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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 October 2011 to 5 December 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
In vitro assay - EPISKIN - performed in accordance with Guideline OECD 439 with no remarkable deviations or deficiencies

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD Guideline 439 In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method (22 July 2010); Commission Regulation (EC) No. 761/2009 amending Regulation (EC) No. 440/2008, B.46 In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test
Principles of method if other than guideline:
EPISKIN reconstructed human epidermis model
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 4,4-biphenol
- Physical state:powder
- Analytical purity:99.9%
- Purity test date: 9 September 2011
- Lot/batch No.: 071111370
- Expiration date of the lot/batch: 11 July 2016
- Storage condition of test material: Ambient room temperature
- Other: description off white to white cream coloured powder

In vitro test system

Test system:
human skin model
Remarks:
cultured human epidermal keratinocytes
Source species:
other: Human
Cell type:
other: human derived epidermis keratinocytes
Cell source:
other: reconstructed human epidermis (RhE) model
Details on test system:
Test model
- Source: EPISKIN - reconstructed human epidermis - supplied by SkinEthic Laboratories, Nice, France
- Received at laboratory: 29 November 2011
- batch: 11-EKIN-044
- Functional controls: Quality controls: histology scoring, magnitute of viability and barrier function (IC50 determination).
Biological safety: absence of HIV1 and 2, Hepatitis B and C antigens, absence of bacteria, fungi and mycoplasma.


IN-LIFE DATES: Experimental start date: 18 October 2011; experimental completion date: 5 December 2011

Test system

Type of coverage:
other: 0.38 cm2 EPISKIN
Preparation of test site:
other: Test system examined on arrival - pale grey temperature indicator and orange pH indicator meaning te system was suitable for use. Culture dishes were incubated for 24 hours after arrival at 37C, at saturated humidity and 5% CO2.
Vehicle:
other: Various media used
Controls:
not required
Amount / concentration applied:
TEST MATERIAL

Negative control - D-PBS 20uL/well; three replicates

Positive control - 5% SDS in water 20uL/well; three replicates

Test material - 4,4-biphenol 20 mg/well; three replicates

Media:
SkinEthic Maintenance Medium: SkinEthic; batch: 11-MAIN3-060
SkinEthic Assay Medium : SkinEthic; batch: 11-ESSC-037 (Preliminary Phase) and 11-ESSC-048 (Main Assay)
Dulbecco’s Phosphate buffered saline (D-PBS): GIBCO; batch: 925780
Sterile water : Bieffe Medital; batch: 08K2901
MTT Reagent : Sigma; batch: MKBB1495
MTT Stock Solution : MTT Reagent 3 mg/ml in D-PBS
Stored at ambient conditions, protected from light; used on the day of dilution
MTT Ready-to-use Solution : MTT Stock Solution diluted 1:10 (v/v) with SkinEthic Assay Medium (final concentration 0.3 mg/ml of MTT)
Stored at ambient conditions, protected from light. Used within 1 hour from preparation
Acidic Isopropanol (0.04 N HCl in isopropanol): 12 N HCL (Analab; batch: 08B22050j) added to 2-propanol (Fluka; batch: 0001434790)
Duration of treatment / exposure:
In the preliminary test, the non-specific reduction of MTT was evaluated. 2ml of MTT Ready-to-Use solution was incubated with 10 mg of the test material for 3 hours (saturated humidity, 5%CO2 at 37°C). The solution was examined after incubation for blue or purple appearance. The colouring potential interaction with the test system was tested by adding 10 mg of test material to 90 uL distilled water and vortex mixed for 15 minutes. The solution colour was evaluated at the end of the incubation time.

In the main assay, the test material and positive and negative controls were applied to live tissue, three replicates. The exposure period was 15 minutes; after exposure each tissue was rinsed with sterile D-PBS and transferred to a new well with 2 ml maintenance medium. The wells were incubated at saturated humidityand 5%CO2 at 37°C for the 42 hour recovery period. At the end of the recovery period samples of incubation medium were reserved (but not subjected to further analyses). The tissue inserts and controls were then incubated with 2 ml MTT ready to use solution for three hours at 37°C (saturated humidity, 5%CO2) .
Tissues were then removed, dried on absorbent paper and a total biopsy obtained using a biopsy punch.
Observation period:
Not applicable
Number of animals:
Reconstructed human epidermal tissue. Three replicate live tissues per treatment
Details on study design:
In the main assay, the test material and positive and negative controls were applied to live tissue, three replicates. The exposure period was 15 minutes; after exposure each tissue was rinsed with sterile D-PBS and transferred to a new well with 2 ml maintenance medium. The wells were incubated at saturated humidity, 5%CO2 at 37°C for the 42 hour recovery period. At the end of the recovery period samples of incubation medium were reserved (but not subjected to further analyses). The tissue inserts and controls were then incubated with 2 ml MTT ready to use solution for three hours at saturated humidity, 5%CO2 at 37°C.
Tissues were then removed, dried on absorbent paper and a total biopsy obtained using a biopsy punch.

The epidermis was separated from the collagen matrix and both elements were placed into a microtube containing 500 uL of acidic isopropanol. The tubes were vortex mixed and preserved for three days at 4°C to allow for formazan extraction. After extraction and centrifugation to remove debris, 200 µL aliquots were read in duplicate for absorbance at 595 nm. OD values were recorded. Isopropanol samples were used as control blanks.

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Value:
119.4
Remarks on result:
no indication of irritation
Remarks:
Mean score from 3 replicates

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The potential of the test item 4,4-Biphenol to be irritant to skin was investigated in an in vitro skin irritation study using a commercial reconstructed human epidermis (RhE) model named EPISKIN. According to the histotoxic potential, when referred to the negative control, the test item is considered to have no effect on the test system (119.4% of mean cell viability when compared to negative control). The negative and positive controls gave the expected results and the study was accepted as valid.

According to the criteria defined in the guidelines for this test (cell viability less than 50%), the test item is not considered to have irritant effect on the skin under the reported experimental conditions.
Executive summary:

The potential for 4,4'-Biphenol to be irritant to the skin was investigated in an in vitro skin irritation study using a commercial reconstructed human epidermis (RhE) model - EPISKINTM. 4,4'-Biphenol as well as positive and negative controls, were tested for their ability to impair cell viability after an exposure period of 15 minutes followed by a 42 hour recovery period. The final endpoint of the assay is the colorimetric measurement of MTT reduction (blue formazan salt) in the test system as an index of cell viability. A preliminary test was carried out to assay the compatibility of the test item with the test system. In particular, the test item was assayed for the ability of reducing MTT and of colouring water per se. No interaction was recorded between the test item and MTT under the normal test condition. No colouring potential was recorded for the test item in contact with water. Therefore, no additional control was added to the main phase for the evaluation of non specific coloration which may have influenced evaluation of results. In the main assay, 4,4'-Biphenol was applied, as supplied, in three replicates at the treatment level of approximately 20 mg/epidermis unit (0.38 cm2 each). Positive and negative controls [a 5% (w/v) sodium dodecyl sulphate solution in water and Dulbecco’s phosphate buffered saline (D-PBS)] were concurrently tested, using the same number of replicates and test conditions, at a dose level of 20 μl/epidermis unit. The negative control gave the expected baseline value and variability, in agreement with the guideline indications. According to the method, the mean value is considered the baseline value of the experiment and thus represents 100% of cell viability. The positive control caused the expected cell death when compared to the negative control (10.1% of cell viability) even with a variability slightly higher than expected (CV% equal to 33.8 instead of less than 18), but the test was considered valid. 4,4'-Biphenol did not induce cell death, the mean viability was 119.4% with reference to the negative control. According to the established criteria (cell viability less than 50%), the test item is considered to have no irritant effect on the skin under the reported experimental conditions.