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

Endpoint summary

Description of key information

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 Freund’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_totalas obtained from SI_nx SI_p, which indicates the total lymph node activation induced by the test chemical. A chemical was regarded as positive if it showed a SI_totalvalue of 3 or more. From the results obtained, SI_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.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

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

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)

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

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:

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

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.

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

 

Various studies have been reviewed to determine the dermal sensitization potential of the test chemical. These include in vivo experimental studies performed on mice, guinea pigs as well as humans for the test chemical. The results are mentioned below:

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 Freund’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_totalas obtained from SI_nx SI_p, which indicates the total lymph node activation induced by the test chemical. A chemical was regarded as positive if it showed a SI_totalvalue of 3 or more. From the results obtained, SI_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.

This is result supported by a Skin Prick Test conducted on a patient who had a history of infectious bronchitis. At the age of 8 years, the patient had taken cefaclor suspension (5 ml) (trade name Panacef, Eli Lilly, Florence, Italy, 250 mg/5 ml) for infectious bronchitis. 6 years later, the child was referred to the allergy clinic to determine the cause of this type of adverse reaction. He was now 14 years old, with allergic rhinitis. Panacef suspension contains excipients such as test chemical and strawberry which might cause adverse reactions. A skin prick test (SPT) to test chemical (10 mg/ml) was carried out on the volar side of the forearm with a plastic lancet (1-mm tip) made by Laboratorio Lofarma (Milan, Italy). A positive control with 10 mg/ml histamine was included. SPT results were assessed as positive (wheal response ≥2+) according to the recommendations of the European Academy of Allergology and Clinical Immunology. The Skin Prick Test result for test chemical was negative (0+). Hence the test chemical was considered to be not sensitizing to the skin.

The above result was supported by the Guinea pig maximization test was conducted according to OECD 406 Guidelines with 0.1% solution of test chemical for 3 weeks. Animals received 10 intradermal injections of a 0.1% solution of the colour. The last six injections were given using Freund’s adjuvant. After 14 days, a further intradermal injection was given as challenge. A second epidermal, occlusive challenge was given after additional 10 days. After a further intradermal challenge, positive reactions could be observed in 11 of 19 erythrosine-treated animals. After the epidermal challenge, none of the 19 animals exhibited signs of positive reactions. Therefore, a further intradermal challenge was performed later on after which 15 of 19 animals reacted positive. Epidermal challenge did not cause any effect on skin; hence, the test chemical was not regarded as a skin sensitizer.

Based on the available studies, the test chemical certainly lacks the potential to cause dermal sensitization reactions when tested on humans, guinea pigs as well as mice. Hence it can be concluded to be non-sensitizing to skin and classified under the category “Not Classified” as per CLP Regulation.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the available studies, the test chemical certainly lacks the potential to cause dermal sensitization reactions when tested on humans, guinea pigs as well as mice. Hence it can be concluded to be non-sensitizing to skin and classified under the category “Not Classified” as per CLP Regulation.