Reaction mass of 2-(3-ethylphenyl)oxirane and 2,2’-(1,3-phenylene)dioxirane and 2,2'-(1,4-phenylene)dioxirane

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was performed between 30 June 2009 and 06 July 2009.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: OECD TG Draft Guideline (Version 7.6)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EU Guideline Testing of Chemicals B46

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 19/08/2008 Date of Signature: 4/03/2009

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of bis(epoxyethyl) benzene and (ethylphenyl) oxirane

IUPAC Name:

Reaction mass of bis(epoxyethyl) benzene and (ethylphenyl) oxirane

Constituent 2

Reference substance name:

Reaction mass of 2-(3-ethylphenyl)oxirane and 2,2’-(1,3-phenylene)dioxirane and 2,2'-(1,4-phenylene)dioxirane

EC Number:

935-853-6

IUPAC Name:

Reaction mass of 2-(3-ethylphenyl)oxirane and 2,2’-(1,3-phenylene)dioxirane and 2,2'-(1,4-phenylene)dioxirane

Details on test material:

- Name of test material (as cited in study report): Reaction mass of bis(epoxyethyl) benzene and (ethylphenyl) oxirane
- Substance type: Organic, Multiconstituent
- Physical state: clear extremely pale yellow liquid
- Analytical purity: 80 - 95%
- Lot/batch No.: 200901317-17
- Stability under test conditions : stable
- Storage condition of test material : approximately 4°C in the dark

Test animals

Species:

other: Reconstituted Hhuman Epidermis Model

Strain:

other: Reconstituted Human Epidermis Model

Details on test animals or test system and environmental conditions:

Not applicable

Test system

Type of coverage:

other: Topical

Preparation of test site:

other: Not applicable

Vehicle:

unchanged (no vehicle)

Remarks:

No vehicle used

Controls:

no

Amount / concentration applied:

TEST MATERIAL

- Amount(s) applied (volume or weight with unit):
10 µL of the test material was applied to the epidermis surface.

- Concentration (if solution):
The test material was used as supplied.

VEHICLE
No vehicle used

Duration of treatment / exposure:

15 Minutes

Observation period:

Not applicable

Number of animals:

Not applicable

Details on study design:

TEST SITE
- Area of exposure:
10 µL of the test material was applied to the epidermis surface..

- % coverage:
The test material was applied topically to the corresponding tissues ensuring uniform covering.

- Type of wrap if used:
Not applicable

REMOVAL OF TEST SUBSTANCE
- Washing (if done):
At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing PBS with Ca2+ and Mg2+. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of PBS to gently remove any residual test material.

- Time after start of exposure:
15 minutes

SCORING SYSTEM:
Quantitative MTT Assessment (percentage tissue viability)
For the test material the relative mean tissue viabilities obtained after the 15 minute treatment followed by the 42 hour post-exposure incubation period were compared to the mean of the negative control treated tissues (n=3). The relative mean viabilities were calculated in the following way:

mean OD540 of test material / mean OD540 of negative control x 100

Classification of irritation potential is based upon relative tissue viability following the 15 minute exposure period followed by the 42 hour post-exposure incubation period according to the following table:
Criteria for in vitro interpretation Classification
Mean tissue viability is ≤50% Irritant (I) R38
Mean tissue viability is >50% Non-Irritant (NI)

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

other: other: % viability

Value:

5.4

Remarks on result:

other:

Remarks:

Basis: mean. Time point: 15 minutes. Max. score: 100.0. Reversibility: other: Not applicable. Remarks: The relative margin viability of the test material treated tissues was 5.4 % after a 15 minutes exposure.. (migrated information)

In vivo

Irritant / corrosive response data:

Irritant

Other effects:

No

Any other information on results incl. tables

RESULTS

Direct MTT Reduction

The MTT solution containing the test material did not turn blue/purple which indicated that the test material did not directly reduce MTT.

Test Material, Positive Control Material and Negative Control Material

The individual and mean OD₅₄₀values, standard deviations and tissue viabilities for the test material, negative control material and positive control material are given in Table 1. The mean viabilities and standard deviations of the test material and positive control, relative to the negative control are also given in Table 1.

The relative mean viability of the test material treated tissues was 5.4% after a 15-minute exposure.

The qualitative evaluation of tissue viability is given in Table 2.

Following the 15-minute exposure the test material treated tissues appeared white which was considered indicative of dead tissue.

Quality Criteria

The relative mean tissue viability for the positive control treated tissues was ≤40% relative to the negative control treated tissues and the standard deviation value of the percentage viability was ≤20%. The positive control acceptance criterion was therefore satisfied.

The mean OD₅₄₀ for the negative control treated tissues was ≥0.6 and the SD value of the percentage viability was ≤20%. The negative control acceptance criterion was therefore satisfied.

Table 1. Mean OD₅₄₀Values and Percentage Viabilities for the Negative Control Material, Positive Control Material and Test Material

Material	OD540of tissues	Mean OD540of triplicate tissues	± SD of OD540	Relative individual tissue viability (%)	Relative mean viability (%)	± SD of viability
Negative Control Material	0.803	0.827	0.042	97.1	100*	5.1
	0.802			97.0
	0.876			105.9
Positive Control Material	0.060	0.046	0.014	7.3	5.6	1.7
	0.033			4.0
	0.046			5.6
Test Material	0.060	0.045	0.014	7.3	5.4	1.7
	0.040			4.8
	0.034			4.1

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

Table 2. Qualitative Evaluation of Tissue Viability (MTT uptake visual evaluation)

Material	Tissue 1	Tissue 2	Tissue 3
Negative Control Material	-	-	-
Positive Control Material	++	++	++
Test Material	++	++	++

MTT visual scoring scheme
-          =         blue tissue (viable)
+         =         blue/white tissue (semi-viable)
++       =         tissue is completely white (dead)

SD=    Standard deviation

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

Applicant's summary and conclusion

Interpretation of results:

irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test material was considered to be Irritant. The symbol “I” and the risk phrase R 38 “IRRITATING TO SKIN” are therefore required.

Executive summary:

Introduction. 

The purpose of this test was to evaluate the skin irritation potential of the test material using the EPISKIN^TM reconstituted human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours. The principle of the assay was based on the measurement of cytotoxicity in reconstituted human epidermal cultures following topical exposure to the test material by means of the colourimetric 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 material treated tissues relative to the negative controls. The concentration of the inflammatory mediator IL-1α in the culture medium retained following the 42 hour post-exposure incubation period is also determined for test materials which are found to be borderline non-irritant based upon the MTT reduction endpoint. This complimentary end-point will be used to either confirm a non-irritant result or will be used to override the non-irritant result.

The EPISKIN^TM model is a three-dimensional reconstituted human epidermis model consisting of adult human-derived epidermal keratinocytes seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after a 13 day culture period comprising of the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

Triplicate tissues were treated with the test material for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for approximately 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-labelled 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. 

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-labelled 96-well plate. The optical density was measured at 540 nm.

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

Results: 

The relative mean viability of the test material treated tissues was 5.4% after a 15‑minute exposure.

Quality criteria: 

The quality criteria required for acceptance of results in the test were satisfied.

Conclusion:  The test material was considered to be Irritant.