4-amino-m-cresol

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 7 September 2016 to 12 September 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

4-amino-m-cresol

EC Number:

220-621-2

EC Name:

4-amino-m-cresol

Cas Number:

2835-99-6

Molecular formula:

C7H9NO

IUPAC Name:

4-amino-m-cresol

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: supplied by HFC Prestige Service, batch No. DRDGAMC110703W
- Expiration date of the lot/batch: 30 June 2018
- Purity test date: 13 May 2016

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient temperature
- Stability under test conditions: not specified
- Solubility and stability of the test substance in the solvent/vehicle: test item used as supplied

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: test item used as supplied
- Preliminary purification step (if any): no purification made
- Final dilution of a dissolved solid, stock liquid or gel: no dilution performed
- Final preparation of a solid: used as supplied

FORM AS APPLIED IN THE TEST (if different from that of starting material)

OTHER SPECIFICS:

In vitro test system

Test system:

artificial membrane barrier model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: reconstructed human derived epidermis model of adult human epidermal keratinocytes

Source strain:

not specified

Justification for test system used:

Following a full validation study the EpiSkinTM reconstructed human epidermis model showed evidence of being a reliable and relevant stand-alone test for predicting rabbit skin irritation when the endpoint is measured by MTT reduction and for being used as a replacement for the Draize Skin Irritation Test for the purpose of distinguishing between Irritating and Non-Irritating test items.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkinTM
- Tissue batch number(s): 16-EKIN-036
- Production date: not specified
- Shipping date: not specified
- Delivery date:06 September 2016
- Date of initiation of testing: 07 September 2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: approximately at 37 deg Celsius
- Temperature of post-treatment incubation (if applicable):at 37 deg Celsius

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 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.
- Observable damage in the tissue due to washing: Not specified
- Modifications to validated SOP: No modifications

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Anthos 2001 microplate reader
- Wavelength: 562 nm (without a reference filter)
- Filter: No reference filter was used
- Filter bandwidth: No reference filter was used
- Linear OD range of spectrophotometer: not specified

NUMBER OF REPLICATE TISSUES: triplicate for each conditions

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Killed tissues
- Procedure used to prepare the killed tissues (if applicable):Water-killed tissues were prepared by placing untreated EPISKINTM tissues in a 12-well plate containing 2.0 mL of sterile distilled water in each well. The tissues were incubated at 37 °C, 5% CO2 in air for 48 ± 1 hours. At the end of the incubation the water was discarded. Once killed the tissues were stored in a freezer (-14 to -30 °C) for up to 6 months. Before use each tissue was thawed by placing in 2.0 mL of maintenance medium for approximately 1 hour at room temperature.
- N. of replicates : 3
- Method of calculation used: Relative mean viability (%) = (mean Optical Density at 562 nm of test item / mean Optical Density at 562 nm of negative control) x 100

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 2 test sequences (pre-test and main test)

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if [complete, e.g. the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.]
- The test substance is considered to be non-corrosive to skin if [complete, e.g. the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.]

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 mg (26.3 mg/cm2)
- Concentration (if solution): pure

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10µL of DPBS
- Concentration (if solution): used as supplied (pure)

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL
- Concentration (if solution): 5% w/v aqueous solution

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3 per conditions

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Main test (test item)

Value:

63.4

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Main test (positive control)

Value:

5.6

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Results 3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Main test (negative control)

Value:

100

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: Not specified
- Direct-MTT reduction: Yes
- Colour interference with MTT: No

DEMONSTRATION OF TECHNICAL PROFICIENCY: SDS used as positive control

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: yes
- Range of historical values if different from the ones specified in the test guideline: Not specifies

Any other information on results incl. tables

Table1 :Mean OD562Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

 

Item	OD562 of tissuestvt	Mean of OD562	OD562 of tissues corrected fortkt-ukt (0.104)	± SD ofOD562	Relative individual tissue viability (%)	Relative mean viability (%)	± SD of Relative meanviability(%)
Negative Control	1.133	1.234		0.099	91.8	100*	8.1
	1.331				107.9
	1.237				100.2
Positive Control	0.082	0.069		0.020	6.6	5.6	1.6
	0.079				6.4
	0.046				3.7
Test Item	0.898	0.885	0.781	0.017	64.4	63.4	1.3
	0.892				63.9
	0.866				61.8

Corrected viability of treatedkilledtissues                  =     0.216 (tkt)-0.112 (ukt) =0.104

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

OD562 = Optical Oensity

SD =       Standard Deviation

tvt =       treated viable tissues

tkt =       treated killed tissues

ukt =       untreated killed tissues

 

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the experimental conditions of the study, incubation of the registered substance 4-amino-m-cresol induced a relative mean viability at 63.3% compared to negative control. Hence, the registered substance was classified as Non-irritant for the skin (EU CLP Not classified for Irritation).

Executive summary:

The purpose of this GLP compliant test was to evaluate the skin irritation potential of the test item using the EPISKINTM reconstructed human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours according to the OECD guideline 439 method.

Triplicate tissues were treated with the test item for an exposure period of 15 minutes. The test item was found to directly reduce MTT and therefore additional non-viable tissues were incorporated into the testing for correction purposes. 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 each tissue was taken for MTT-loading. The maintenance 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 a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues.

The relative mean viability of the test item treated tissues was 63.3% after the 15-Minute exposure period and 42-Hours post-exposure incubation period.

Under the experimental conditions of the study, incubation of the registered substance 4-amino-m-cresol induced a relative mean viability at 63.3% compared to negative control. Hence, the registered substance was classified as Non-irritant for the skin (EU CLP Not classified for Irritation).