Registration Dossier

Toxicological information

Repeated dose toxicity: oral

Currently viewing:

Administrative data

Endpoint:
repeated dose toxicity: oral
Remarks:
combined repeated dose and carcinogenicity
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 publication

Data source

Reference
Reference Type:
publication
Title:
Repeated oral toxicity study of the test chemical
Author:
Drake et al
Year:
1977
Bibliographic source:
Food and cosmetics toxicology

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: Refer below principle
Principles of method if other than guideline:
A repeated dose study was performed to investigate the effect of the test chemical in CFW strain mice when administered orally for 80 wk.
GLP compliance:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material: C.I Briliant black BN
- Molecular formula: C28H21N5O14S4.4Na
- Molecular weight: 867.6873 g/mol
- Substance type: Organic
- Physical state: Solid
- Purity: No data available
- Impurities (identity and concentrations): essentially tetrasodium 8-acetamido-2-(7- sulpho-4-p- sulphophenylazo- 1-naphthylazo)- 1-naphthol-3,5-disulphonate; dye content*, rain. 82%; subsidiary dyes, max 4%; matter volatile at 135°C, max 10%; matter insoluble in water, max 0.1%; matter soluble in diisopropyl ether, max 0.2%; chloride and sulphate (as sodium salts), max 8%; copper, max 10ppm; arsenic, max 1 ppm; lead, max 10 ppm; heavy metals (as sulphides)

Test animals

Species:
mouse
Strain:
other: CFW strain
Details on species / strain selection:
No data
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: From a specified-pathogen-free colony
- Age at study initiation: No data available
- Weight at study initiation: The mice were weighed at the start of the experiment (exact weight not mentioned)
- Fasting period before study: No
- Housing: They were caged in groups of 15 in a room
- Diet (e.g. ad libitum): Oxoid pasteurized breeding diet,ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: No data available

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21±1°C
- Humidity (%): 50-60%
- Air changes (per hr): No data available
- Photoperiod (hrs dark / hrs light): No data available

Administration / exposure

Route of administration:
oral: feed
Vehicle:
not specified
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The test chemical was mixed with feed at dose level of 0, 0.1, 0.25, 0.5 or 1.0% (0, 130, 325, 650, 1300 mg/kg bw/d)

DIET PREPARATION
- Rate of preparation of diet (frequency): No data
- Mixing appropriate amounts with (Type of food): No data
- Storage temperature of food: No data

VEHICLE
- Justification for use and choice of vehicle (if other than water): Oxoid pasteurized breeding diet
- Concentration in vehicle: 0, 0.1, 0.25, 0.5 or 1.0% (0, 130, 325, 650, 1300 mg/kg bw/d)
- Amount of vehicle (if gavage): No data
- Lot/batch no. (if required): No data
- Purity: No data
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
No data
Duration of treatment / exposure:
80 wk
Frequency of treatment:
Daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 0.1, 0.25, 0.5 or 1.0% (0, 130, 325, 650, 1300 mg/kg bw/d)
Basis:
no data
No. of animals per sex per dose:
Total: 180 male and 180 female mice
0 mg/kg bw/d: 60 male and 60 female mice
130 mg/kg bw/d: 30 male and 30 female mice
325 mg/kg bw/d: 30 male and 30 female mice
650 mg/kg bw/d: 30 male and 30 female mice
1300 mg/kg bw/d: 30 male and 30 female mice
Control animals:
yes, concurrent vehicle
Details on study design:
No data available
Positive control:
No data available

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Frequently dring the study period
- Cage side observations checked in table [No.?] were included.: general condition and behaviour, the animals were also observed for ill health

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

BODY WEIGHT: Yes
- Time schedule for examinations: start of the experiment, at wk 3 and then at intervals of 2 wk until wk 73 of the experiment

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

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

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

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

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At wk 28 and 55 from the caudal vein of ten males and ten females from the control group and from the groups of 0.5 and 1.0% dietary levels. At 80 wk, blood samples were collected from the aorta of all surviving mice during the autopsy.
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: 20 animals (10 male and 10 female)
- Parameters were examined: haemoglobin concentration and packed cell volume, as well as for counts of erythrocytes and leucocytes. In addition, the methaemoglobin concentrations were determined in the samples collected at 80 wk.

CLINICAL CHEMISTRY: No data
- Time schedule for collection of blood: No data
- Animals fasted: No data
- How many animals: No data
- Parameters were examined: No data

URINALYSIS: Yes
- Time schedule for collection of urine: At 28 wks at 6-hr period from three groups of five mice of each sex from the controls and the groups on the two highest dietary levels (0.5 and 1.0%) of Black PN.
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Parameters were examined: protein, reducing substances, bile salts and blood as well as for colour, pH and microscopic constituents

NEUROBEHAVIOURAL EXAMINATION: No data
- Time schedule for examinations: No data
- Dose groups that were examined: No data
- Battery of functions tested: sensory activity / grip strength / motor activity / other: No data
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, The animals were killed by exsanguination from the aorta under sodium pentobarbitone anaesthesia following an overnight period without food. At autopsy, macroscopic abnormalities were recorded and the brain, heart, liver, spleen, kidneys, adrenal glands and gonads were weighed.

HISTOPATHOLOGY: Yes, samples of the brain, heart, liver, spleen, kidneys, adrenal glands and gonads and of salivary glands, pituitary, thyroid, thymus, various lymph nodes, pancreas, urinary bladder, lungs, stomach, duodenum, ileum, colon, caecum, rectum, striped muscle (hind limb), spinal cord, uterus, aortic arch and any other tissue that appeared abnormal were preserved in 10% buffered formalin. Paraffin-wax sections of these tissues were stained with haematoxylin and eosin. All tissues from the control mice and from those fed diet containing 1% Black PN were examined histologically. At the lower dose levels, the examination was confined to the liver, kidney and any tissues seen to be abnormal at autopsy.
Other examinations:
No data
Statistics:
chi-square test, Student's t test

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
The ingestion of the test chemical had no effect on the condition or behaviour of the animals.
Mortality:
no mortality observed
Description (incidence):
No statistically significant differences between the number of deaths in the control mice and those given the test chemical were noted
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Throughout the study the body weights of mice of both sexes were similar in all groups
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:
effects observed, treatment-related
Description (incidence and severity):
The haematological examinations revealed only inconsistent isolated changes of statistical significance. At wk 28 the haemoglobin concentrations and red blood cell counts were lower in female mice fed diet containing if 0.5 or 1.0% than in the controls. There were no comparable findings in the males. The total white cell count of the male animals fed 1.0% in the diet was higher than that of the controls at this time, but there was no comparable change in this measurement in the females, or in either sex at any other time. At wk 55, the haemoglobin concentrations of male animals fed 0.5% of the coiouring in the diet were significantly (P < 0.05) lower than the control values. However, the corresponding value at the higher dietary level was not affected and there were no differences from the control value in the females. Also at wk 55, the erythrocyte counts of females fed 1.0%were lower (P < 0.01) than those of the controls, but this finding was again isolated. There were no statistically significant differences between the control and test samples taken at wk 80.
Clinical biochemistry findings:
not specified
Urinalysis findings:
no effects observed
Description (incidence and severity):
No abnormal constituents were detected in the urine from the control or treated mice.
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
There were only scattered differences in mean organ weights between treated and control animals. A lower brain weight in females, compared with the control value, was the only difference affecting animals fed 1%. This difference, which did not occur in the males, was only marginally significant and there was no significant difference when the weights were expressed relative to body weight By contrast, the relative brain weight of females fed 0.25% was higher than the control figure. Liver weights of female but not of male mice fed 0.25% were lower than control values, but again this was an isolated finding and there were no significant differences in relative liver weights. Kidney weights of male animals only were significantly lower than control values at the two lowest levels of treatment (0.1 and 0.25%), but a significant difference in relative kidney weights of males occurred only at the 0.5% level, at which a higher value was recorded for the treated mice. The only other significant differences occurred in the relative heart weights, which were raised in male mice fed 0.5% and in females fed 0.25%.
Gross pathological findings:
not specified
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
The incidence of histological findings was similar in all groups of mice, including the controls
Histopathological findings: neoplastic:
not specified
Description (incidence and severity):
Most of the tumours in the study occurred with either a comparable or a greater incidence in the control groups than in the treated mice. Several isolated tumours were identified in mice given the lower levels of Black PN, without comparable findings in the controls or in the highest dose group. They were a mammary fibroadenoma (in
a female on 0.1%), a uterine fibromyoma (0.19%) and a squamous-cell carcinoma of the skin (female, 0.5%). The only tumour found at the highest dietary level
without comparable control findings was a squamous- cell carcinoma of the skin in a male mouse fed 1%.
Other effects:
not specified

Effect levels

Dose descriptor:
NOAEL
Effect level:
1 300 other: mg/kg/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Body weight, weight gain, organ weight and histopathology.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 1. Cumulative death rate in mice fed diets containin 0.01% test chemical for 80 wk

Week

Total no. of deaths

Males

Females

0

0.1

0.25

0.5

1.0

0

0.1

0.25

0.5

1.0

24

3

1

0

3

0

1

0

0

0

0

48

8

1

3

5

3

8

1

3

3

2

64

11

4

7

9

5

15

9

9

6

7

72

16

6

9

13

6

24

12

13

12

10

78

20

10

10

16

7

28

19

13

15

12

 

Table 2: Mean body weights of mice fed diets containing 0.1% test chemical for 80 wk

Week

Total no. of deaths

Males

Females

0

0.1

0.25

0.5

1.0

0

0.1

0.25

0.5

1.0

0

21

22

21

22

22

18

18

18

18

19

15

38

35

37

37

38

28

29

28

28

28

39

41

39

40

39

40

32

33

32

33

32

57

43

40

40

37

41

35

34

32

33

33

73

42

41

41

38

42

36

35

33

36

34

 

Table 3. Results of haematological examinations of mice fed diets containing 0.01% test chemical for 80 wk

Sex and dietary level (%)

No. of mice examined

Hb (g/100 mL)

Met Hb (% of Hb)

PCV (%)

RBC (106/mm3)

Total leucocytes (103/ mm3)

Week 28

Males

 

 

 

 

 

 

0

10

14.2

-

43

7.51

11.4

0.5

10

14.4

-

48

8.05

12.4

1.0

10

15.5

-

47

8.04

14.8*

Females

 

 

 

 

 

 

0

10

15.9

-

49

9.01

14.8

0.5

10

14.0**

-

48

7.54--

12.1

1.0

10

13.1**

-

48

7.41**

12.6

Week 80

Males

 

 

 

 

 

 

0

39

11.9

3.61

35

6.67

4.5

0.1

15

11.9

4.51

36

6.85

4.2

0.25

18

11.5

4.90

35

6.67

3.2

0.5

10

11.7

3.86

34

6.62

5.4

1.0

17

12.0

3.81

35

6.90

3.8

Females

 

 

 

 

 

 

0

27

13.5

6.03

39

7.32

5.5

0.1

10

13.0

4.68

40

7.64

5.7

0.25

10

14.1

7.39

42

8.21

5.4

0.5

13

13.3

4.89

41

7.87

3.9

1.0

15

14.4

5.67

42

8.09

3.8

 

Table 4. Relative organ weights of mice fed diets containing 0.1% test chemical for 80 wk

Dose

No. of mice examined

Relatve organ weight (g/10 g bw)

Terminal body weight (g)

Brain

Heart

Liver

Spleen

Kidneys

Adrenals

Gonads

 

Males

0

40

1.07

0.64

5.47

0.38

1.67

27.5

0.45

35

0.1

20

1.21

0.59

5.84

0.45

1.50

27.8

0.41

36

0.25

20

1.15

0.63

5.88

0.44

1.62

27.1

0.45

33

0.5

12

1.32

0.71*

5.80

0.51

1.77*

29.6

0.49

32

1.0

23

1.28

0.63

5.29

0.39

1.60

28.5

0.48

35

 

Females

0

32

1.53

0.57

5.80

0.51

1.46

40.6

84.2

29

0.1

11

1.53

0.61

5.87

0.56

1.56

37.8

84.8

28

0.25

17

1.72*

0.64*

5.29

0.41

13.51

45.9

100.8

26

0.5

15

1.55

0.56

5.43

0.54

1.41

42.8

101.2

27

1.0

18

1.53

0.53

5.27

0.47

1.40

42.3

87.8

30

*P < 0.05.

Table 5. Incidence of histolooical findings (excluding tumours) in mice fed diets containin 0 0-1% test chemical for 80 wk

Tissue and finding

No. of mice affected

Males

Females

0

0.1

0.25

0.5

1.0

0

0.1

0.25

0.5

1.0

No. of mice examined

54

27

28

26

29

58

28

28

28

29

Lung

 

 

 

 

 

 

 

 

 

 

Chronic inflammatory infiltration

10

10

7

9

7

9

9

6

5

5

Congestion

1

1

0

2

1

3

3

0

1

1

Liver

 

 

 

 

 

 

 

 

 

 

Abscess

0

1

0

1

0

1

0

0

0

0

Degeneration (focal)

0

0

2

0

0

3

0

0

1

0

Macrophages (focal aggregations)

0

2

1

0

0

0

0

0

0

0

Cysts

1

0

0

0

0

0

1

0

0

0

Kidney

 

 

 

 

 

 

 

 

 

 

Glomerulonephrosis

2

0

5

1

1

2

1

0

0

0

Pyelonephritis

1

0

0

0

0

0

0

0

0

0

Lymphoid hyperplasia

1

1

1

0

1

0

0

0

0

0

Urethra

 

 

 

 

 

 

 

 

 

 

Chronic inflammation

1

1

2

0

0

0

0

0

0

0

Testes

 

 

 

 

 

 

 

 

 

 

Atrophy

1

0

0

1

0

-

-

-

-

-

Ovaries

 

 

 

 

 

 

 

 

 

 

Follicular cyst

-

-

-

-

-

3

1

1

0

5

Uterus

 

 

 

 

 

 

 

 

 

 

Cystic

-

-

-

-

-

1

0

0

2

2

Lymphoid tissue Reactive hyperplasia in

 

 

 

 

 

 

 

 

 

 

Spleen

2

0

2

1

1

0

1

1

0

0

Thymus

0

0

0

0

1

2

0

1

0

0

Lymphnodes

2

1

0

0

0

1

3

1

1

0

 

Table 6. Incidence of tumours in mice fed diets containing 0.1% test chemical for 80 wk

Tissue and finding

No. of mice affected

Males

Females

0

0.1

0.25

0.5

1.0

0

0.1

0.25

0.5

1.0

No. of mice examined

54

27

28

26

29

58

28

28

28

29

Lung

 

 

 

 

 

 

 

 

 

 

Adenoma

9

1

8

5

7

14

6

3

5

6

Mammary tissue

 

 

 

 

 

 

 

 

 

 

Fibroadenoma

-

-

-

-

-

0

1

0

0

0

Carcinoma

-

-

-

-

-

1

0

1

0

1

Adenoma

-

-

-

-

-

20

13

12

9

9

Uterus

 

 

 

 

 

 

 

 

 

 

Fibrosarcoma

-

-

-

-

-

0

1

0

0

0

Skin

 

 

 

 

 

 

 

 

 

 

Squamous cell carcinoma

0

0

0

0

1

0

0

0

1

0

Subcutaneous tissue

 

 

 

 

 

 

 

 

 

 

Fibroma

1

0

0

0

0

0

0

0

0

0

Ovary

 

 

 

 

 

 

 

 

 

 

Carcinoma

-

-

-

-

-

1

0

0

0

0

Lymphoid tissue

 

 

 

 

 

 

 

 

 

 

Lymphosarcoma

2

0

1

1

1

1

1

0

1

1

 

Applicant's summary and conclusion

Conclusions:
The no observed adverse effect level (NOAEL) for the test chemical in mice is considered to be 1 % (1300 mg/kg/day)
Executive summary:

Repeated dose toxicity test were performed on mice with different concentrations from 0.1, 0.25, 0.5 or 1.0% (130, 325, 650, 1300 mg/kg bw/d) test chemical for 80 wk. 30 males and 30 females was used for the treatment and group of 60 mice of each sex as control. During the study period the animals were observed for clinical signs, mortality, hematology, urine analysis was performed and the animals were subjected to gross and histopathology. The general condition and behaviour of the animals were observed frequently and any mouse that showed signs of ill-health was isolated, to be returned to its cage on recovery or to be killed if its condition deteriorated. The mice were weighed at the start of the experiment, at wk 3 and then at intervals of 2 wk until wk 73 of the experiment. Blood sample were taken at wk 28 and 55. At 80 wk, blood samples were collected from the aorta of all surviving mice during the autopsy. For haematology blood samples were collected and for urine sample from 6-hr period from three groups of five mice of each sex from the controls and the groups on the two highest dietary levels of the test chemical. Histopathology was also conducted. The ingestion of the test chemical had no effect on the condition or behaviour and mortality of the animals. The haematological examinations revealed only inconsistent isolated changes of statistical significance. At wk 28 the haemoglobin concentrations and red blood cell counts were lower in female mice fed diet containing if 0.5 or 1.0% test chemical than in the controls. There were no comparable findings in the males. The total white cell count of the male animals fed 1.0% test chemical in the diet was higher than that of the controls at this time, but there was no comparable change in this measurement in the females, or in either sex at any other time. At wk 55, the haemoglobin concentrations of male animals fed 0.5% of the coiouring in the diet were significantly (P < 0.05) lower than the control values. However, the corresponding value at the higher dietary level was not affected and there were no differences from the control value in the females. Also at wk 55, the erythrocyte counts of females fed 1.0% test chemical were lower (P < 0.01) than those of the controls, but this finding was again isolated. There were no statistically significant differences between the control and test samples taken at wk 80. There were only scattered differences in mean organ weights between treated and control animals. A lower brain weight in females, compared with the control value, was the only difference affecting animals fed 1%. This difference, which did not occur in the males, was only marginally significant and there was no significant difference when the weights were expressed relative to body weight By contrast, the relative brain weight of females fed 0.25% test chemical was higher than the control figure. Liver weights of female but not of male mice fed 0.25% were lower than control values, but again this was an isolated finding and there were no significant differences in relative liver weights. Kidney weights of male animals only were significantly lower than control values at the two lowest levels of treatment (0.1 and 0.25%), but a significant difference in relative kidney weights of males occurred only at the 0.5% level, at which a higher value was recorded for the treated mice. The only other significant differences occurred in the relative heart weights, which were raised in male mice fed 0.5% and in females fed 0.25%. The incidence of histological findings was similar in all groups of mice, including the controls. Most of the tumours in the study occurred with either a comparable or a greater incidence in the control groups than in the treated mice. Several isolated tumours were identified in mice given the lower levels, without comparable findings in the controls or in the highest dose group. They were a mammary fibroadenoma (in a female on 0.1%), a uterine fibromyoma (0.19%) and a squamous-cell carcinoma of the skin (female, 0.5%). The only tumour found at the highest dietary level without comparable control findings was a squamous- cell carcinoma of the skin in a male mouse fed 1%. Based on these considerations, the no observed adverse effect level (NOAEL) for the test chemical in mice is considered to be 1 % (1300 mg/kg/day).