Disodium 2,5-dichloro-4-[4-[[5-[[(dodecyloxy)carbonyl]amino]-2-sulphonatophenyl]azo]-4,5-dihydro-3-methyl-5-oxo-1H-pyrazol-1-yl]benzenesulphonate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From February 19 to 25, 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

22 July 2010

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Acid Yellow 218

IUPAC Name:

Acid Yellow 218

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Remarks:

CBA/Ca Ola Hsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: TOXI-COOP ZRT. H-1103, Budapest, Cserkesz u. 90.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 10-12 weeks old
- Weight at study initiation: 19.9 – 23.9 g ( The weight variation in animals involved in the study did not exceed ± 20 % of the mean weight).
- Housing: Grouped caging (4 animals/cage)
- Diet (e.g. ad libitum): ssniff® Rat/Souris-Elevage E complete ad libitum
- Water (e.g. ad libitum): tap water from watering bottles ad libitum
- Acclimation period: 14 days
- Indication of any skin lesions: Only healthy animals (and not showing any sign of skin lesion) were used

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 – 70 %
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.
- IN-LIFE DATES: From: To:

Study design: in vivo (LLNA)

Vehicle:

dimethyl sulphoxide

Concentration:

50 %, 25 %, 10 % and 5 % (w/v) as formulation (apparently solutions)

No. of animals per dose:

4 animals for treatment group

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: soluble in DMSO and formulated at 50 %, 25 % and 10 % (w/v)
- Irritation: the effect on the ear thicknesses observed in the 50 % (w/v) dose group was not considered as obvious sign of a significant irritation.
- Systemic toxicity: No mortality, significant, treatment related effect on body weights or any other sign of systemic toxicity were observed.
- Ear thickness measurements: an increase of > 25 % of ear thickness (compared to the initial values) was observed in the 50 % (w/v) dose group for one of the two animals on Day 6 (30 % or 25 % increase was observed on the left or right ear, respectively). No significantly (> 25 %) increased ear thicknesses were observed for the other animals in this dose group or in the other dose groups on the days of measurement (Day 3 or Day 6).
- Erythema scores: No erythema or any other local effect was observed in any dose group during the test.

MAIN STUDY
The test item was examined in the main test at concentrations of 50 %, 25 %, 10 % and 5 % (w/v).
An appropriate positive control (α-Hexylcinnamaldehyde, HCA), and furthermore two negative control groups dosed with the vehicles of the test and positive control groups, respectively, were employed.
The positive control item [25 % (w/v) HCA in Acetone: Olive oil 4:1 (v/v) mixture (AOO)] induced significant stimulation over the relevant control (SI = 5.6) thus confirming the validity of the assay.

ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: the test item is considered as a skin sensitizer, if:
Exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than recorded in control mice, as indicated by the stimulation index (SI ≥ 3). However, the strength of the dose-response, the statistical significance and the consistency of the solvent/vehicle and positive control responses may also be used when determining whether a borderline result is declared positive.

TREATMENT PREPARATION AND ADMINISTRATION: Each mouse was topically treated with 25 μL of the appropriate formulations of the test item, the positive control substance or the vehicles using a pipette, on the dorsal surface of each ear. After the treatment animals were returned to their cages. Each animal was dosed once a day for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

6.1

Test group / Remarks:

Group treated at 50 % (w/v) test item concentration

Results 2

Parameter:

SI

Value:

2.7

Test group / Remarks:

Group treated at 25 % (w/v) test item concentration

Results 3

Parameter:

SI

Value:

1.9

Test group / Remarks:

Group treated at 10 % (w/v) test item concentration

Results 4

Parameter:

SI

Value:

1.5

Test group / Remarks:

Group treated at 5 % (w/v) test item concentration

Results 5

Parameter:

EC3

Value:

> 25

Test group / Remarks:

Due to the unreliable SI value at the 50 % (w)v) test concentration no EC3 value for the test item was calculated in this LLNA. On the other hand, it is evident from the test results that the theoretical EC3 value is > 25 %

Any other information on results incl. tables

Body Weight Measurement

Body weight decrease by > 5 % was observed in the following test groups: positive control group (1 of the 4 animals with 7 % decrease) and in the 25 % (w/v) dose group (1 of the 4 animals with 9 % decrease). No body weight decrease by > 5 % was observed in the other groups. In spite of these observations no significant, dose related effect on the body weights was believed during the test.

Clinical Observations, Signs of Irritation

No mortality was observed in any treatment group. The following symptoms of a possible systemic toxicity were observed in the 50 % (w/v) dose group (4 of the 4 animals): hunched back posture, increased respiratory rate on Day 2 (after the treatment); hunched back posture, narrow eyes and restlessness on Day 3 (after the treatment). All animals were symptom-free before the treatments on these days as well as on the other days. Loss of hair on the head was also observed in this dose group as a sign of a local or systemic effect. No local or systemic effects were observed in the other dose groups.

Irritation was monitored by erythema scoring in all test groups and by ear thickness measurements in the DMSO control group and in the test item treated groups. No erythema was observed in any test group during the test. Significantly increased ear thickness values (i. e. an increase of ≥ 25 % compared to the initial value) were observed in the 50 % (w/v) dose group both on Day 3 and on Day 6: the minimum or maximum increase was 15 % or 40 %, respectively. No significantly increased ear thicknesses were observed in the other groups.

Other Observations

Test item residuals were observed on the ears of animals. Amount of the residual was dose related and was not completely removed by normal animal grooming hence the residuals were observed from the first treatment to the end of the test (from Day 1 to Day 6).

Proliferation Assay

Visually larger lymph nodes compared to the relevant vehicle controls (AOO or DMSO) were observed in the positive control group and in the 50 % (w/v) dose group. The appearance of the lymph nodes was normal in both vehicle control groups (AOO or DMSO) and in the other test item treated groups.

Significant lymphoproliferative response (indicated by an SI ≥ 3) compared to the concurrent control (DMSO) was noted for Acid Yellow 218 at 50 % (w/v) concentration. No SI ≥ 3 was observed at the other test concentrations. The observed stimulation index (SI) values were 6.1, 2.7, 1.9 and 1.5 at test item concentrations of 50 %, 25 %, 10 % and 5 % (w/v), respectively. Dose-related response was observed.

Significance of the dose-response was evaluated by linear regression by using SI values of all four test concentrations and also by using three SI values (not including the 50 % (w/v) test concentration).

Significant dose-related response was observed (p = 0.019, r2 = 0.96) when all SI values were used. No statistical significance was observed when three concentrations were evaluated (p = 0.058, r2 = 0.99) although the response was dose-related.

Reliability of the Test

The positive control group animals were treated with 25 % (w/v) HCA solution (formulated in AOO) concurrent to the test item groups. No mortality, cutaneous reactions or signs of toxicity were observed in the positive control group.

A significant lymphoproliferative response (SI ≥ 3) was noted for HCA (SI = 5.6). The results of the positive control item demonstrated an appropriate performance of the test in accordance with the relevant guidelines and confirmed the validity of the assay.

Interpretation of Observations

The maximum dose selection was based on results of the formulation evaluation and the dose range finding test (DRF).

The test item was soluble in most of the standard LLNA vehicles. The maximum soluble concentration was 50 % (w/v) in Dimethyl sulfoxide (DMSO). Since no significant adverse effects (systemic toxicity or irritation) were observed in the dose range finding test up to this maximum concentration, the test item was examined in the main test as 50 %, 25 %, 10 % and 5 % (w/v) formulations in the selected vehicle.

Based on the positive control result the test was valid.

In this LLNA the observed stimulation index values were 6.1, 2.7, 1.9 and 1.5 at test item concentrations of 50 %, 25 %, 10 % and 5 % (w/v), respectively.

No confounding effects of irritation or systemic toxicity were observed in the 25 %, 10 % or 5 % (w/v) dose groups hence the proliferation values related to these test concentrations are considered reliable.

Symptoms of a possible systemic toxic effect were observed at the 50 % (w/v) test concentration however it was not expected (based on the preliminary test results). The symptoms were not excessive, the animals recovered in all cases and no effect on the body weights were observed in this dose group. The significantly increased ear thicknesses may indicate irritation effect of the test item at 50 % (w/v) concentration although it is believed that the observed test item residuals could have effect on the outcome of the ear thickness measurements, especially on Day 3.

Based on all these it is concluded that the proliferation value obtained at the 50 % (w/v) concentration was influenced by effects other than sensitisation hence is not reliable. On the other hand, only its magnitude is questionable hence it should not be excluded from the evaluation of the dose-response correlation.

The SI value of 2.7 observed at 25 % (w/v) concentration was close to the threshold value of 3. The response was clearly dose-related and biologically relevant. The lack of the statistical significance is probably a consequence of an inadequate number of data points in case of evaluation of three test concentration.

Based on the overall results the significantly increased lymphoproliferation at the 50 % (w/v) concentration as well as the dose-related response are considered as evidence that Acid Yellow 218 is a skin sensitizer.

Chemicals can be classified according to their relative skin-sensitization potency using EC3 value (dose calculated to induce a stimulation index of 3) calculated by linear interpolation using data points lying immediately above and below the SI value of 3 on the LLNA dose-response curve according to published method.

Due to the unreliable SI value at the 50 % (w)v) test concentration no EC3 value for the test item was calculated in this LLNA. On the other hand, it is evident from the test results that the theoretical EC3 value is > 25 % hence the test item can be ranked among weak skin sensitizers according to the published data for classification of contact allergens

Applicant's summary and conclusion

Interpretation of results:

other: Category 1B (indication of skin sensitising potential) based on CLP criteria

Conclusions:

Skin sensitizer

Executive summary:

The aim of this study was to evaluate the skin sensitization potential of Acid Yellow 218 following dermal exposure in the Local Lymph Node Assay, according to the OECD guideline 429.

Preliminary tests were performed according to the relevant guidelines to find an appropriate vehicle and the maximum applicable concentration.

The test item was soluble in most of the standard LLNA vehicles. The maximum soluble concentration was 50 % (w/v) in Dimethyl sulfoxide (DMSO).

No significant adverse effects (systemic toxicity or irritation) were observed in the dose range finding test up to this maximum concentration. According to this the test item was examined in the main test at concentrations of 50 %, 25 %, 10 % and 5 % (w/v).

An appropriate positive control (α-Hexylcinnamaldehyde, HCA), and furthermore two negative control groups dosed with the vehicles of the test and positive control groups, respectively, were employed.

The positive control item [25 % (w/v) HCA in Acetone: Olive oil 4:1 (v/v) mixture (AOO)] induced significant stimulation over the relevant control (SI = 5.6) thus confirming the validity of the assay.

No mortality was observed during the test. No significant treatment related effect on body weights was observed in any test group. Body weight decrease by > 5 % were observed in some cases but without dose relevance and the mean body weights did not decrease significantly in any test groups. Although it was not expected, signs of systemic toxic effect of the test item (hunched back posture, increased respiratory rate, narrow eyes and restlessness) as well as loss of hair (on the head) were observed in the 50 % (w/v) dose group. The symptoms were not excessive, the animals recovered in all cases and no effect on the body weights were observed in this dose group.

Sign of irritation (significantly increased ear thickness values) was also observed in the 50 % (w/v) dose group. On the other hand, it is considered that the ear thickness measurement could have influenced by the test item residuals observed during the whole test on the ears of animals in the test item treated groups.

Significantly increased lymphoproliferation [indicated by a Stimulation Index (SI) ≥ 3] compared to the relevant control (DMSO) was noted for Acid Yellow 218 at 50 % (w/v) test concentration. No SI ≥ 3 was observed in the other dose groups. The observed stimulation index values were 6.1, 2.7, 1.9 and 1.5 at test item concentrations of 50 %, 25 %, 10 % and 5 % (w/v), respectively.

It is concluded that the proliferation value obtained at the 50 % (w/v) concentration was influenced by effects other than sensitisation hence is not reliable. On the other hand, only its magnitude is questionable hence it should not be excluded from the evaluation of the dose-response correlation. The SI value of 2.7 observed at 25 % (w/v) was close to the threshold value of 3. Significance of the dose-response was evaluated by linear regression by using SI values of all four test concentrations and also by using three SI values (not including the 50 % (w/v) test concentration).

Significant dose-related response was observed (p = 0.019, r2 = 0.96) when all SI values were used. No statistical significance was observed when three concentrations were evaluated (p = 0.058, r2 = 0.99).

The response was clearly dose-related and biologically relevant. The lack of the statistical significance is considered as consequence of an inadequate number of data points in case of evaluation of three test concentrations.

Based on the overall results the significantly increased lymphoproliferation at the 50 % (w/v) concentration as well as the dose-related response are considered as evidence that Acid Yellow 218 is a skin sensitizer.

Chemicals can be classified according to their relative skin-sensitization potency using EC3 value (dose calculated to induce a stimulation index of 3) calculated by linear interpolation using data points lying immediately above and below the SI value of 3 on the LLNA dose-response curve according to published method.

Due to the questionable magnitude of the SI value at the 50 % (w)v) test concentration no EC3 value for the test item was calculated in this LLNA. On the other hand, it is evident from the test results that the theoretical EC3 value is > 25 % hence the test item can be ranked among weak skin sensitizers (10 ≤ EC3 ≤ 100) according to the published data for classification of contact allergens.

Conclusion

In conclusion, under the conditions of the present assay, Acid Yellow 218 tested at the maximum feasible concentration of 50 % (w/v, based on the solubility) and also at concentrations of 25 %, 10 % or 5 % (w/v) as adequate homogeneous formulations (solution, prepared with Dimethyl sulfoxide as vehicle) was shown to have skin sensitization potential (skin-sensitizer) in the Local Lymph Node Assay.