2-Naphthalenol, heptyl-1-[2-[3-methyl-4-[2-(3-methylphenyl)diazenyl]phenyl]diazenyl]-

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from August 9 to September 2, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction products of 3-Methylaniline with heptyl naphthalen-2-ol

Cas Number:

1619917-05-3

Molecular formula:

Not applicable (UVCB Substance)

IUPAC Name:

Reaction products of 3-Methylaniline with heptyl naphthalen-2-ol

Test material form:

solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- batch No.of test material: 78-231-15
- Expiration date of the batch: July 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature, protected from light
- Stability in Solvent or Vehicle at RT: Stable in acetone

In vitro test system

Test system:

human skin model

Cell type:

other: normal human epidermal keratinocytes

Justification for test system used:

This test uses the EPISKIN-SM™ reconstructed human epidermis model (SkinEthic) which consists of normal human epidermal keratinocytes (NHEK) and therefore represents in vitro the target organ of the species of interest and closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis.
This test method is able to detect chemicals that cause skin irritation, i.e. produce reversible damage to the skin and allows for hazard identification in accordance with UN GHS “Category 2”. Depending on the regulatory framework it can also be used to identify non-classified chemicals .

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN-SM™ (SkinEthic)

Firstly, 5 µL distilled water (aqua dest.) was applied to the epidermal surface in order to improve further contact between the powder and the epidermis. The water was gently spread on the surface.
After warming the test item to 37°C, approximately 10 ± 2 mg (26.3 mg/cm²) of the test item were formed to a flat disc which was placed atop the wetted epidermis surface.

The test was carried out with the reconstituted three-dimensional human skin model EPISKIN-SM™ (SkinEthic). This skin model consists of normal human epidermal keratinocytes (NHEK) which have been cultured to form a multilayered, highly differentiated model of the human epidermis. The NHEK are cultured on chemically modified, collagen-coated cell culture inserts. A highly differentiated and stratified epidermis model is obtained after 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

The EPISKIN-SMTM tissues were provided as kits (SkinEthic), consisting of the following components relevant for this study:
1x EPISKIN-SM™ plate containing 12 reconstructed epidermis units (area: 0.38 cm2); each reconstructed epidermis is attached to the base of a tissue culture insert with an O-ring set and maintained on nutritive agar for transport (Lot No.: 16-EKIN-033, 16-EKIN-035)
1x 12-well assay plate
1x flask of sterile maintenance medium (basic medium for incubations, Lot No.: 16-MAIN3-055, 16-MAIN3-060)
1x flask of sterile assay medium (basic medium for use in MTT assays, Lot No.: 16-ESSC-036, 16-ESSC-038)
Validity controls as provided by the supplier (SkinEthic):
Morphology:
Histology scoring (HES stained vertical paraffin sections, n = 6):
Specification ≥ 19.5
First Experiment:
Result: 22.9 ± 0.4, CV = 1.6%
Second Experiment:
Result: 22.5 ± 0.4, CV = 2.0%
Well-differentiated epidermis consisting of a basal layer, several spinous and granular layers and a thick stratum corneum.
Barrier function:
IC50 determination (SDS concentration, MTT test, n = 14):
Specification ≥ 1.5 mg/mL
Result: 1.9 mg/mL (First Experiment)
Result: 1.8 mg/mL (Second Experiment)


PREDICTION MODEL / DECISION CRITERIA
To check the non-specific MTT-reducing capability of the test item 10 mg of the test item was mixed per 2 mL MTT medium and incubated for 3 h at 37  1 °C in the dark. If the mixture turned blue/purple, the test item was presumed to have reduced MTT and the part of absorption due to the non-specific reduction of MTT (NSMTT) was determined by using killed tissues. For quantitative correction of results three killed tissues were treated with 10 mg of the test item (KT) and with the negative control (DPBS; KU), respectively. All steps were performed exactly as described in the chapter below. NSMTT was calculated relative to the negative control of living tissues (NK) per treatment period according to the following formula:
NSMTT [%] = [(ODKT - ODKU)/ODNK] * 100
If non-specific MTT reduction was ≤ 30% relative to the negative control of living epidermis, the true MTT metabolic conversion (TODTT) of the test item treated living tissues (TM) was corrected according to the following formula:
TODTT = ODTM - (ODKT - ODKU)
If non-specific MTT reduction was > 30% relative to the negative control of living epidermis, the test item was considered as incompatible with the test method.

To check the colouring potential of the test item 10 mg of the test item was mixed per 90 µL aqua dest. and per 90 µL isopropanol each in a transparent recipient for 15 min. If colouring was detected by unaided eye-assessment and the chemical in water and/or isopropanol absorbs light in the range of 570 ± 30 nm, the test item was checked for its tissue-colouring potential. For quantitative correction of results the test was performed using two additional living tissues treated with 10 mg of the test item (TVT) and one living tissue treated with 10 µL of the negative control (DPBS; UVT), respectively. All steps were performed exactly as described in the chapter below, except for the MTT-staining of the test item treated tissues, which was incubated in medium without MTT. The non-specific colour of additional viable tissues (NSCliving) was then calculated according to the following formula:
NSCliving [%] = [ODTVT/ODUVT]*100
If NSCliving was ≤ 5% relative to the negative control of living epidermis, no correction of the results was necessary.
If NSCliving was > 5% and ≤ 30% relative to the negative control of living epidermis, the true MTT metabolic conversion (TODTT) was corrected according to the following formula:
TODTT = ODTM – ODTVT
If NSCliving was > 30% relative to the negative control of living epidermis, the test item was considered as incompatible with the test method.
For test items which act as non-specific MTT-reducers and show non-specific colouring of living tissues, a third control for non-specific colour in killed tissues (NSCkilled) was performed to avoid a possible double-correction for colour interference. Therefore, two killed tissues were treated with 10 mg of the test item (TKT) and one killed tissue was treated with the negative control (DPBS; UKT), respectively. All steps were performed exactly as described in the chapter below, except for the MTT-staining of the test item treated with tissues, which was incubated in medium without MTT. The non-specific colour of additional viable tissues (NSCkilled) was then calculated according to the following formula:
NSCkilled [%] = [ODTKT/ODUKT]*100
The true tissue viability was then calculated as the percent tissue viability obtained with living tissues minus NSMTT minus NSCliving plus NSCkilled.

Upon receipt of the EPISKIN-SMTM, the transport and the temperature indicator were inspected. The tissues were transferred into 12-well plates containing 2 mL pre-warmed maintenance medium per well. The 12-well plates were incubated in a humidified incubator at 37  1 °C, 5.0% CO2 for at least 24 h.
After this pre-incubation the tissues were treated with each dose group in triplicate, starting with the negative control. Start time was recorded with dosing of the first tissue. Then the tissues were incubated at room temperature for 15  0.5 min. The positive control was re-spread after 7 min contact time. Afterwards, the disc-like test item was removed from the tissues and the tissues were washed with DPBS to remove any residual test item. Excess DPBS was removed by blotting bottom with blotting paper. The inserts were placed in a prepared 12-well plate containing 2 mL pre-warmed fresh maintenance medium and post-incubated at 37  1 °C, 5.0% CO2 for 42  1 h.
After this incubation period excess medium was removed by blotting bottom on absorbent paper before the inserts were transferred in a prepared 12-well plate containing 2 mL pre-warmed MTT medium and further incubated for 3 h ± 5 min at 37  1 °C, 5.0% CO2.
After the 3 h MTT incubation period the tissues were placed on blotting paper to dry. Afterwards a total biopsy of the epidermis using the special biopsy punch was performed and the epidermis was separated from the collagen matrix with the aid of forceps. Both parts (epidermis and collagen matrix) were transferred into suitable tubes and 500 µL of acidic isopropanol were added. The plugged tubes were mixed using a vortex mixer. Extraction was carried out protected from light at least 4 h at room temperature with vortexing of each tube at the middle of the incubation period.
At the end of the formazan extraction period the tubes were mixed by vortexing until solution colour became homogeneous.
If any visible cell/tissue fragments were in suspension, the tubes were centrifuged to eliminate the fragments and avoid further possible interference with the absorbance readings.
Per each tissue 2 x 200 µL aliquots of the extract were transferred into a 96-well plate and OD was measured at 570 nm using a filter band pass of maximum ± 30 nm in a plate spectrophotometer.

Irritant potential of the test item was predicted from the relative mean tissue viabilities compared to negative control tissues concurrently treated with DPBS. The test item is considered to be irritant to skin in accordance with regulation EC 1272/2008 [18] and UN GHS [16], [17] “Category 2”, if the tissue viability after 15 min of exposure and 42 h of post-incubation is less or equal to 50%. Further testing is required to resolve between UN GHS categories 1 and 2 and decide on the final classification of the test substance [9]. The test substance may be considered as non-irritant to skin in accordance with UN GHS “No Category” if the tissue viability after exposure and post-treatment incubation is higher than 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Duration of treatment / exposure:

15 min of application

Duration of post-treatment incubation (if applicable):

42-h post incubation period

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

overall meal tissue viability of six test item treated tissues

Value:

87.6

Remarks on result:

no indication of irritation

Any other information on results incl. tables

Irritant potential of the test item was predicted from the relative mean tissue viabilities obtained compared to the corresponding negative control tissues concurrently treated with DPBS.

The test item showed no irritant effects. In the first experiment, the mean relative tissue viability (% negative control) was> 50% (77.8%) after 15 min treatment and 42 h post-incubation. The test was repeated since the standard deviation of the corresponding negative control tissues was exceeding the 19% threshold value (18.5%). In the second experiment, the mean relative tissue viability (% negative control) wasreduced to 97.3%. All single values were far beyond the 50% threshold value (117.1% / 94.0% / 80.9%), but the standard deviation between the three identically test item treated tissues was slightly exceeding the 18% threshold value (18.3). This was accepted, since the overall mean relative tissue viability of six test item treated tissues indicates with 87.6% a clearly non-irritant effect.

The mean OD₅₇₀of each six blank values was< 0.1. The mean absolute OD₅₇₀of each three negative control tissues was³ 0.6 and ≤ 1.5. The mean relative tissue viability (% negative control) of each positive control group was£ 40% (6.2% and 5.3%). The maximum standard deviation of viability of replicate tissues of the control dose groups in the second experiment was notexceeding18% (1.1% and 10.6%).

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In this study under the given conditions the test item showed no irritant effects. The test item is therefore classified as “non-irritant” in accordance with UN GHS “No Category”.

Executive summary:

In the present study theskin irritant potential of RED 2596 was analysed. The EPISKIN-Standard Model™ (EPISKIN-SM^TM), a reconstituted three-dimensional humanepidermismodel, was used as a replacement for the Draize Skin Irritation Test (OECD TG 404[8]) to distinguish between UN GHS “Category 2” skin irritating test substances and not categorized test substances (“No Category”) which may be considered as non-irritant. Hereby, the test item was applied topically. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT after a 15 min exposure and 42 h post-incubation period and compared to those of the concurrent negative controls.

In this study under the given conditions the test item showed no irritant effects. The relative mean tissue viability after 15 min of exposure and 42 h post-incubation was> 50%. The test item is therefore classified as “non-irritant” in accordance with UN GHS “No Category”.