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Diss Factsheets

Toxicological information

Skin irritation / corrosion

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Administrative data

skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
From 2012-05-22 to 2012-05-28
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
The study was conducted in accordance with OECD Guideline 439. However, despite the fact that the present study generally followed Good Laboratory Practice principles, not all the elements required in GLP are contained and no study specific Quality Assurance procedures were performed. Additionally, some of the requirements of the OECD guidelines are not met (see Material and Methods section).

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
- No verification of the barrier properties, stability of tissues treated with negative control over the exposure duration unproved
GLP compliance:
The study followed Good Laboratory Practice principles, however no study specific Quality Assurance procedures were performed and the report may not contain all of the elements required by GLP

Test material

Constituent 1
Chemical structure
Reference substance name:
bis(ethyl (1R,2S)-1-amino-2-ethenylcyclopropane-1-carboxylate); sulfuric acid
EC Number:
Cas Number:
Molecular formula:
bis(ethyl (1R,2S)-1-amino-2-ethenylcyclopropane-1-carboxylate); sulfuric acid
Test material form:
solid: particulate/powder
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): JNJ-31052047-ABI or T3325
- Substance type: Isolated process intermediate
- Physical state: Solid
- Analytical purity: 100.1 % (w/w)
- Lot/batch No.:00549620
- Expiration date of the lot/batch: 12 May 2012
- Storage condition of test material: Room temperature (ca. 20°C), dark

Test animals

other: Human-derived epidermal keratinocytes : 99-KERA-037, 04-KERA-12, 02-KERAMR-005, 09-KERA-003
Details on test animals or test system and environmental conditions:
The EPISKIN Skin Irritation Test-42 hours using human epidermis skin constructs was supplied by SkinEthic Laboratories, Lyon, France and consists of reconstructed epidermis of normal human keratinocytes. The Quality Controls performed on the tissue material confirm the well-differentiation of the epidermis, consisting of a basal layer, several spinous and granular layers and a thick stratum corneum. This product was prepared and packaged using aseptic techniques. On receipt, the inserts with tissue on agarose were stored at room temperature until use. The kit was used within the expiry date indicated by the supplier (expiry date: 28 May 2012). The maintenance medium was pre-warmed to 37ºC. The tissues were removed from the agar and placed into wells of 12 well plates containing 2 mL pre-warmed maintenance medium per well. The tissues were incubated for a minimum of 24 hours at 37 ± 2°C in a humidified atmosphere of 5% CO2 in air.

Test system

Type of coverage:
other: not applicable
Preparation of test site:
other: not applicable
other: See Any other information on materials and methods incl. tables
Amount / concentration applied:
A weight of 10±2 mg of the substance was dispensed over each tissue using glass weighing boats. The tissues were wetted with 5 µL of distilled water prior to application of the test substance.
Duration of treatment / exposure:
After incubation of at least 24 hours maintenance, triplicate EPISKIN tissues were treated with the test item, positive, and negative controls for 15± 0.5 minutes at room temperature. A maximum of four samples were applied in a block with a minimum of 1 minute intervals between each application of substance. On application of 10 μL, the positive control was spread over the tissue for approximately 30 seconds and then respread with a curved flat spatula after 7 minutes application time.
Observation period:
Each tissue was then rinsed with 25 mL sterile Dulbeccos Phosphate Buffered saline (DPBS) to remove residual substance. Inserts with tissues were blotted on absorbent paper to remove remaining DPBS. Each insert was then transferred to a well containing 2 mL maintenance medium and incubated for 42 ± 1 hour at 37 ± 2°C in a humidified atmosphere of 5% CO2 in air. After 42 ± 1 hour, each insert was transferred to a well containing 2 mL of 0.3 mg/mL MTT and incubated for 3 hours ± 5 minutes at 37 ± 2°C in a humidified atmosphere of 5% CO2 in air. At the end of 3 hours ± 5 minutes, all the inserts were blotted on absorbent paper. The tissue was removed from the insert using a biopsy punch and the epidermis was separated from the collagen matrix using forceps. The epidermis and the collagen matrix were placed together in a micro tube. When all the tissues had been punched, the tissues (epidermis and collagen matrix) were vortexed with 500 μL of acidic isopropanol (0.04 N HCl final concentration). The tissues were then extracted by storing at 2-8 ºC, protected from light, for 70 hours. After formazan extraction, duplicate 200 μL aliquots of the extractant from each micro tube were pipetted into the wells of flat-bottomed 96-well plates. The extractant was mixed by vortexing prior to taking the aliquots. The absorbance was read at 540 nm [with six wells containing acidified isopropanol (0.04 N HCl final concentration) as a blank].
Number of animals:
Not applicable -> triplicates EPISKIN tissues
Details on study design:
The negative control was sterile Dulbecco’s Phosphate Buffered Saline (DPBS) with magnesium and calcium.
The positive control was 5% Sodium Dodecyl Sulphate (SDS) in distilled water.

Results and discussion

In vivo

Irritant / corrosive response data:
With a mean tissue viability of 98.5 ± 3.2% after 15 minutes exposure to JNJ-31052047-ABI, this substance was predicted as non-irritant to the skin.

Any other information on results incl. tables

The mean absorbance of the triplicate negative control values was 0.723 which was between the minimum and maximum values of 0.6 and 1.5. The standard deviation (SD) of the % viability was 16.0 which was below the maximum value of 18.

The percentage mean viability of the positive control was 25.2 ± 5.5 of the negative control. These were below the maximum acceptance values of 40% viability and SD of 18.

Applicant's summary and conclusion

Interpretation of results:
not irritating
Migrated information Criteria used for interpretation of results: EU
Under the experimental conditions of this in vitro study, T003063 is observed to be non irritating to the skin.