Hydrogen [4-[4-(diethylamino)-2',4'-disulphonatobenzhydrylidene]cyclohexa-2,5-dien-1-ylidene]diethylammonium, sodium salt

Endpoint summary

Description of key information

Repeated Dose toxicity (Oral): The No observed adverse effect level taken from a peer reviewed publication is reported to be 375 mg/kg bw when rats were repeatedly exposed to the test chemical (C.I. Food Blue 3) for a 90 day period.

Repeated Dose toxicity (Inhalation): The estimated vapour pressure of the chemical hydrogen [4-[4-(diethylamino)-2',4'-disulphonatobenzhydrylidene]cyclohexa-2,5-dien-1-ylidene]diethylammonium, sodium salt is very low  i.e. 1.145239e-31 Pascal. With such low vapour pressure, repeated exposure by the inhaltion route is highly unlikely. Thus, this end point was considered for waiver since no adverse effect by this route are expected.

Repeated dose toxicity (Dermal): The chemical hydrogen [4-[4-(diethylamino)-2',4'-disulphonatobenzhydrylidene]cyclohexa-2,5-dien-1-ylidene]diethylammonium, sodium salt (Synonym C.I. Food Blue 3) is dominantly used as a food color. THus, repeated exposure by the dermal route is highly unlikely. Hence this end point was considered for waiver since no adverse effect by this route are expected..

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Data is from peer reviewed journal

Qualifier:

according to guideline

Guideline:

other: Data is from Journal with permission

Principles of method if other than guideline:

Chronic repeated dose toxicity was studied for Blue VRS in rats orally.

GLP compliance:

no

Species:

rat

Strain:

other: SPF Carworth Farm E

Sex:

male/female

Details on test animals or test system and environmental conditions:

Details on test animalTEST ANIMALS- Source: No data available- Age at study initiation: No data available- Weight at study initiation: No data available- Fasting period before study: No data available- Housing: 5/cage- Diet (e.g. ad libitum): Spillers Small Laboratory Animal diet, ad libitum- Water (e.g. ad libitum): Water ad libitum- Acclimation period: No data availableENVIRONMENTAL CONDITIONS- Temperature (°C): No data available- Humidity (%):No data available- Air changes (per hr): No data available- Photoperiod (hrs dark / hrs light): No data availableIN-LIFE DATES: From: To: No data available

Route of administration:

oral: feed

Vehicle:

other: Spillers Small Laboratory Animal diet

Details on oral exposure:

Details on oral exposurePREPARATION OF DOSING SOLUTIONS: No data availableDIET PREPARATION- Rate of preparation of diet (frequency): Daily - Mixing appropriate amounts with (Type of food): Spillers Small Laboratory Animal food - Storage temperature of food: No data availableVEHICLE- Justification for use and choice of vehicle (if other than water): Spillers Small Laboratory Animal diet - Concentration in vehicle: 0.0, 0.3, 0.75, 1.5 and 3.0% - 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:

not specified

Details on analytical verification of doses or concentrations:

No data

Duration of treatment / exposure:

90 days

Frequency of treatment:

Daily

Remarks:

Doses / Concentrations:0.0, 0.3, 0.75, 1.5 and 3.0 % (0, 150, 375, 750 and 1500 mg/kg/day)Basis:nominal in diet

No. of animals per sex per dose:

Total : 1800 % : 15 male and 15 female 0 % : 15 male and 15 female003 % : 15 male and 15 female0.75 % : 15 male and 15 female1.5 % : 15 male and 15 female3.0 % : 15 male and 15 female

Control animals:

yes, concurrent vehicle

Details on study design:

No data

Positive control:

No data

Observations and examinations performed and frequency:

Observations and examinations performed & frequencyCAGE SIDE OBSERVATIONS: Yes - Time schedule: Daily - Cage side observations checked in table [No.?] were included: Mortality were observed DETAILED CLINICAL OBSERVATIONS: Yes - Time schedule: Daily BODY WEIGHT: Yes - Time schedule for examinations: WeeklyFOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes FOOD EFFICIENCY:- 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: Weekly At 6 week and on terminally - Anaesthetic used for blood collection: Cardiac puncture No data available - Animals fasted: No data- How many animals: All the treated animals were examined. - Parameters checked in table [No.?] were examined: Erythrocytes, Packed cell volume, Hemoglobin concentration in all teeated animals and total and Dtfferentlal Leucocytes count in 0, 1.5 and 3.0 % dose groups were examined. CLINICAL CHEMISTRY: Yes - Time schedule for collection of blood: At 6 week and on terminally - Animals fasted: No data available - How many animals: All the treated animals were examined. - Parameters checked in table [No.?] were examined: Serum urea was examined. URINALYSIS: Yes - Time schedule for collection of urine: at week 12, a 6-hr period of water deprivation and during a 4-hr period beginning 16 hr after a water load of 25 ml/kg. - Metabolism cages used for collection of urine: : No data available - Animals fasted: : No data available - Parameters checked in table [No.?] were examined: pH, Specific gravity and Volume were examined. 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:Absolute and relative organ weights were examined.Organ examined. Liver, Left and Right Kidneys, Brain, Heart, Spleen, Adrenals and Gonads were examined.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes Gross abnormalities were examined. HISTOPATHOLOGY: YesOrgan examined. Liver, Left and Right Kidneys, Brain, Heart, Spleen, Adrenals and Gonads were examined.

Other examinations:

No data available

Statistics:

No data available

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Mortality: No effect on servival were observed in treated rat as compared to control. Clinical signs: No effect were observed on health of treated rat as compared to control.

Mortality:

mortality observed, treatment-related

Description (incidence):

Mortality: No effect on servival were observed in treated rat as compared to control. Clinical signs: No effect were observed on health of treated rat as compared to control.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Impairment of growth was observed in male rats during the first 4 weeks in 1.5 and 3.0 % dose group.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption: Reduced food intake was observed in 1.5 and 3.0 % treated rats. Compound intake: Reduced in compound intake was observed in 1.5 and 3.0 % treated rats.

Food efficiency:

not specified

Description (incidence and severity):

Reduced

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not specified

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

No departure from normality was observed; red cell morphology was also unaffected.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

No effect on kidney function investigated during the last week of treatment or on serum urea nitrogen determined terminally

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

The urine of all the animals receiving Blue VRS in the diet was bluish-green and slightly more acid than that of the controls. Proteinurea was comparable in test and control groups and no reducing substances were detected in the urine of any group.

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

No effect on the absolute or relative (g/100 g body weight) organ weights was observed

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Fatty changes in the liver of 1.5 and 0.3 % treated female rat were observed.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not specified

Details on results:

Histopathology:Increase number of active acini in the thyroids of 1.5 & 0.3% treated male and female rats were observed. Blue material in the lumen of convoluted tubules associated with a coloured line observed at the cortlcomedullary junction of kidney in male of 3.0% treated dose group. Retardation of growth in males at the 1.5 and 3.0 % dose group and occasional fatty change in the livers of some of the females at the 3.0 % dose group. Increase in the number of active follicles in the thyroids of both males and females of the treated groups but this is not necessarily attributable to Blue VRS. Histological changes associated with acinal activity occur in response to stress of various kinds.

Dose descriptor:

NOAEL

Effect level:

375 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effect on survival, clinical sign, body weight, food consumption, compound intake, haematology, clinical chemistry, organ weights, gross pathology and histopathology.

Critical effects observed:

not specified

Conclusions:

The no-observed-adverse-effect-level (NOAEL) was considered to be 375 mg/kg/day (0.75 %) when SPF Carworth Farm E male and female rats were treated with Blue VRS.

Executive summary:

In a Chronic repeated dose toxicity study, SPF Carworth Farm E male and female rats were treated with Blue VRS at a concentration of 0.0, 0.3, 0.75, 1.5 and 3.0 % (0, 150, 375, 750 and 1500 mg/kg/day) orally. Impairment of growth, Reduction in food consumption, compound intake and bluish-green and slightly more acid urine were observed in 1.5 and 3.0 % treated male and female as compared to control. In addition, Fatty changes in the liver of female rat and increase number of active acini in the thyroids of male rat of 1.5 and 3.0 % treated animals. Blue material in the lumen of convoluted tubules associated with a coloured line observed at the cortcomedullary junction of kidney in male of 3.0 % treated dose groups but this was not necessarily attributable to Blue VRS. Histological changes associated with acinal activity occur in response to stress of various kinds were noted. Therefore, the no observed adverse effect level (NOAEL) was considered to be 375 mg/kg/day (0.75 %) when SPF Carworth Farm E male and female rats were treated with Blue VRS orally for 90 days.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

375 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

Data from K2 publication.

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

repeated dose toxicity: inhalation

Data waiving:

exposure considerations

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

repeated dose toxicity: dermal

Data waiving:

exposure considerations

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Repeated dose toxicity: oral

Various publications were reviewed to determine the toxic nature of the test compound Blue VRS upon repeated application by the oral route of exposure. The studies ate reviewed as below:

In a Chronic repeated dose toxicity study by Hall et al (1967), SPF Carworth Farm E male and female rats were treated with Blue VRS at a concentration of 0.0, 0.3, 0.75, 1.5 and 3.0 % (0, 150, 375, 750 and 1500 mg/kg/day) orally. Impairment of growth, Reduction in food consumption, compound intake and bluish-green and slightly more acid urine were observed in 1.5 and 3.0 % treated male and female as compared to control. In addition, Fatty changes in the liver of female rat and increase number of active acini in the thyroids of male rat of 1.5 and 3.0 % treated animals. Blue material in the lumen of convoluted tubules associated with a coloured line observed at the cortcomedullary junction of kidney in male of 3.0 % treated dose groups but this was not necessarily attributable to Blue VRS. Histological changes associated with acinal activity occur in response to stress of various kinds were noted. Therefore, the no observed adverse effect level (NOAEL) was considered to be 375 mg/kg/day (0.75 %) when SPF Carworth Farm E male and female rats were treated with Blue VRS orally for 90 days.

Based on the SSS QSAR prediction (2016) done for repeated dose toxicity via oral route, the NOAEL value is estimated to be 615.1199 mg/kg bw/d for 24 months daily exposure for the test compound hydrogen [4-[4-(diethylamino)-2', 4’-disulphonatobenzhydrylidene] cyclohexa-2, 5-dien-1-ylidene]diethylammonium, sodium salt on rats. Thus based on this value it can be concluded that hydrogen [4-[4-(diethylamino)-2', 4’-disulphonatobenzhydrylidene] cyclohexa-2, 5-dien-1-ylidene]diethylammonium, sodium salt is not very toxic in nature via oral route of exposure and hence is not likely to classify as an repeated oral toxicant.

In a Combined repeated dose & carcinogenicity study (EFSA Panel, 2013), male and female mice were treated with Patent Blue V at a concentration of 0, 0.1, 0.3 and 1 % (equivalent to 0, 150, 500 and 1500 mg/kg body weight/day) orally. Slightly increased in mortality, significant decrease in body weight of male mice and significant reduction in haemoglobin, haematocrit, red blood cell counts and reticulocyte count, increase in the total number of white blood cells in male and female rat were observed at 1 % dose group as compared to control. Increase in relative heart and caecal weights were observed in female at 1 % dose group. In addition, non-neoplastic lesions of lungs, kidneys and liver were observed. Adrenocortical adenoma and carcinoma, adenoma of mammary gland were noted and were found to be dose independent. The observed changes were mainly isolated findings, of commonly occurring tumours showing no dose-response relationship. Therefore, NOAEL was considered to be 0.3 % (500 mg/kg body weight /day) when male and female mice were treated with Patent Blue V orally for 32 weeks.

 

Repeated dose toxicity: Inhalation

The estimated vapour pressure of the chemical hydrogen [4 -[4-(diethylamino)-2',4'-disulphonatobenzhydrylidene]cyclohexa-2,5-dien-1-ylidene]diethylammonium, sodium salt is very low i.e. 1.145239e-31 Pascal. With such low vapour pressure, repeated exposure by the inhaltion route is highly unlikely. Thus, this end point was considered for waiver.

 

Repeated dose toxicity: Dermal

The chemical hydrogen [4-[4-(diethylamino)-2',4'-disulphonatobenzhydrylidene]cyclohexa-2,5-dien-1-ylidene]diethylammonium, sodium salt (Synonym C.I. Food Blue 3) is dominantly used as a food color. Thus, repeated exposure by the dermal route is highly unlikely. Hence this end point was considered for waiver.

On the basis of the available data and the applied waivers, the test chemical is not likely to classify as a toxicant upon repeated exposure by the oral, dermal and inhalation route of exposure.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:

The no-observed-adverse-effect-level (NOAEL) was considered to be 375 mg/kg/day (0.75 %) when SPF Carworth Farm E male and female rats treated with Blue VRS.

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:

The estimated vapour pressure of the chemical hydrogen [4-[4-(diethylamino)-2',4'-disulphonatobenzhydrylidene]cyclohexa-2,5-dien-1-ylidene]diethylammonium, sodium salt is very low  i.e. 1.145239e-31 Pascal. With such low vapour pressure, repeated exposure by the inhaltion route is highly unlikely. Thus, this end point was considered for waiver.

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:

The chemical hydrogen [4-[4-(diethylamino)-2',4'-disulphonatobenzhydrylidene]cyclohexa-2,5-dien-1-ylidene]diethylammonium, sodium salt (Synonym C.I. Food Blue 3) is dominantly used as a food color. Thus, repeated exposure by the dermal route is highly unlikely. Hence this end point was considered for waiver.

Justification for classification or non-classification

Based on the available data and the applicable waivers, it can be concluded that the chemical C.I. Food Blue 3 is not likely to cause repeated dose toxicity by the oral, inhalation or dermal route of exposure. Thus, the chemical was not considered for classification.