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

Repeated dose toxicity: oral

Administrative data

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

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 journal

Data source

Reference

Reference Type:

publication

Title:

Acute (Mouse and Rat) and Short-term (Rat) Toxicity Studies on Blue VRS

Author:

D. E. Hall and I. F. Gaunt, Madge Farmer and P. Grasso

Year:

1967

Bibliographic source:

Fd Cosmet. Toxicol Vol. 5, pp. 165-170, 1967

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: Refer below principle

Principles of method if other than guideline:

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

GLP compliance:

no

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

204-934-1

EC Name:

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

Cas Number:

129-17-9

Molecular formula:

C27H32N2O6S2.Na

IUPAC Name:

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

Details on test material:

- Name of test material: Blue VRS
- Molecular formula: C27H31N2O6S2.Na
- Molecular weight: 566.672g/mole
- Substance type: Organic
- Physical state: Solid
- Impurities: 5.5 %

Specific details on test material used for the study:

- Name of test material: Blue VRS
- Molecular formula: C27H31N2O6S2.Na
- Molecular weight: 566.672g/mole
- Substance type: Organic
- Physical state: Solid
- Impurities: 5.5 %

Test animals

Species:

rat

Strain:

other: SPF Carworth Farm E

Details on species / strain selection:

No data

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST 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 available


ENVIRONMENTAL CONDITIONS
- Temperature (°C): No data available
- Humidity (%):No data available
- Air changes (per hr): No data available
- Photoperiod (hrs dark / hrs light): No data available

IN-LIFE DATES: From: To: No data available

Administration / exposure

Route of administration:

oral: feed

Details on route of administration:

No data

Vehicle:

other: Spillers Small Laboratory Animal diet

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: No data available

DIET 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 available

VEHICLE
- 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

Doses / concentrations

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 : 180
0 % : 15 male and 15 female
0 % : 15 male and 15 female
003 % : 15 male and 15 female
0.75 % : 15 male and 15 female
1.5 % : 15 male and 15 female
3.0 % : 15 male and 15 female

Control animals:

yes, concurrent vehicle

Details on study design:

No data

Positive control:

No data

Examinations

Observations and examinations performed and frequency:

Observations and examinations performed & frequency
CAGE 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: Weekly

FOOD 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: Yes
Organ examined.
Liver, Left and Right Kidneys, Brain, Heart, Spleen, Adrenals and Gonads were examined.

Other examinations:

No data available

Statistics:

No data available

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

No effect were observed on health of treated rat as compared to control.

Mortality:

no mortality observed

Description (incidence):

No effect on survival were observed in 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 % (750 and 1500 mg/Kg/day) dose group.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Reduced food intakeand compound intake was observed in 1.5 and 3.0 % (750 and 1500 mg/Kg/day) treated rats.

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

no effects observed

Description (incidence and severity):

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

Clinical biochemistry findings:

no effects observed

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

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

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 % (750 and 1500 mg/Kg/day) treated female rat were observed.

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Increase number of active acini in the thyroids of 1.5 & 0.3% (375 and 150 mg/Kg/day) 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% (1500 mg/kg/day) treated dose group.

Retardation of growth in males at the 1.5 and 3.0 % (750 and 1500 mg/Kg/day) dose group and occasional fatty change in the livers of some of the females at the 3.0 % (1500 mg/Kg/day) 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.

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Effect levels

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.

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table 1. Mean values of body weight, food consumption and coloring intake of rats fed Blue VRS at 0-3% of the diet for 12 wk

Dietary level (%)	Body weight (g) at end ofwk				Food consumption (g/rat/day) at end of wk				Intake of colourmg (g/kg/day)* at end of wk
	0§	4	8	12	0§	4	8	12	0§	4	8	12
	Males
0.0	135	273	364	414	10.7	8.7	4.8	3.6	-	-	-	-
0.0	134	283	365	415	10.8	8.3	5.2	4.0	-	-	-	-
0.3	134	278	366	405	10.3	8.1	4.7	4.5	0.32	0.24	0.14	0.14
0.75	133	275	357	397	10.4	8.2	4.8	4.1	0.78	0.62	0.36	0.31
1.5	134	261	333	373	10.5	7.6	5.1	3.9	1.56	1.14	0.77	0.58
3.0	135	266	347	386	9.6	6.8	5.0	3.9	2.87	2.65	1.51	1.16
	Females
0.0	125	202	235	254	11.4	7.9	5.9	5.0	-	-	-	-
0.0	124	197	227	242	11.3	8.2	6.2	5.7	-	-	-	-
0.3	123	194	224	241	10.6	8.8	6.0	5.0	0.32	0.26	0.18	0.15
0.75	125	202	234	252	11.0	7.4	6.0	5.2	0.82	0.56	0.46	0.39
1.5	125	199	228	244	10.8	7.8	5.8	5.1	1.61	1.17	0.87	0.74
3.0	13	192	223	238	8.3	9.5	5.7	5.0	2.49	2.84	1.70	1.49

*Colouring retake is calculated from data on body weight and food consumption.

§Day 1 of feeding.

Values are the means for groups of 15 animals. Although weekly measurements were made of growth food consumption and coloring intake, only values recorded at 4-wk intervals are included in the Table.

Table 2. Terminal haematological response in rats fed Blue VRS at 0-3 % of the diet for 90 days

Dietary level (%)	Erythrocytes (106/mm3)	Packed cell volume (%)	Haemoglobin concentration (g/100mL)	Leucocytes
				Total (103/mm3)	Differential count (%)*
					N	E	L	M
	Males
0.0	8.82	44	14.5	12.4	12	2	80	6
0.0	8.59	45	14.8	9.2	17	1	76	6
0.3	8.42	45	14.7	6.5	-	-	-	-
0.75	8.83	45	14.8	7.9	-	-	-	-
1.5	8.51	45	14.8	7.9	-	-	-	-
3.0	8.57	45	14.9	11.9	17	3	76	4
	Females
0.0	8.03	42	13.5	4.6	12	3	77	7
0.0	7.75	42	13.5	5.3	12	3	80	5
0.3	8.16	42	13.4	5.0	-	-	-	-
0.75	7.98	41	13.3	5.3	-	-	-	-
1.5	8.39	42	13.3	4.1	13	2	78	7
3.0	7.71	41	13.1	4.8	10	2	82	6

N = Neutrophils E: EOslnophlls L = Lymphocytes M = Monocytes

*Differential counts were not made at the 0 3 or 0-75 % levels.

Values obtained at 6 wk which are not included in the Table showed no significant differences between the treated and control groups. Basophils were absent m the differential leucocyte count

Table 3. Serum and urine analyses of rats fed Blue VRS at 0-3 % of the diet for 90 days

Dietary level (%)	Serum urea (mg/100mL)	Urine
		pH	Concentration test*
			Specific gravity§			Volume (mL)
			Initial	0-6hrs	16-20hrs	0-6 hr	16-20 hr
	Males
0.0	16.5	8.0	2.4	4.8	8.4	4.5	1.6
0.0	16.5	8.0	4.0	6.8	10.6	3.1	1.1
0.3	-	7.0	2.9	5.8	9.5	3.6	1.0
0.75	-	6.8	2.9	5.4	8.7	4.8	1.4
1.5	-	7.0	4.5	6.5	9.0	3.4	1.5
3.0	18.4	7.2	1.0	6.9	8.6	4.4	1.1
	Females
0.0	13.9	6.9	3.2	8.1	10.6	1.5	0.3
0.0	13.9	6.4	9.5	10.6	10.6	0.6	0.2
0.3	-	5.8	2.5	7.6	10.6	1.5	0.5
0.75	-	5.8	5.1	5.8	10.6	2.1	0.3
1.5	-	5.8	2.7	6.6	10.6	2.7	0.7
3.0	17.4	6.0	3.9	7.8	10.6	2.4	0.5

*Urine concentration test revolved the measurement of the specific gravity and volume of the urine excreted over a 6-hr period of water deprivation and during a 4-hr period beginning 16 hr after a water load of 25 ml/kg.

§Expressed as (specific gravity x 100)-100.

The results are the mean values of two urine collections from cages containing five animals.

Protein urea was comparable in test and control groups and no reducing substances were detected in the urine of any group.

 

Table 4. Absolute organ weights of rats fed Blue VRS at 0-3 % of the diet for 90 days

Dietary level (%)	Terminal Nody weight (g)	Absolute organ weight (g)
		Liver	Kidneys		Brain	Heart	Spleen	Adrenals	Gonads
			Left	Right
Males
0.0	428	13.75	1.25	1.30	1.96	1.35	0.84	0.032	0.38
0.0	431	14.54	1.35	1.40	2.07	1.37	0.83	0.031	0.43
0.3	418	13.70	1.30	1.32	1.96	1.32	0.84	0.034	0.45
0.75	406	12.90	1.21	1.24	1.96	1.32	0.79	0.031	0.45
1.5	388*	12.66	1.22	1.24	1.97	1.30	0.75	0.030	0.44
3.0	399*	12.40	1.24	1.26	1.96	1.34	0.79	0.030	0.44
Females
0.0	262	7.44	0.86	0.87	1.81	0.92	0.55	0.037	0.060
0.0	257	7.86	0.83	0.86	1.83	0.93	0.55	0.034	0.059
0.3	249	6.90	0.80	0.81	1.85	0.93	0.55	0.035	0.056
0.75	258	7.45	0.85	0.87	1.83	0.92	0.54	0.034	0.056
1.5	249	7.19	0.83	0.84	1.84	0.93	0.51	0.034	0.053
3.0	247	7.68	0.84	0.86	1.81	0.92	0.52	0.034	0.055

Values are the means for groups of 15 animals.

Value significantly different from that of the control group: *P<0.05

The relative organ weights (g/100 g body weight) are not included in the Table since no significant differences were found between the values of the test and control groups

Applicant's summary and conclusion

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.