Disodium 2-(2,4,5,7-tetraiodo-6-oxido-3-oxoxanthen-9-yl)benzoate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

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 scientific journal

Data source

Reference

Reference Type:

publication

Title:

Application of Sensitive Mouse Lymph Node Assay for Detection of Contact Sensitization

Author:

Yoshiaki Ikarashi, Toshie Tsuchiya and Akitada Nakamura

Year:

1996

Bibliographic source:

JOURNAL OF APPLIED TOXICOLOGY, VOL. 16(4), 349-354 (1996)

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: as mentioned below

Principles of method if other than guideline:

In the SLNA as well as the LLNA, the lymph node cell (LNC) proliferation of test chemical is measured by incorporation of radioactive (3H) methyl thymidine (3HTdR).

GLP compliance:

not specified

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Disodium 2-(2,4,5,7-tetraiodo-6-oxido-3-oxoxanthen-9-yl)benzoate

EC Number:

240-474-8

EC Name:

Disodium 2-(2,4,5,7-tetraiodo-6-oxido-3-oxoxanthen-9-yl)benzoate

Cas Number:

16423-68-0

Molecular formula:

C20H8I4O5.2Na

IUPAC Name:

disodium 2-(2,4,5,7-tetraiodo-6-oxido-3-oxoxanthen-9-yl)benzoate

Details on test material:

- Name of test material (as cited in study report):
D&C Red 3
- Molecular formula (if other than submission substance): C20-H6-I4-O5.2Na
- Molecular weight (if other than submission substance): 879.8424 g/mol
- Substance type: Organic
- Physical state: Solid

In vivo test system

Test animals

Species:

mouse

Strain:

Balb/c

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Japan SLC Inc, Shizuoka,Japan
- Age at study initiation: 6-8 weeks old
- Weight at study initiation: No data available
- Housing: No data available
- Diet (e.g. ad libitum): No data available
- Water (e.g. ad libitum): No data available
- 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

Study design: in vivo (LLNA)

Vehicle:

other: Intradermal - Saline; Topical - DMSO

Concentration:

Intradermal Injection - 0.25% in saline
Topical Application - 5% in DMSO

No. of animals per dose:

Groups of mice (n= 3)

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility:
- Irritation:
- Systemic toxicity:
- Ear thickness measurements:
- Erythema scores:

MAIN STUDY : Groups of mice (n= 3) were initially injected intradermally with a total of 50 µl of test chemical-FCA emulsion into two sites of the abdominal skin at both sides of the ventral midline. Five days after injection, 25µl of test chemical in vehicle was applied to both sides of each ear for three consecutive days. Control mice were treated by intradermal injection of vehicle-FCA emulsion into the abdomen and then after 5 days they were exposed to vehicle alone on the ears for three consecutive days. The next day following the final topical application, auricular lymph nodes were excised and pooled for each experimental group. A single-cell suspension of LNC was prepared by mechanical disaggregation through a sterile 200-mesh gauge and washed once with Hank's balanced salt solution. The LNCs were resuspended in RPMI- 1640 culture medium supplemented with 25 mM N-2-hydroxyethylpiperazine-N'- 2-ethanesulphonic acid (HEPES), 100 U ml-I penicillin, 100 µg/ml streptomycin and 10% fetal calf serum, and the total LNC number was determined using an automated cell counter. The LNC suspensions (1 x 106 cells) were seeded into 96-well culture plates (five wells per group) and cultured with 0.5 µCi [3H]methyl thymidine ('HTdR) for 24 h at 37°C in a humidifed atmosphere of 5% CO, in air. Culture was terminated by a semiautomatic cell harvester, and the 3HTdR incorporation was determined by liquid scintillation counting. The increases in LNC number and 3HTdR incorporation relative to controls were derived for each experimental group and expressed as SIn and SIp, respectively

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method:
- Criteria used to consider a positive response: SItotal as obtained from SIn x SIp, which indicates the total lymph node activation induced by the test chemical. A chemical was regarded as positive (a sensitizer) if it showed an SItotal value of 3 or more.

TREATMENT PREPARATION AND ADMINISTRATION:

Results and discussion

In vivo (LLNA)

Results

Parameter:

SI

Value:

> 1.15 - <= 1.55

Test group / Remarks:

test group

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA :

DETAILS ON STIMULATION INDEX CALCULATION :SIn (stimulation index of LNC number) = 1.35; SIp (stimulation index of LNC proliferation) = 1.15; SI total (stimulation index of total lymph node response) = 1.55

EC3 CALCULATION : The EC3 could not be calculated as the SI values were less than 3.

CLINICAL OBSERVATIONS:

BODY WEIGHTS

Any other information on results incl. tables

Results of the SLNA (sensitive mouse lymph node assay) with a range of dyes

                          Topical application

CONCENTRATION	Vehicle	SIn	SIP	SItotal
5 %	DMSO	1.35	1.15	1.55

 

DMSO = dimethylsulphoxide

SI_n= stimulation index of LNC number;

SI_P= stimulation index of LNC proliferation;

SI_total= stimulation index of total lymph node response.

Applicant's summary and conclusion

Interpretation of results:

other: not sensitising

Conclusions:

The test material was considered as not sensitizing to the skin of BALB/c strain mice in the sensitive mouse lymph node assay (SLNA)

Executive summary:

A sensitive mouse lymph node assay (SLNA) was performed on Female BALB/c strain mice, 6-8 weeks old to assess the skin sensitization potential of the test chemical.A sensitive mouse lymph node assay (SLNA) developed for the identification of contact allergens and succeeded in enhancement of the sensitivity of the LLNA using Freud’s complete adjuvant (FCA) and two application phases: intradermal injection and repeated topical application. In this method, a group of 3 mice were intra dermally injected with 50 µl of test chemical –FCA emulsion into two sites of the abdominal skin at both sides of the ventral line. After 5 days 25 µl of test chemical in vehicle was applied to both sides of each ear for three consecutive days. Control mice were treated by intradermal injection of vehicle-FCA emulsion into the abdomen and then after 5 days they were exposed to vehicle alone on the ears for three consecutive days. The next day following the final topical application, auricular lymph nodes were excised and a single-cell suspension of LNC was prepared by mechanical disaggregation through a sterile 200-mesh gauge and washed once with Hank's balanced salt solution. The LNCs were resuspended in RPMI- 1640 culture medium supplemented with different supplements and the total LNC number was determined using an automated cell counter.The LNC suspensions (1 x 10⁶cells) were seeded into 96-well culture plates (five wells per group) and cultured with 0.5 µ Ci [³H] methyl thymidine (³HTdR) for 24 h at 37°C in a humidified atmosphere of 5% CO₂, in air. Culture was terminated by a semiautomatic cell harvester, and the ³HTdR incorporation was determined by liquid scintillation counting. The increases in LNC number and³HTdR incorporation relative to controls were derived for each experimental group and expressed as SI_nand SI_P, respectively; SI_total as obtained from SI_nxSI_P, which indicates the total lymph node activation induced by the test chemical.A chemical was regarded as positive if it showed anSI_total value of 3 or more. From the results obtainedSI_totalvalue for the test chemical was calculated to be 1.55. Hence, it was concluded that the test chemical elicited negative responses under the conditions used and can be considered to be non-sensitizing to mice.