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Toxicological information

Repeated dose toxicity: oral

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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:
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

Reference
Name:
Unnamed
Type:
Constituent
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 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

4

8

12

4

8

12

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.