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Toxicological information

Carcinogenicity

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Administrative data

Endpoint:
carcinogenicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: data from peer - reviewed journals

Data source

Reference
Reference Type:
publication
Title:
SKIN PAINTING STUDIES IN MICE ON 11 FD&C AND D&C COLORS: F D&C Green NO. 3, Red No. 2, Red No. 4, Yellow No. 6, and External D&C No. 7, D&C Orange No. 4, Violet No. 2, Red No. 17, Red No. 34, and Yellow No. 7
Author:
Dr. STEVEN CARSON
Year:
1984
Bibliographic source:
J. Toxicol. Cut. & Ocular Toxicol. 3(3), 309-331 (1984)

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
Principles of method if other than guideline:
Skin painting studies in SwissWebster mice were carried out using FD&C Red 2 to determine the dermal toxicity and carcinogenicity of the test chemical.
GLP compliance:
not specified

Test material

Constituent 1
Chemical structure
Reference substance name:
Trisodium 3-hydroxy-4-(4'-sulphonatonaphthylazo)naphthalene-2,7-disulphonate
EC Number:
213-022-2
EC Name:
Trisodium 3-hydroxy-4-(4'-sulphonatonaphthylazo)naphthalene-2,7-disulphonate
Cas Number:
915-67-3
Molecular formula:
C20H14N2O10S3.3Na
IUPAC Name:
trisodium 3-hydroxy-4-(4'-sulphonatonaphthylazo)naphthalene-2,7-disulphonate
Constituent 2
Reference substance name:
trisodium (4E)-3-oxo-4-[(4- sulfonato-1- naphthyl)hydrazono]naphthalene- 2,7-disulfonate
IUPAC Name:
trisodium (4E)-3-oxo-4-[(4- sulfonato-1- naphthyl)hydrazono]naphthalene- 2,7-disulfonate
Constituent 3
Reference substance name:
Amaranth dye
IUPAC Name:
Amaranth dye
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Name of test material (as cited in study report): Amaranth dye
Molecular formula (if other than submission substance): C20H11N2Na3O10S3
Molecular weight (if other than submission substance): 604.47
Smiles notation (if other than submission substance): c1ccc2c(c1)c(ccc2S(=O)(=O)[O])N=Nc3c4ccc(cc4cc(c3O)S(=O)(=O)[O-])S(=O)(=O)[O-].[Na+].[Na+].[Na+]
InChl (if other than submission substance): 1S/C20H14N2O10S3.3Na/c23-20-18(35(30,31)32)10-11-9-12(33(24,25)26)5-6-13(11)19(20)22-21-16-7-8-17(34(27,28)29)15-4-2-1-3-14(15)16;;;/h1-10,23H,(H,24,25,26)(H,27,28,29)(H,30,31,32);;;/q;3*+1/p-3
Substance type: Organic
Physical state: Solid

Test animals

Species:
mouse
Strain:
Swiss Webster
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
Source: no data
Age at study initiation: no data
Weight at study initiation: mean body weights 17 to 25 g
Fasting period before study: no data
Housing: Mice of same sex were housed 5/cage
Diet (e.g. ad libitum):Purina Laboratory Chow, ad libitum
Water (e.g. ad libitum): fresh water, ad libitum

Administration / exposure

Route of administration:
dermal
Type of inhalation exposure (if applicable):
not specified
Vehicle:
water
Details on exposure:
TEST SITE
Area of exposure: dorsal area of each animal
% coverage: An area of approximately 6 cm2
Type of wrap if used: no data
Time intervals for shavings or clipplings: Subsequent periodic clipping was performed according to the rate of hair growth.

REMOVAL OF TEST SUBSTANCE
Washing (if done): no data
Time after start of exposure: no data

TEST MATERIAL
Amount(s) applied (volume or weight with unit): 0.1 ml of color solution
Concentration (if solution): 0.1 ml of color solution containing 1.0% of the respective color on an actual pigment basis
Constant volume or concentration used: yes/no: yes
For solids, paste formed: yes/no: no
PREPARATION OF DOSING SOLUTIONS:
Materials were prepared fresh each week by homogenizing in a Waring blender and kept under magnetic stirrer during treatment. The dosage was
applied with an automatic syringe and uniformly distributed on the exposed skin with a rubber applicator.
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
no data
Duration of treatment / exposure:
19.5 months
Frequency of treatment:
Once a week for 18 - 19.5 months
Post exposure period:
no data
Doses / concentrations
Remarks:
Doses / Concentrations:
0.1 ml solution
Basis:
no data
No. of animals per sex per dose:
50 male and 50 female mice
Control animals:
yes, concurrent vehicle
Details on study design:
no data
Positive control:
no data

Examinations

Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS: Yes / No / No data: Yes
Time schedule: Daily

DERMAL IRRITATION (if dermal study): Yes / No / No data: Yes
Time schedule for examinations: daily

BODY WEIGHT: Yes / No / No data: Yes
Time schedule for examinations: 3,6, 9, 12, 15, 18 and 19.5 months

NEUROBEHAVIOURAL EXAMINATION: Yes / No / No data: Yes
Time schedule for examinations: Daily
Dose groups that were examined: test group
Battery of functions tested: sensory activity / grip strength / motor activity / other: no data

OTHER: Each mouse was observed daily for behavior, survival and visible or palpable growth. Records included observations on the incidence, size, and description of any such growth. Mean body weight was determined at 3,6, 9, 12, 15, 18 and 19.5 months
Sacrifice and pathology:
GROSS PATHOLOGY:
Yes (see table) / No / No data : Yes
HISTOPATHOLOGY:
Yes (see table) / No / No data: Yes
Other examinations:
no data

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
effects observed, treatment-related
Details on results:
Clinical sign and mortality: There were no significant differences in survival rate in groups that had color applied
Body weight and weight gain: No adverse reactions or pathological changes were observed after 19.5 months dermal application of test chemical
Food consumption & compound intake: Unusually high or low body weights observed late in the study for some of the groups, were due to only a few surviving animals which reduced the biological significance of the averages.
Neuro behavior:During the study, there were no abnormalities observed in behavior of mice in the experimental or control groups nor were there any significant differences in rate of growth
Histopathology: non-neoplastic: There was no significant difference in the incidence of this lesion (lymphoma) among the test and control groups. There was no indication of treatment related pathology since most lesions were also evident in the controlsTissues from any abnormal mass were examined histologically in a follow- up sequence of microscopic preparations and examinations. The total numbers of animals examined were increased to include all in whom gross changes.
Relevance of carcinogenic effects / potential:
non - carcinogenic in nature

Effect levels

Dose descriptor:
NOAEL
Effect level:
0.1 other: ml test solution
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse reactions or pathological changes were observed after 19.5 months
Remarks on result:
other: Effect type: carcinogenicity (migrated information)

Any other information on results incl. tables

Table 1: Skin Painting studies in mice (growth rate)

 

Group

Test chemical

Sex

Mean body weighta

Month

0

3

6

12

18

19.5

A

FD&C Red 2

M

18

33

36

35

33

35

A

FD&C Red 2

F

18

30

34

36

31

21

A

Distilled Waterb

M

17

33

37

38

36

36

A

Distilled Waterb

F

18

29

33

33

36

36

 

a- Treatment groups-50 persex.

b- Control group size- 100 malesand100 females

 

Table 2: Skin Painting Studies in mice (% survival and survival rate)

Test group

Sex

Month

6

12

18

19.5

%Sa

SIb

%Sa

SIb

%Sa

SIb

%Sa

SIb

Distilled Water

M

77

92

32

73

7

54

3

80

Distilled Water

F

90

98

40

79

8

60

7

56

FD&C Red 2

M

76

87

34

71

10

53

6

51

FD&C Red 2

F

80

94

44

80

18

62

8

59

 

a - %S denotes percent survival.

b - SI,survival index: %ratio of mousedayssurvived compared to anticipated number ofdaysif all the surviving animals lived to the end of the experiment.

 

Table 3: Histopathological Observations in Mice in Skin Painting Studies with FD&C Dyes

Organ

Color

None

(Distilled Water)

FD&C Red 2

No of mice examined/sex

46M

44F

18 M

20F

Liver

Malignant lymphoma – all grades

 

20

28

9

10

Spleen

Malignant lymphoma – all grades

 

21

22

9

7

Increased number of megakaryocytes

 

8

7

5

4

Kidneys

Malignant lymphoma – all grades

 

28

28

4

10

Leukemic and round cell infiltration

 

1

1

4

4

Leukocyte aggregation

 

4

 

2

 

Salivary Glands

Malignant lymphoma – all grades

 

 

2

1

 

Lymph nodes

Malignant lymphoma – all grades

 

7

7

1

3

Lungs

Malignant lymphoma – all grades

 

9

16

8

7

Leukocytic aggregations and perivascular infiltration

 

3

2

1

 

Inflammation, pneumonia, bronchitis

 

14

7

6

4

Congestion

 

3

1

1

 

Necrotic changes

 

5

 

5

 

Hemorrhage

 

3

 

1

1

Thymus

Malignant lymphoma – all grades

 

5

9

1

4

Skin

Malignant lymphoma

 

6

6

 

1

Growths

Subcutaneous adenocarcinoma

 

3

 

 

3

Applicant's summary and conclusion

Conclusions:

Repeated dermal application of 0.1 ml of dye solution applied to the dorsal area of Swiss Webster mice weekly for 19.5 months failed to increase the incidence of neoplasia resulting from the Amaranth dye when compared to the findings in the concurrent control water groups.
Hence No Observed Adverse Effect Level (NOAEL) for Amaranth dye was concluded to be 0.1 ml of test solution when applied dermally to mice for 19.5 months
Executive summary:

Skin painting studies in Swiss Webster mice were carried out using FD&C Red 2 to determine the dermal toxicity and carcinogenicity of the test chemical.

Amaranth dye was dissolved in water for application to the depilated skin of mice once a week for approximately 19.5 months. 50 male and 50 female Swiss Webster mice were used as test animals.100 female and 100 male mice were used in each control group. Swiss-Webster mice with an initial weight ranging from 17 to 25 g were used in this study. All groups were equally divided as to sex. Mice of the same sex were housed five per cage and were allowed free access to pellets of Purina Laboratory Chow and fresh water. Initially, the hair on the dorsal area of each animalwasclipped with an animal clipper free of lubricating oil. Subsequent periodic clipping was performed according to the rate of hair growth.Anarea of approximately 6 cm2 was treated twice weekly.

Materials were prepared fresh each week by homogenizing in a Waring Blender and kept under magnetic stirrer during treatment. The dosage was applied with an automatic syringe and uniformly distributed on the exposed skin with a rubber applicator. Once each week for the duration of the study 0.1 ml of the solvent or of the color solution containing 1.0% of the Amaranth dye on an actual pigment basis was applied to the depilated area of the mice. Each mouse was observed daily for behavior, survival and visible or palpable growth. Records included observations on the incidence, size, and description of any such growths.

All surviving animals were terminated after approximately 19.5 months, when a marked increase of geriatric mortality became apparent. All mice were necropsied after they died or were sacrificed. Organs were fixed in 10% formalin solution after recording any gross pathological findings. The initial microscopic examinations of the tissues listed below involved approximately 50% of the treated animals in this group, but was enlarged in a supplemental series to include histological examinations carried out on all tumors and all grossly abnormal organs or tissues.

The following tissues were preserved in 10% formalin:

Brain, Pituitary. Thyroid, Spleen,Thymus, Liver, Kidney, Adrenal, Stomach, Small intestines, Large intestines, Urinary bladder, Axillary lymph node, Kidney, ovary, Skin from area of treatment, Any tissue masses, Grossly abnormal organs or tissues

 

There were no significant differences in survival rate in groups that had the color applied.No adverse reactions or pathological changes were observed after 19.5 months dermal application of test chemical.Unusually high or low body weights observed late in the study for some of the groups, were due to only a few surviving animals which reduced the biological significance of the averages. During the study, there were no abnormalities observed in behavior of mice in the experimental or control groups nor were there any significant differences in rate of growth.

 

Repeated dermal application of 0.1 ml of dye solution applied to the dorsal area of Swiss Webster mice weekly for 19.5 months failed to increase the incidence of neoplasia resulting from the Amaranth dye when compared to the findings in the concurrent control water groups.

Hence No Observed Adverse Effect Level (NOAEL) for Amaranth dye was concluded to be 0.1 ml of test solution when applied dermally to mice for 19.5 months.