Disodium 6-hydroxy-5-[(2-methoxy-4-sulphonato-m-tolyl)azo]naphthalene-2-sulphonate

Endpoint summary

Description of key information

Local lymph node assay (LLNA) of the test chemical was performed on female CBA/J mice by measuring the cell proliferation in the draining lymph nodes after topical application onto the ears.The mean stimulation indices of 0.6, 0.7, 0.9 and 0.9 were obtained when the test chemical was tested at 0.5, 1, 2, 4 % (w/v) in DMSO, while when the  test chemical was tested at 0.5, 1, 2, 4 % in aqua/acetone (1:1) mixed with olive oil (4:1),  the mean stimulation indices of 1.2, 1.0, 0.9 and 0.9 were obtained.As no relevant increase in the mean stimulation indices was observed (all figures were well below the trigger value of three), Hence it is considered that the test chemical was not skin sensitizer in mice by using Local lymph node assay (LLNA).

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:

4 (not assignable)

Rationale for reliability incl. deficiencies:

secondary literature

Justification for type of information:

Data is from safety assessment reports

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Principles of method if other than guideline:

The skin sensitization study of the test chemical was performed on mice by using Mice local lymph node assay (LLNA)

GLP compliance:

not specified

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA:J

Sex:

female

Vehicle:

other: aqua/acetone (1:1) and olive oil (4:1) and DMSO

Concentration:

a) 0.5, 1, 2, 4 % (w/v) in DMSO
b) 0.5, 1, 2, 4 % (w/v) in aqua/acetone (1:1) mixed with olive oil (4:1)

No. of animals per dose:

Total:15
Test group :5 female
control group :5 female
positive control group :5 female

Details on study design:

Details on study design

PRE-SCREEN TESTS:
- Compound solubility: No data available
- Irritation: No data available
- Systemic toxicity: No data available
- Ear thickness measurements: No data available
- Erythema scores: No data available

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay (LLNA)
- Criteria used to consider a positive response: measuring the cell proliferation in the draining lymph nodes after topical application onto the ears.

TREATMENT PREPARATION AND ADMINISTRATION: 25 μl of 0 (vehicle only), 0.5, 1, 2 and 4 %of test chemical in either DMSO or a mixture of aqua/acetone (1:1) with olive oil (4:1) were applied on three consecutive days to the surface of the ear of 5 female CBA/J mice per group.
After application, the ears were dried for about 5 minutes by means of a hair dryer.
A positive control (p-phenylenediamine at 1 % in DMSO) was investigated in parallel under identical tests conditions.

Positive control substance(s):

other: p-phenylenediamine at 1 % in DMSO

Statistics:

The mean dpm per treated group and the stimulation index (test item compared to the concurrent vehicle control)

Positive control results:

an increase in the stimulation index by a Factor of 7.8.

Key result

Parameter:

SI

Value:

0.9

Test group / Remarks:

5

Remarks on result:

other: not sensitizing

Cellular proliferation data / Observations:

CLINICAL OBSERVATIONS: Animals were observed daily before and at least once after dosing.
BODY WEIGHTS: Body weight was determined on day 1 and day 5.
mean stimulation index For
a)test substance as 0.5, 1, 2, 4 % (w/v) in DMSO mean stimulation indices of 0.6, 0.7, 0.9 and 0.9 were obtained
b) test substance as 0.5, 1, 2, 4 % in aqua/acetone (1:1) mixed with olive oil (4:1) mean stimulation indices of1.2, 1.0, 0.9 and 0.9 were obtained
c) The positive control (PPD, 1 % in DMSO) caused an increase in the stimulation index by a factor of 7.8.

Interpretation of results:

other: Not sensitizing

Conclusions:

The mean stimulation indices of 0.6, 0.7, 0.9 and 0.9 were obtained when the test chemical was tested at 0.5, 1, 2, 4 % (w/v) in DMSO, while when the  test chemical was tested at 0.5, 1, 2, 4 % in aqua/acetone (1:1) mixed with olive oil (4:1),  the mean stimulation indices of 1.2, 1.0, 0.9 and 0.9 were obtained. As no relevant increase in the mean stimulation indices was observed (all figures were well below the trigger value of three), Hence it is considered that the test chemical was not skin sensitizer in mice by usingLocal lymph node assay (LLNA).

Executive summary:

Local lymph node assay (LLNA) of the test chemical was performed on female CBA/J mice by measuring the cell proliferation in the draining lymph nodes after topical application onto the ears.

In control group only 25µl of vehicle while in test group , 0.5, 1, 2, 4 % (w/v) test substance dissolved in DMSO and 0.5, 1, 2, 4 % in aqua/acetone (1:1) mixed with olive oil (4:1).Positive control p-phenylenediamine at 1 % in DMSO was applied on three consecutive days to the surface of the ear of 5 female CBA/J mice per group. After application, the ears were dried for about 5 minutes by means of a hair dryer.

 

All the animals were observed twice daily for any clinical signs and morbidity/mortality before and after given dose.Body weight was determined on day 1 and 5.On day 5 the mice received an intravenous injection of 250 μl phosphate buffered saline containing 20.8 μCi of [H³] methyl thymidine. After 5hr animals were died by giving CO2-inhalation and the draining auricular lymph node was removed and weighed. The cell suspension for each animal prepared. The cells were predicated by TCA. The liquid scintillation counting as disintegration per minute (dpm) was used to determined radioactivity due to incorporation of [H3] methyl thymidine in the pellets. The stimulation index was calculated by comparing test material with concurrent vehicle control.

 

The mean stimulation indices of 0.6, 0.7, 0.9 and 0.9 were obtained when the test chemical was tested at 0.5, 1, 2, 4 % (w/v) in DMSO, while when the  test chemical was tested at 0.5, 1, 2, 4 % in aqua/acetone (1:1) mixed with olive oil (4:1),  the mean stimulation indices of 1.2, 1.0, 0.9 and 0.9 were obtained.

 

As no relevant increase in the mean stimulation indices was observed (all figures were well below the trigger value of three), Hence it is considered that the test chemical was not skin sensitizer in mice by usingLocal lymph node assay (LLNA).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Various studies have been reviewed to evaluate the degree of dermal sensitization caused by the test chemical in living organisms. These include in vivo experimental studies performed on guinea pigs, mice, humans for the test chemical. The results are summarized below:

 

Local lymph node assay (LLNA) of the test chemical was performed on female CBA/J mice by measuring the cell proliferation in the draining lymph nodes after topical application onto the ears. In control group only 25µl of vehicle while in test group , 0.5, 1, 2, 4 % (w/v) test substance dissolved in DMSO and 0.5, 1, 2, 4 % in aqua/acetone (1:1) mixed with olive oil (4:1).Positive control p-phenylenediamine at 1 % in DMSO was applied on three consecutive days to the surface of the ear of 5 female CBA/J mice per group. After application, the ears were dried for about 5 minutes by means of a hair dryer. All the animals were observed twice daily for any clinical signs and morbidity/mortality before and after given dose.Body weight was determined on day 1 and 5.On day 5 the mice received an intravenous injection of 250 μl phosphate buffered saline containing 20.8 μCi of [H3] methyl thymidine. After 5hr animals were died by giving CO2-inhalation and the draining auricular lymph node was removed and weighed. The cell suspension for each animal prepared. The cells were predicated by TCA. The liquid scintillation counting as disintegration per minute (dpm) was used to determined radioactivity due to incorporation of [H3] methyl thymidine in the pellets. The stimulation index was calculated by comparing test material with concurrent vehicle control. The mean stimulation indices of 0.6, 0.7, 0.9 and 0.9 were obtained when the test chemical was tested at 0.5, 1, 2, 4 % (w/v) in DMSO, while when the  test chemical was tested at 0.5, 1, 2, 4 % in aqua/acetone (1:1) mixed with olive oil (4:1),  the mean stimulation indices of 1.2, 1.0, 0.9 and 0.9 were obtained. As no relevant increase in the mean stimulation indices was observed (all figures were well below the trigger value of three), Hence it is considered that the test chemical was not skin sensitizer in mice by using Local lymph node assay (LLNA).

This is supported by the results of Draize- Shelanski Repeat Insult Patch Test performed on 200 human subjects to determine the degree of dermal sensitization caused by the test chemical. The test chemical was applied to the subject's volvar forearms as an aqueous solution for 10 alternate days, for 24-hour periods, followed by a 14-day rest period. Challenge treatment were then applied under occlusion to fresh skin sites on the subjects scapular backs for 24 hours. The test chemical did not produce any skin allergic reaction in the induction as well as challenge treatment.

Hence, the test chemical was considered to be not sensitizing to skin.

The above results are supported by a prophetic patch test was performed to evaluate the dermal sensitization potential of the test chemical.

The test chemical was applied either as a neat or as a 25% aqueous solution to the skin of 200 human subjects. The initial exposure to the compound was for 72 hours, and this was followed by a 24-hour application 10 to 14 days later. None of the subjects exhibited compound induced irritation or sensitization. Hence, the test chemical can be considered to be not sensitizing to skin.

Based on the available data, it can be concluded that the test chemical can be considered to be not sensitizing to skin. Thus it can be further 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 data, it can be concluded that the test chemical can be considered to be not sensitizing to skin. Thus it can be further classified under the category “Not Classified” as per CLP regulation.