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

Carcinogenicity

Description of key information

Non carcinogenic NOAEL was considered to be 530 and 660 mg/kg body weight/day in males and females when CD-1 male and female mice treated with Green S orally in diet for 2 years. 

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records

Reference

Endpoint:

carcinogenicity: oral

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 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Principles of method if other than guideline:

Long term chronic and carcinogenicity study of Green S orally in mice.

GLP compliance:

not specified

Specific details on test material used for the study:

- Name of test material (as cited in study report): Green S
- 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:
Purity: Dye 82% (80%)
- Impurities (identity and concentrations): 20 %; 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, 4ppm (2ppm); chromium, 6ppm (8ppm); zinc, 9ppm (5ppm); iron, 30ppm (35ppm); cadmium, < 1 ppm; mercury, 0.2 ppm (< 1 ppm); free aromatic amines (as aniline), 29ppm (17ppm); 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:

mouse

Strain:

CD-1

Details on species / strain selection:

No data

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Source: Specified-pathogen-free colony (Charles River (UK) Ltd, Margate, Kent)
- Age at study initiation: 4-5 week old
- Weight at study initiation: Female : 20.2-20.6 g
- Fasting period before study: No data available
- Housing: Animals were housed in stainless-steel and plastic solid-floored cages with autoclaved softwood sawdust as bedding. The cages were held on mobile racks.
- Diet (e.g. ad libitum): Basal diet (Spratt's Laboratory Diet No. 2-Spratt's Patent Ltd, Barking, Essex)
- Water (e.g. ad libitum): No data available
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3°C
- Humidity (%): 50-70%
- 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: feed

Type of inhalation exposure (if applicable):

not specified

Vehicle:

other: Basal diet (Spratt's Laboratory Diet No. 2)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Green S was incorporated by dry-blending into the basal diet (Spratt's Laboratory Diet No. 2-Spratt's Patent Ltd, Barking, Essex) to give the required concentration.

DIET PREPARATION
- Rate of preparation of diet (frequency): Once every 6 week
- Mixing appropriate amounts with (Type of food): Basal diet (Spratt's Laboratory Diet No. 2)
- Storage temperature of food: No data available

VEHICLE
- Justification for use and choice of vehicle (if other than water): Basal diet (Spratt's Laboratory Diet No. 2)
- Concentration in vehicle: 0, 0.033, 0.33 and 0.66%
- Amount of vehicle (if gavage): No data available
- Lot/batch no. (if required): No data available
- Purity: No data available

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

by using a colorimetric estimation of aqueous diet extracts.

Duration of treatment / exposure:

2 year (106 weeks)

Frequency of treatment:

Daily

Post exposure period:

No data available

Remarks:

0, 49.5, 495 and 530-660 mg/kg bw/day

No. of animals per sex per dose:

Total: 510
0 mg/kg bw : 105 male, 105 female
49.5 mg/kg bw: 65 male, 65 female
495 mg/kg bw: 65 male, 65 female
660 mg/kg bw: 65 male
530 mg/kg bw: 65 female

Control animals:

yes, concurrent vehicle

Details on study design:

- Rationale for animal assignment (if not random): Animals were assigning to treatment group by using random number chart.

Positive control:

No data available

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily
- Cage side observations checked in table [No.?] were included: Morbidity and mortality were observed.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily

BODY WEIGHT: Yes
- Time schedule for examinations: On day 1 and weekly thereafter

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: Yes
- Time schedule for collection of blood: At 14, 28 and 51 week and at the end of study.
- Anaesthetic used for blood collection: No data available
- Animals fasted: No data available
- How many animals: 20 animals from each group
- Parameters checked in table [No.?] were examined: Hemoglobin concentration, erythrocyte count, total leucocyte count, packed cell volume, reticulocyte count and differential leucocyte count were examined.

CLINICAL CHEMISTRY: No data available
- Time schedule for collection of blood: No data available
- 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: No data available

OTHER:
Organ weigth:
Brain, caecum (full and empty), heart, kidneys, liver, spleen, stomach and testes were weighed.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Any abnormalities were observed.

Non-neoplastic and neoplastic lesions were observed

Samples of organs were preserved in 10% buffered formalin, along with any other tissue found to be abnormal. One eye was fixed in Davidson's fluid for 24 hr before transfer to 10% buffered formalin.

HISTOPATHOLOGY: Yes

Samples of all tissues taken, except nasal bones from the 0.033 and 0.33% groups, were processed for wax embedding. Sections 5pro thick were prepared, stained with haematoxylin and eosin and examined by light microscopy.

Organ examined:
Brain, caecum (full and empty), heart, kidneys, liver, spleen, stomach, testes, adrenal gland, aorta, bladder, colon, diaphragm, duodenum, ears, epididymis, eye, Harderian gland, ileum, lungs, lymph nodes, mammary gland, skeletal muscle, nasal
bones, nerve, oesophagus, ovaries, pancreas, pituitary gland, prostate, rectum, salivary gland, seminal vesicles, skin, spinal cord, thymus, thyroid, trachea, uterus, vagina and vena cava were examined.

Other examinations:

Numbers of live and dead pups and gross internal examination were performed.

Statistics:

Statistical analysis of Mortality data were analysed according to the method of Peto & Pike (1973). Body weight, organ weight and haematology data were subject to analysis of variance and t test. Litter observations were analysed using Fisher's exact test. Incidences of histopathological findings were compared using the methods of Peto et al. (1980). In all analyses a probability level of less than 5% was taken as statistically significant.

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

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:

no effects observed

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:

effects observed, non-treatment-related

Gross pathological findings:

effects observed, treatment-related

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Details on results:

Mortality: No effect was observed on survival of all treated mice as compared to control.

Clinical signs: No effect was observed on general condition of all treated mice as compared to control.

Body weight and weight gaiin: No effect was observed on body weight of male mice in all treated groups.
In female mice, significant decrease was observed in body weight on week 9 in 0.66 % dose and on week 87 in 0.33 % dose group, but from this point onwards the difference was reversed and differences were not statistically significant.

Haematology Very few statistically significant differences were observed on 14, 28, 51 and 106 week in treated and control.
The differences present did not occur consistently at all times of examination or in both sexes.

Organ Weights: Increase in absolute stomach weight was observed in female mice at 0.66 % dose but was not present when the weights were expressed relative to body weight.
No other consistent or dose-related statistically significant differences between the treated and control groups.

Gross Pathology: Green coloration of the gastro-intestinal tract was observed in all the treated mice as compared to control.

Histopathology: 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 were observed in male mice at 0.66 % were observed as compared to control.
Pneumonia in Lung at 0.66 % and Hepatitis of Liver in female at 0.33 % was observed as compared to control.

Histopathology: neoplastic Cyst adenoma in Harderian Gland, Interstitial-cell tumors in Testes were observed in 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.

OTHER FINDINGS: No effects were observed on Numbers of live and dead pups of treated mice as compared to control.

Dose descriptor:

NOAEL

Effect level:

660 mg/kg bw/day

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: neoplastic

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

Dose descriptor:

NOAEL

Effect level:

530 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: neoplastic

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

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

Tumour incidence in mice fed diets containing 0-1100 mg/Kg/day % Gree

	No. of animals affected in each group
	Males				Females
	0	55	550	1100	0	55	550	1100
Total no. of mice
Examined	85	50	50	50	85	50	50	50
With primary tumours	56	35	29	38	57	36	35	30
With malignant tumors	25	10	11	13	232	18	19	16
Benign tumors	45	31	22	30	39	21	20	16
Total no. of malignant tumours	25	10	11	13	26	18	20	16
Total no. of benign tumours	57	51	26	41	56	24	27	18

Statistical analysis (Peto et al. 1980) of the number of animals with primary tumours, total number of animals with malignant tumours and total number with benign tumours did not show any positive or negative trends.

Conclusions:

Non carcinogenic NOAEL was considered to be 530 and 660 mg/kg body weight/day in males and females when CD-1 male and female mice treated with Green S orally in diet for 2 years.

Executive summary:

In a Long term chronic and carcinogenicity study, CD-1 male and female mice treated with Green S in the concentration of 0, 0.033, 0.33 and 0.66 % orally in diet (0, 49.5, 495 and 530 – 660 mg/kg bw). 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 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. 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 in Liver and oedema in rectum and Pneumonia in Lung at 530 mg/kg bw and Hepatitis of Liver at 495 in female mice were observed. In addition, neoplastic cyst adenoma in Harderian Gland, Interstitial-cell tumors in Testes were observed in 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 and considered as non carcinogenic. Therefore, NOAEL was considered to be 530 and 660 mg/kg body weight/day in males and females when CD-1 male and female mice treated with Green S orally in diet for 2 years. 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

660 mg/kg bw/day

Study duration:

chronic

Species:

mouse

Quality of whole database:

Data is Klimisch 2 and from peer -reivewed journal

Carcinogenicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Carcinogenicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the above study 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 Hydrogen [4-[4-(dimethylamino)-α-(2-hydroxy-3,6-disulphonato-1-naphthyl)benzylidene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium, monosodium salt can be considered as Not classified for carcinogenicity. 

Additional information

Carcinogenicity - Oral:

In a study, 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 carcinogenicity based on in vivo experiments in rodents, i.e. most commonly in mice 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 Long term chronic and carcinogenicity study by Brantomet al(Food Chem. Toxic. Vol. 25, No. 12, pp. 977-983, 1987), CD-1 male and female mice were treated with Green S in the concentration of 0, 49.5, 495 and 530 – 660 mg/kg bw orally in diet. 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 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. 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 in Liver and oedema in rectum and Pneumonia in Lung at 530 mg/kg bw and Hepatitis of Liver at 495 in female mice were observed. In addition, neoplastic cyst adenoma in Harderian Gland, Interstitial-cell tumors in Testes were observed in 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 and considered as non carcinogenic. Therefore, non carcinogenic NOAEL was considered to be 530 and 660 mg/kg body weight/day in males and females when CD-1 male and female mice treated with Green S orally in diet for 2 years. 

Thus, based on the above study 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 Hydrogen [4-[4-(dimethylamino)-α-(2-hydroxy-3,6-disulphonato-1-naphthyl)benzylidene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium, monosodium salt can be considered as Not classified for carcinogenicity.