Reaction mass of [5-methyl-2-[[4-(4-methylanilino)-9,10-dioxo-1-anthryl]amino]phenyl]sulfonyloxypotassium and [5-methyl-2-[[4-(4-methyl-2-potassiooxysulfonyl-anilino)-9,10-dioxo-1-anthryl]amino]phenyl]sulfonyloxypotassium

Endpoint summary

Description of key information

Not skin sensitising

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

From 9 to 23 October, 2002

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Justification for type of information:

The read across approach is detailed into the document attached to the IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 24 June 2002

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Netherlands.
- Females: nulliparous and non-pregnant.
- Age at study initiation: 8 - 12 weeks beginning of acclimatization.
- Weight at study initiation: 18.5 g -22.4 g beginning of acclimatization period.
- Housing: in groups of 4 in Makrolon type-3 cages with standard softwood bedding.
- Diet: pelleted standard Kliba 3433, batch no. 57102 mouse maintenance diete, ad libitum.
- Water:community tap water from ltingen, ad libitum.
- Acclimation period: under test conditions after health examination.
- Indication of any skin lesions: only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C.
- Relative humidity: 30 - 70 %
- Air changes: 10 - 15 air changes per hour.
- Photoperiod: 12 hour fluorescent light / 12 hour dark cycle with at least 8 hours music during the light period.

Vehicle:

other: ethanol:water, 1:1 (v/v)

Concentration:

2.5, 5 and 10 % (w/v)

No. of animals per dose:

three groups of four female

Details on study design:

TEST ITEM PREPARATION
- Preparation: the test item was placed into a volumetric flask on a tared Mettler balance and the vehicle (ethanol:water, 1:1 (v/v)) was quantitatively added. The weigh/volume dilutions were prepared individually using a magnetic stirrer as homogenizer.
- Homogeneity: homogeneity in the vehicle was maintained during treatment with the magnetic stirrer.
- Time: the preparations were made shortly before each dosing occasion.

TOPICAL APPLICATION
- Aera of aplication: topical (epidermal) application to the dorsal surface of each ear lobe (left and right).
- Application volume: 25 µl, was spread over the entire dorsal surface (Ø - 8 mm) of each ear lobe once daily for three consecutive days.
- Control: mice treated with an equivalent volume of the relevant vehicle alone.

RADIO-LABELLED
- Administration: 5 days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (3H-methyl thymidine).
- Sacrifice: approximately 5 hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per group.
- Cell suspension: single cell suspensions of lymph node cells were prepared from pooled lymph nodes which were washed subsequently.
- Incubation: cell suspension was incubated with trichloroacetic acid overnight.
- Proliferative capacity: the proliferative capacity of pooled lymph node cells was determined by the incorporation of 3H-methyl thymidine measured in a β-scintillation counter.

RANGE-FINDING
- Number of animals: 2 mice.
- Test cocnentrations: 1, 2.5, 5 and 10 % (w/v)
- Irritaiton: no irritation effects were observed at the concentrations applied.
- Top dose: the top dose is the highest technically achievable concentration whilst avoiding systemic toxicity and excessive local irritation.

INTERPRETATION OF RAW DATA
The proliferative response of lymph node cells is expressed as the number of radioactive disiniegrations per minute per lymph node (DPM/NODE) and as the ratio of 3HTdR incorporated into lymph node cells of test lymph nodes relative to that recorded for control lymph nodes (STIMULATION INDEX). Before DPM/NODE values were determined, mean scintillation-background DPM was subtracted from test and control raw data.
A test item is regarded as a sensitizer in the LLNA if the following criteria are fulfilled:
- First, that exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the STIMULATION INDEX.
- Second, that the data is compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.
The decision to select a stimulation index of 3 as an arbitrary indication of sensitizing activity was made on the basis of investigations performed with a wide range of chemicals.

OBSERVATIONS
- Mortality / Viability: twice daily from acclimatization start to the termination of in-life phase.
- Body weights: at acclimatization start and prior to necropsy.
- Clinical signs (local/ systemic): daily from acclimatization start to the termination of in-life phase. Especially the treatment sites were recorded carefully.

Parameter:

SI

Value:

1.7

Variability:

No deaths occurred

Test group / Remarks:

10 %

Parameter:

SI

Value:

1.5

Variability:

No deaths occurred

Test group / Remarks:

5 %

Parameter:

SI

Value:

1.8

Variability:

No deaths occurred

Test group / Remarks:

2.5 %

Cellular proliferation data / Observations:

The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3H-methyl thymidine measured on a β-scintillation counter. A test item is regarded as a sensitizer if the exposure to at least one concentration resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the stimulation index.

VIABILITY / MORTALITY
No deaths occurred during the study period

CLINICAL SIGNS
No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period.

BODY WEIGHTS
The body weight of the animals, recorded at the start of acclimatization period and prior to necropsy, was within the range commonly recorded for animals of this strain and age.

Interpretation of results:

other: not classified, according to the CLP Regulation (EC) No 1272/2008

Conclusions:

The test item was found to be a non-sensitizer when tested up to 10 % (wlv) in ethanol:water, 1:1 (v/v).

Executive summary:

In order to study a possible allergenic potential of test substance, three groups of four female mice were each treated with the test item at concentrations of 2.5, 5 and 10 % (w/v) in ethanol:water, 1:1 (v/v) by topical application to the dorsum of each ear lobe (left and right) on three consecutive days. A control group of four mice was treated with the vehicle (ethanol:water, 1:1 (v/v)) only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (3H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes which were washed subsequently and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3H-methyl thymidine measured in a pscintillation counter.

No test item-related clinical signs were observed. All treated animals survived the scheduled study period.

Stimulation indicies of 1.8, 1.5 and 1.7 were determined with the test item at concentrations of 2.5, 5 and 10 % (w/v) in ethanol:water, 1:1 (v/v). An exact EC3 value could not be calculated because this calculation requires data lying immediately above and below S.l. value of 3.

Conclusion

The test item was found to be a non-sensitizer when tested up to 10 % (wlv) in ethanol:water, 1:1 (v/v).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

There is no data about the skin sensitisation potential of Acid Green 025, thus, the available information on the structural analogous Similar Substance 01 have been taken into consideration; the read across approach can be considered as appropriate and suitable to assess the property under investigation (details about the approach are reported into the IUCLID section 13).

In order to study a possible allergenic potential of Similar Substance 01, three groups of four female mice were each treated with the test item at concentrations of 2.5, 5 and 10 % (w/v) in ethanol:water, 1:1 (v/v) by topical application to the dorsum of each ear lobe n three consecutive days. Five days after the first topical application, the mice were injected intravenously with radio-labelled thymidine (3H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed. No test item-related clinical signs were observed. All treated animals survived the scheduled study period.

Stimulation indicies of 1.8, 1.5 and 1.7 were determined with the test item at concentrations of 2.5, 5 and 10 % (w/v) in ethanol:water, 1:1 (v/v). An exact EC3 value could not be calculated because this calculation requires data lying immediately above and below S.l. value of 3.

Justification for classification or non-classification

According to the CLP Regulation (EC) No 1272/2008, 3.4 Respiratory or skin sensitisation section, skin sensitizer means a substance that will lead to an allergic response following skin contact.

Based on the Local lymph node assay (LLNA) results, a substance in considered a skin sensitizer when the EC3 value resulted to be higher than 2 %.

The LLNA assay failed to calculate an EC value higher than 2 %; Stimulation Index resulted to be lower than 3 for all of the tested concentrations.

In conclusion, Acid Green 025 is expected to not meet the criteria to be classified as skin sensitizer, according to the CLP Regulation (EC) No 1272/2008.