Concrete of honeycomb cells of the bee by hexane extraction

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 September to 13 October 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2018

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:

adopted 28 July 2015

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

23 July 2009

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

German GLP Compliance Program (inspected on July 13-16, 2015 / Signed on September, 2015)

Test material

Test material information

Constituent 1

Reference substance name:

Concrete of honeycomb cells of the bee by hexane extraction

EC Number:

946-615-6

IUPAC Name:

Concrete of honeycomb cells of the bee by hexane extraction

Test material form:

solid

Details on test material:

- Appearance: pale brown solid, wax-like

Specific details on test material used for the study:

- Storage conditions: At room temperature, protected from moisture (Dry area, unopened containers, optimum temp. 11-25 °C / 52-77 °F)

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

Following the REACH top-down strategy, the EPISKIN™ Reconstructed Human Epidermis Model method was used to assess skin irritation as recommended in the OECD test guideline No. 439.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN™ Reconstructed Human Epidermis Model Kit, SkinEthic Laboratories, Lyon, France
- Tissue batch number(s): 17-EKIN-041
- Production date: not reported
- Shipping date: not reported
- Delivery date: 10 October 2017
- Expiry date: 16 October 2017
- Date of initiation of testing: 10 October 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation (if applicable): 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: After the end of the treatment interval the inserts were immediately removed from the 12-well plate. The tissues were gently rinsed with PBS to remove any residual test material. Excess PBS was removed by gently shaking the inserts and blotting the bottom with blotting paper.
- Observable damage in the tissue due to washing: none reported
- Modifications to validated SOP: none reported

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Versamax® Molecular Devices, 85737 Ismaning, Germany, version 4.7.1
- Wavelength: 570 nm
- Filter: without reference filter
- Linear OD range of spectrophotometer: not reported

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Barrier function: IC50 = 1.9 mg/ml ( ≥ 1.5 mg/ml)
- Morphology: Well-differenciated epidermis consisting of a basal layer, several spinous and granular layers and a thinck stratum corneum
- Contamination: absence of bacteria, fungus and mycoplasma
- Reproducibility: All of the values for the negative and positive control groups fell within the historical ranges of the testing laboratory obtained in the previous ten months. This was taken to show the correct functioning of the test system. Envigo CRS GmbH changed the negative control for the test system “In vitro Skin Irritation Assay using RHE supplied by SkinEthik” from deionized water to PBS. The test is performed at Envigo CRS GmbH a long time prior to OECD 439 guideline, where deionized water or PBS are suggested, and at that time, SkinEthik requested deionized water to be used as negative control in the protocol. All of Envigo CRS GmbH’s historical data are based on deionized water. In the current SkinEthik protocol, PBS is suggested as negative control. Therefore, Envigo CRS GmbH decided to change the negative control to be in accordance with the SkinEthik protocol, but the number of performed assays using PBS as negative control is still too low to issue historical data. SkinEthik confirmed, that use of deionized water in the past is acceptable, since the acceptability ranges for the OD values according to the OECD guideline are met.

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE: Not a MTT reducer

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritating to skin if relative mean tissue viability is ≤ 50% after 15 minutes of exposure.
- The test substance is considered to be non-irritating to skin if relative mean tissue viability is > 50% after 15 minutes of exposure.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- The test item was used as supplied (undiluted).
- Amount(s) applied (volume or weight with unit): In order to spread approximately 10 ± 5 mg (26 mg/cm2) of the test item onto triplicate EpiSkin™ tissues, 14-15 mg of the test item were weighed to compensate for the test item remained on the applying instrument (curved flat micro spatula). The tissues were wetted with 5 μL of deionised water prior to application of the test substance.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10 μL
- Concentration (if solution): PBS

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 μL
- Concentration (if solution): 5% SLS solution in deionised water

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

Triplicate tissues for test substance, negative and positive controls

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

15 minute exposure period and 42 h post-exposure incubation period

Value:

96.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100%

Positive controls validity:

valid

Remarks:

2.7%

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: none reported
- Direct-MTT reduction: no
- Colour interference with MTT: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD570 for the negative control treated tissues was 1.149 and the standard deviation value of the viability was 9.6%.
- Acceptance criteria met for positive control: The relative mean tissue viability for the positive control treated tissues was 2.7% relative to the negative control treated tissues and the standard deviation value of the viability was 23.3%. Therefore the relative Standard deviation of the positive control was greater than 18 %. However, this can be explained by the low OD values measured which implies higher relative standard variations. Moreover the values are lower than the historical positive control data of the laboratory and the results are not bordeline, therefore the relative standard deviation calculated for positive control tissues is not considered to affect the overall conclusion of the study.
- Acceptance criteria met for variability between replicate measurements: The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 11.5%.
- Reference to historical values: All of the values for the negative and positive control groups fell within the historical ranges of the testing laboratory obtained in the previous 10 months. This was taken to show the correct functioning of the test system.

Any other information on results incl. tables

Table 7.3.1/1: EpiSkin™ results

Test Group	Mean Absorbance 570 nm blank corrected	Mean Absorbance of 3 Tissues	Relative Viability [%] Tissue 1, 2, 3*	Relative Standard Deviation [%]	Mean Rel. Viability [%]**
Negative Control	1.255	1.149	109.2	9.6	100.0
	1.034		90.0
	1.158		100.8
Positive Control	0.040	0.031	3.5	23.3	2.7
	0.028		2.4
	0.026		2.3
Test Item	1.116	1.104	97.1	3.5	96.1
	1.062		92.4
	1.135		98.8

*   Relative viability [rounded values]

**  Mean relative viability [rounded values]

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the experimental conditions of this study, the test substance is not classified for skin irritation according to Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

Executive summary:

An in vitro skin irritation study was performed according to the OECD Guideline 439 and in compliance with GLP, using the EPISKIN^TM reconstructed human epidermis model.

Triplicate tissues were treated with the test item for an exposure period of 15 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 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 plastic vials containing acidified 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 duplicate 200 µL samples were transferred to the appropriate wells of a pre‑labeled 96‑well plate. The optical density was measured at 570 nm.

 

The relative mean viability of the test item treated tissues was 96.1% after the 15‑Minute exposure period and 42‑Hours post‑exposure incubation period.

The relative mean tissue viability for the positive control treated tissues was 2.7% relative to the negative control treated tissues and the standard deviation value of the viability was 23.3%.  Therefore the relative Standard deviation of the positive control was greater than 18 %. However, this can be explained by the low OD values measured which implies higher relative standard variations. Moreover the values are lower than the historical positive control data of the laboratory and the results are not bordeline, therefore the relative standard deviation calculated for positive control tissues is not considered to affect the overall conclusion of the study.

The mean OD570 for the negative control treated tissues was 1.149 and the standard deviation value of the viability was 9.6%. The negative control acceptance criteria were therefore satisfied.

The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 3.5%. The test item acceptance criterion was therefore satisfied.

All of the values for the negative and positive control groups fell within the historical ranges of the testing laboratory obtained in the previous ten months. This was taken to show the correct functioning of the test system.

 

Under the experimental conditions of this study, the test substance is not classified for skin irritation according to Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

This study is considered as acceptable and satisfies the requirement for skin irritation endpoint.