2-[tert-butyl(phenylmethyl)amino]-1-[4-hydroxy-3-(hydroxymethyl)phenyl] hydrochloride

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 December 2014 - 06 December 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Performed in a GLP laboratory in accordance with OECD and EU guidelines with minor deviations that were not considered not to affect the purpose or integrity of the study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

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)

Deviations:

yes

Remarks:

The control groups for this study were shared with study 41402573. The control groups raw data will be filed with study 41402564.

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

The control groups for this study were shared with study 41402573. The control groups raw data will be filed with study 41402564.

GLP compliance:

yes (incl. QA statement)

Remarks:

OECD Principles on GLP (revised 1997, ENV /MC/CHEM(98) 17); and are in accordance with, and implement, the requirements of Directives 2004/9/EC and 2004/10/EC.

Test material

Test material information

Constituent 1

Reference substance name:

2-[tert-butyl(phenylmethyl)amino]-1-[4-hydroxy-3-(hydroxymethyl)phenyl] hydrochloride

EC Number:

246-014-2

EC Name:

2-[tert-butyl(phenylmethyl)amino]-1-[4-hydroxy-3-(hydroxymethyl)phenyl] hydrochloride

Cas Number:

24085-08-3

Molecular formula:

C20H25NO3.HCl

IUPAC Name:

2-[tert-butyl(phenylmethyl)amino]-1-[4-hydroxy-3-(hydroxymethyl)phenyl] hydrochloride

Test material form:

other: Off white solid

Details on test material:

Store at room temperature in the dark

Test animals

Species:

other: SKINETHIC™ Reconstructed Human Epidermis Model

Strain:

other: human primary keratinocytes

Test system

Type of coverage:

open

Preparation of test site:

other: none

Vehicle:

water

Controls:

other: SKINETHIC™ Reconstructed Human Epidermis Model

Amount / concentration applied:

Quadruplicate tissues were treated with 16 mg of the test item for an exposure
period of 42 minutes. The test item was applied topically to the corresponding tissues
ensuring uniform covering. 10 µL of sterile water was topically applied to the epidermal
surface in order to improve further contact between the solid and the epidermis.
Quadruplicate tissues treated with 16 µL of DPBS served as the negative controls and
quadruplicate tissues treated with 16 µL of SDS 5% w/v served as the positive controls.

Duration of treatment / exposure:

42 minutes

Observation period:

42 hours

Number of animals:

Quadruplicate tissues were treated with test item, negative and positive controls.

Details on study design:

MTT Salt Metabolism, Cell Viability Assay
The MTT assay, a colorimetric method of determining cell viability, is based on reduction of the yellow tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt by mitochondrial succinate dehydrogenase
in viable cells.
One limitation of the assay is possible interference of the test item with MTT. A test item may directly reduce MTT, thus mimicking dehydrogenase activity of the cellular mitochondria. This property of the test item is only a problem, if at the time of the MTT
test (after rinsing) there are still sufficient amounts of the test item present on or in the tissues. In this case, the true metabolic MTT reduction and the false direct MTT reduction can be differentiated and quantified.

Test for Direct MTT Reduction
As specified, a test item may interfere with the MTT endpoint, if it is able to directly reduce MTT and at the same time is present on or in the tissues when the MTT viability test is performed. To identify this possible interference, the test item is checked for the
ability to directly reduce MTT according to the following procedure:

16 mg of the test item was added to 300 µL of a 1.0 mg/mL MTT solution freshly prepared in assay medium. The solution was incubated in the dark at 37 °C, 5% CO2 in air for 3 hours. 16 µL sterile water was tested concurrently to act as a control. If the MTT solution containing the test item turned blue, the test item was presumed to have reduced the MTT and the determination of skin irritation potential would be performed in parallel on viable and freeze-killed tissues for quantitative correction of the results.

The test item was shown to directly reduce MTT in the direct MTT reduction test. There was a possibility that if the test item could not be totally rinsed off the tissues, any residual test item present on or in the tissue may directly reduce MTT and could have given rise to a false negative result. Therefore, the determination of skin irritation potential was performed in parallel on viable and freeze-killed tissues.

This step was a functional check which employs freeze-killed tissues that possess no metabolic activity but absorb and bind the test item like viable tissues.

Freeze-killed tissues were prepared by placing untreated SkinEthicTM tissues in a freezer overnight.

In addition to the normal test procedure, described in the main test, the MTT reducing test item was applied to three freeze-killed tissues for each exposure period. In addition, three freeze-killed tissues for each exposure period remained untreated. The untreated freeze-killed tissue demonstrates a small amount of MTT reduction due to residual reducing enzymes within the tissue.

Main Test

0.3 mL of maintenance medium, at room temperature, was pipetted into 4 wells of new sterile 24 well plates using one plate per test item or control. 2.0 mL of growth medium at room temperature was pipetted into 4 wells of new sterile 6-well plates using one plate per test item or control.

The 6-well pre-incubation plates were removed from the incubator and using sterile forceps the tissue inserts were carefully transferred to the prefilled wells of the 24-well plate. Quadruplicate tissues were treated with 16 mg of the test item for an exposure period of 42 minutes. The test item was applied topically to the corresponding tissues ensuring uniform covering. 10 µL of sterile water was topically applied to the epidermal surface in order to improve further contact between the solid and the epidermis. Quadruplicate tissues treated with 16 µL of DPBS served as the negative controls and quadruplicate tissues treated with 16 µL of SDS 5% w/v served as the positive controls. After dosing, the plates (with lids on) were kept in the biological safety cabinet at room temperature for 42 minutes.

At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test item. The rinsed tissues were transferred to the 6-well post exposure incubation plates prefilled with 2.0 mL of growth medium. The rinsed tissues were incubated at 37 oC, 5% CO2 in air for 42 hours.

Using aseptic techniques 0.3 mL of 1.0 mg/mL MTT solution, freshly prepared in maintenance medium, was pipetted into the wells of a 24-well plate. The tissues were transferred to the MTT filled wells, being careful to remove any excess growth medium from the bottom of the tissue insert by blotting on absorbent paper. Three tissues per group were incubated for 3 hours at 37 °C, 5% CO2 in air. The remaining treated tissues were retained for possible histology. The medium from underneath the treated RHE tissues was retained for possible measurement of mediators and/or enzyme releases such as cytokine or LDH release. Following the 42-Hour post exposure incubation period the growth culture medium was homogenized by gentle agitation for 2 minutes. 3 x 500 µL

for each tissue was transferred to a pre-labeled eppendorf tube and frozen at approximately -20 oC. At the end of the 3 Hour incubation period each tissue was placed onto absorbent paper to remove residual MTT solution and transferred into a new pre- labeled 24-well plate containing 800 µL Isopropanol. A further 700 µL was pipetted on top of each tissue in order to completely immerse the tissue. The plate was sealed with a suitable plate sealer to prevent Isopropanol evaporation and covered to protect from light. The sealed plate was incubated at room temperature for 2 hours on a plate shaker, with gentle agitation, in order to extract the formazan crystals out of the MTT-loaded tissues.

At the end of the formazan extraction period each tissue was pierced with a pipette fitted with a 1000 µL tip and the extraction solution forced vigorously up and down to produce a homogenous solution. For each tissue, triplicate 200 µL samples were transferred to the appropriate wells of a pre-labeled 96-well plate. 200 µL of isopropanol alone was added to the six wells designated as ‘blanks’. The optical density was measured (quantitative viability analysis) at 562 nm (without a reference filter) using the Anthos 2001 microplate reader

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

other: other: Tissue viability

Value:

ca. 100

Remarks on result:

other:

Remarks:

Basis: mean Negative control. Time point: 42 minutes exposure. Reversibility: no data. (migrated information)

Results 2

Irritation / corrosion parameter:

other: other: Tissue viability

Value:

ca. 1.2

Remarks on result:

other:

Remarks:

Basis: mean Positive control. Time point: 42 minutes exposure. Reversibility: no data. (migrated information)

Results 3

Irritation / corrosion parameter:

other: other: Tissue viability

Value:

94.2

Remarks on result:

other:

Remarks:

Basis: mean Test item. Time point: 42 minutes exposure. Reversibility: no data. (migrated information)

Any other information on results incl. tables

Table 1: Mean OD562 Values and Percentage Viabilities for the Negative Control Item, Positive Control Item and Test Item:

 

 

Item   OD562 of tissues tvt OD562 Tissues corrected for tkt-ukt   Mean OD ±SD   Individual tissue viability(%)   Relative mean viability (%) ± SD of Relative mean viability(%)   Negative Control Item⊕ 2.166         2.039 106.2     100∗       5.4   1.961   96.2   1.989   97.5     Positive Control Item⊕   0.029         0.025   1.4       1.2       0.2   0.024   1.2   0.023   1.1       Test Item   1.911   1.896       1.921   93.0       94.2       1.6   1.973   1.958   96.0   1.924   1.909   93.6

Corrected Mean OD562 = 0.031(tkt) – 0.016(ukt) = 0.015

 

 

 

tvt =   treated viable tissues

tkt =   treated killed tissues

ukt =  untreated killed tissues

SD= Standard deviation

∗=     The mean viability of the negative control tissues is set at 100%

⊕ =    Control groups shared with Harlan study number 41402564

Applicant's summary and conclusion

Interpretation of results:

not classified

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test item was classified as non-irritant. The following classification criteria apply:
EU DSD & CLP Not classified for Irritation.
UN GHS Not classified for Irritation (category 3 can not be determined).

Executive summary:

The purpose of this test was to evaluate the skin irritation potential of the test item using the SKINETHICTM reconstructed human epidermis model after a treatment period of 42 minutes followed by a post-exposure incubation period of 42 hours. The principle of the assay was based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item by means of the colorimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls. Histopathological analysis maybe conducted if considered necessary. The concentration of the cytokine or LDH release in the culture medium retained following the 42-Hour post-exposure incubation period may also be determined under certain circumstances.

Quadruplicate tissues were treated with the test item or control items for an exposure period of 42 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. At the end of the post-exposure incubation period three tissues for each group were taken for MTT-loading. The remaining treated tissues were retained for possible histology. The growth medium from beneath each tissue was transferred to pre-labeled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT-loading the inserts were removed and immersed in isopropanol for extraction of formazan crystals out of the MTT-loaded tissues.

At the end of the formazan extraction period each tube was mixed thoroughly and triplicate 200 µL samples were transferred to the appropriate wells of a pre-labeled 96-well plate. The optical density was measured at 562 nm.

Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

The relative mean viability of the test item treated tissues was 94.2% after the 42-Minute exposure period and 42 hour post-exposure incubation period. It was considered unnecessary to proceed with tissue histology or analysis of inflammatory mediators.

The test item was classified as non-irritant. The following classification criteria apply: EU DSD & CLP Not classified for Irritation.

UN GHS Not classified for Irritation (category 3 can not be determined).