wistar female rats were treated with Green S orally by gavage for 19 days.

Endpoint conclusion

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 from peer reviewed publication

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Repeated dose toxicity oral:

Various peer reviewed publications were reviewed to determine the toxic nature of Hydrogen [4-[4-(dimethylamino)-α-(2-hydroxy-3, 6-disulphonato-1-naphthyl)benzylidene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium, monosodium salt. Six studies were reviewed to decide the toxic nature of test chemical. The results are based on the basis of one subacute one subchronic, one chronic and three combined repeated dose repro-devlop. screen studies. The details are mentioned as below:

In a study by Clode ( Food Chemical Toxicology, 1987), Combined repeated dose repro-devp. screen was performed for Green S orally in female rats. Wistar female rats were treated with Green S in the concentration of 0, 250, 500 and 1000 mg/kg/day orally by gavage for 19 days. The animals were observed for changes in body weight and gross pathology. No effect was observed on body weight of treated rats as compared to control. Similarly, no effects were observed on reproductive system and the foetuses of treated female rats. 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. Therefore, No Observed Adverse Effect level (NOAEL) was considered to be 1000 mg/kg/day when Wistar female rats were treated with Green S orally by gavage for 19 days.

 

Repeated dose subacute toxicity study was also performed by Clode et al ( Food Chemical Toxicology, 1987) to determine the toxic nature of Green S in rats. Wistar male and female rats were treated with Green S in the concentration of 0, 250, 500 and 1500 mg/kg/day orally in diet for 2 weeks. The animals were observed for clinical signs, mortality, body weight and food intake, hematology, clinical chemistry, urinalysis, gross and histopathological changes. No effects were observed in hematology of treated rats. Green staining of fur, extremities and faeces in all treated rats were observed. Higher levels of urinary protein and in dilution test, Specific gravity and Cell excretion were significantly decreased and urine volume was increased in 1500 mg/kg/day treated male rats and urine volume was decreased in female rat. In addition, Green colouring of gastro-intestinal tract were observed in all the treated animals and Increases in the numbers of prominent lymph nodes of the gastro-intestinal tract were observed in female rats treated with 1500 mg/kg/day as compared to control. Therefore, No Observed Adverse Effect level (NOAEL) was considered to be 500 mg/kg/day when Wistar male and female rats were treated with Green S for 2 weeks.

 

In the same study by Clode et al ( Food Chemical Toxicology, 1987), repeated dose subchronic toxicity study was performed, male and female Wistar rats were treated with Green S in the concentration of 0, 250, 500 and 1500 mg/kg/day orally in diet. The animals were observed for clinical signs, mortality, body weight and food intake, hematology, clinical chemistry, urinalysis, gross and histopathological changes. Green staining of fur, extremities and faeces was observed in all treated rats. Total Leucocytes counts were decreased, Higher levels of urinary protein and increased Specific gravity of urine were observed in male rats treated with 500 mg/kg/day. Increased Reticulocytes in male and female rats and packed cell volume in female rats was observed in 1500 mg/kg/day. In addition, Green colouring of gastro-intestinal tract were observed in all the treated animals and increases in the numbers of prominent lymph nodes of the gastro-intestinal tract were observed in female rats treated with 1500 mg/kg/day as compared to control. Therefore, No Observed Adverse Effect level (NOAEL) was considered to be 500 mg/kg/day when Wistar male and female rats were treated with Green S for 6 weeks.

 

In the same study by Clode et al ( Food Chemical Toxicology, 1987), repeated dose chronic toxicity study was performed, male and female Wistar rats were treated with Green S in the concentration of 0, 250, 500 and 1500 mg/kg/day orally in diet. The animals were observed for clinical signs, mortality, body weight and food intake, hematology, clinical chemistry, urinalysis, gross and histopathological changes. Green staining of fur, extremities and faeces in all treated rats. Statistically significant increased in mean body weight,Food consumption and water consumption were observed in 1500 mg/kg/day treated rats as compare to control. Similarly, Decreasedglucose concentration and lactic dehydrogenase activity in the serum, change in urine volume and Specific gravity in concentration and dilution test and increased levels of urinary protein, positive reaction for ketones and traces of blood were observed in 1500 mg/kg/day treated male and female rats. In addition, significant change in absolute kidney, stomach and empty caecum weight and relative stomach and caecum weight were observed in 1500 mg/kg/day treated rats. Green colouring of gastro-intestinal tract and mild degree of thyroid degeneration in female rats and slight increase inprotein casts in kidneys were observed in male rats when treated with 1500 mg/kg/day as compared to control. Therefore, the No Observed Adverse Effect level (NOAEL) was considered to be 500 mg/kg/day whenWistar male and female rats were treated with Green S orally in diet for 13 weeks.

Combined repeated dose repro-devp. Screen was performed by Mooorhouse et al ( Food Chemical Toxicology, 1987) to determine the toxic nature of Green S upon repeated oral exposure for 60 days. Wistar male and female rats were treated in the concentration of 0, 50, 500 and 1000 mg/kg body weight/day orally in diet. The animals were observed for mortality, clinical signs, body weight, food and compound intake, organ weight, gross and histopathology. 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. 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. In addition, Occasional focal inflammatory lesions were seen in the liver, heart, kidney and stomach and significantly 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. Therefore, No Observed Adverse Effect Level (NOAEL) is 500 mg/kg body weight /day when male and female Wistar rats were treated with Green S.

 

In a Combined repeated dose repro-devp. Screen & carcinogenicity by Brantom et al ( Food Chemical Toxicology, 1987), CD-1 male and female mice were treated with Green S in the concentration of 0, 0.033, 0.33 and 0.66 % (0, 55, 550 and 1100 mg/kg/day)) orally in diet for 2 years. The animals were observed for clinical signs and mortality, body weight changes and food intake, food and compound intake, hematology, gross pathology and histopathological observations were made. No effects were observed on survival, clinical sign and body weight of treated mice as compared to control. Very few statistically significant differences were observed in hematology on 14, 28, 51 and 106 week, 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 0.66 % (1100 mg/Kg/day) 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. It is possible that such differences could arise from the study design. Non – neoplastic hyperplasia in Caecum, Luminal casts in Epididymis, Focal epithelial hyperplasia of nephron in Kidney, Necrotic/inflammatory foci inLiver and oedema in rectum and Pneumonia in Lung at 0.66 % (1100 mg/Kg/day) and Hepatitis of Liver at 0.33 % (630 mg/Kg/day) in female mice were observed. In addition, neoplastic cyst adenoma in Harderian Gland, Interstitial-cell tumors in Testes were observed in male miceas 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, No Observed Adverse Effect Level (NOAEL) was considered to be 0.66 % (530 and 660 mg/kg body weight/day in males and females) when CD-1 male and female mice treated with Green S by oral route.

 

Based on the studies summarized, Green S (EC name: Hydrogen [4-[4-(dimethylamino)-α-(2-hydroxy-3, 6-disulphonato-1-naphthyl)benzylidene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium, monosodium salt) is not likely to classify as a toxicant upon repeated exposure by oral route.

Justification for classification or non-classification

Based on the studies summarized, Green S (EC name: Hydrogen [4-[4-(dimethylamino)-α-(2-hydroxy-3, 6-disulphonato-1-naphthyl)benzylidene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium, monosodium salt) is not likely to classify as a toxicant upon repeated exposure by oral route.