1,3-bis(2-methylphenyl)guanidinium m-[[p-anilinophenyl]azo]benzenesulfonate

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 28 November 2012 to 30 November 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Report date:

2013

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)

Version / remarks:

2010

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

1

Reference substance name:

1-3(Diphenylguanidinium) m-[[p-anilinophenyl]azo]benzenesulfonate

EC Number:

941-151-0

Molecular formula:

C31H28N6SO3

IUPAC Name:

1-3(Diphenylguanidinium) m-[[p-anilinophenyl]azo]benzenesulfonate

Test material form:

solid: particulate/powder

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

other: reconstructed epidermis of normal human keratinocytes.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: SkinEthic RHE/S/17
- Tissue batch number(s): 12 022A 1103
- Certificate of analysis of reconstructed human epidermis date: 2012-11-26
- Delivery date: recevied on 2012-11-27
- Expiration date : 2012-12-03
- Date of initiation of testing: 2012-11-28


TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 25 x 1 mL with PBS
- Observable damage in the tissue due to washing: 1 of the replicate was compromised.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hr

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION:

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin if the tissue viability after 42 minutes exposure and 42 hrs of post-treatment incubation is < than 50%.
- The test substance is considered to be non-corrosive to skin if the tissue viability after 42 minutes exposure and 42 hrs of post-treatment incubation is > than 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 16 mg

NEGATIVE CONTROL : PBS - Pan BIOTECH GmbH - in the same experimental conditions

POSITIVE CONTROL : 5% SDS CAS 151-21-3 (0.5 g SDS in a 10 mL volumetric flask qsp 10 mL distilled water), in the same experimental conditions.

Duration of treatment / exposure:

42 min

Duration of post-treatment incubation (if applicable):

42 hrs

Number of replicates:

3 replicates

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

replicate 1 (skin #7)

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: aberrants values

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

replicate 2 (skin #8)

Value:

ca. 76

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

replicate 3 (skin #9)

Value:

ca. 96.6

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: mean valibility = 100 %
- Acceptance criteria met for positive control: mean viability = 1.2 %

Any other information on results incl. tables

Individual and average values of OD after 42 minutes of exposure:

	Skin	OD	Mean OD/disc (Mean of the 3 OD measurements)	Mean OD/ product	Viability %	Mean Viability %	SD	Conclusion
Negative Control	1	1.041 1.100 1.138	1.093	1.031*	106.0	100.0	5.9
	2	0.961 1.039 1.092	1.030		99.9
	3	0.908 0.976 1.029	0.971		94.1
Positive Control	4	0.012 0.014 0.013	0.013	0.013	1.3	1.2	0.1	Irritant
	5	0.011 0.013 0.012	0.012		1.2
	6	0.013 0.012 0.014	0.013		1.3
Test Item	7	0.216 0.243 0.250	0.236 – Aberrant values
	8	0.763 0.761 0826	0.783	0.890	76.0	86.3*	14.6*	Non irritant
	9	0.918 1.030 1.040	0.996		96.6

* The result is based on two tissues replicates (n° 8 and 9) instead of three as initially scheduled in the study plan. The rinsing of the epidermises in view to eliminate the test item after treatment was difficult and required more rinsing with PBS than usually. The first treated epidermis was compromised during this rinsing step and was not taken into account for the classification of the test item.

                 

Negative control and positive control are in the expected range.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item must not be classified, in accordance with the Regulation EC No. 1272/2008.

Executive summary:

The aim of the study was to evaluate the possible irritating effects of the test item after topical administration on in vitro human reconstructed epidermis (RHE® model). The test item was applied, as supplied, at the dose of 16 mg, to 3 Reconstructed Human epidermis small during 42 minutes, followed by a 42 hours postincubation period at 37°C, 5% C02.

The elimination of the test item on the epidermis after treatment was difficult. Even after a rinse with 35 mL of PBS, there was remaining test item. The first epidermis was compromised during rinsing and was not taken into account for the classification of the test item.

The experimental protocol was established in accordance with OECD guideline No. 439 adopted 22 July 2010 and the Test method B.46 of Council regulation No. 761/2009 dated 23 July 2009 (EU Journal L220)-ATP Council regulation No. 4401/2008 of 30 May 2008 (E. U. Journal L142).

The mean viability ofthe epidermis skins was 86.3%, versus 1.2% in the positive control (5% Sodium Dodecyl Sulfate ).

The results obtained, under these experimental conditions, enable to conclude that in accordance with the Regulation EC No. 1272/2008, the test item must not be classified. No signal word or hazard statement is required.