Trisodium 3-hydroxy-4-(4'-sulphonatonaphthylazo)naphthalene-2,7-disulphonate

Repeated dose toxicity: dermal

Administrative data

Endpoint:

chronic toxicity: dermal

Remarks:

combined repeated dose and carcinogenicity

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: FD&C Green NO. 3, Red No. 2, Red No. 4, Yellow No. 6, and External D8.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 Swiss Webster mice were carried out using FD&C Red 2 to determine the dermal toxicity and carcinogenicity of the test chemical

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

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-sulfonato-1-naphthyl)diazenyl]naphthalene-2,7-disulfonate

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 also known as FD&C Red 2
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
Acclimation period:No data

Administration / exposure

Type of coverage:

open

Vehicle:

water

Details on exposure:

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.

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
USE OF RESTRAINERS FOR PREVENTING INGESTION: yes/no: no

Analytical verification of doses or concentrations:

no

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

Doses / concentrations

Remarks:

Doses / Concentrations:
0.1 ml of dye 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:

other: 100 male and 100 female mice were used as controls

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 palable 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:

Sacrifice and pathology
GROSS PATHOLOGY: Yes (see table) / No / No data: Yes
HISTOPATHOLOGY: Yes (see table) / No / No data: Yes

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Dermal irritation:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

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:

not specified

Clinical biochemistry findings:

not specified

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

effects observed, treatment-related

Details on results:

Clinical signs and mortality: There were no significant differences in survival rate in groups that had color applied
Dermal Irritation: No adverse reactions or pathological changes were observed after 19.5 months dermal application of test chemical
Body weight and weight gain: 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.
Neurobehaviour: 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
Gross pathology: 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 controls

Effect levels

Dose descriptor:

NOAEL

Effect level:

0.1 other: ml of the 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

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

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

 

Group	Test chemical	Sex	Mean body weight^a
			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 Water^b	M	17	33	37	38	36	36
A	Distilled Water^b	F	18	29	33	33	36	36

 

a- Treatment groups-50 per sex.

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
		%S^a	SI^b	%S^a	SI^b	%S^a	SI^b	%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 mouse days survived compared to anticipated number of days if all the surviving animals lived to the end of the experiment.

Applicant's summary and conclusion

Conclusions:

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. Mice were painted once weekly in an area that precluded oral exposure with 0.1 ml of the solution or suspension containing 1.0% of the FD&C Red 2 to a depilated 6 cm2 area. Survival, body weight, and palpable growths were followed for the 19.5 month period in the test and control groups.
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.
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 inSwissWebster 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.

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.