Diethyl sulphate, compound with 2-(heptadec-8-enyl)-4,5-dihydro-1H-imidazole-1-ethanol (1:1)

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-01-15 to 2018-03-01

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

2016-07-29

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: 4.2.2 EpiSkinTM Skin Corrosivity Test INVITTOX n° 118, ECVAM DB-ALM: INVITTOX protocol

Version / remarks:

March 1997

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: EpiSkinTM SOP, Version 1.8 (February 2009), ECVAM Skin Irritation Validation Study: Validation of the EpiSkinTM test method 15 min-42 hours for the predication of acute skin irritation of chemicals.

Version / remarks:

Feebruary 2009

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Diethyl sulphate, compound with 2-(heptadec-8-enyl)-4,5-dihydro-1H-imidazole-1-ethanol (1:1)

EC Number:

271-272-8

EC Name:

Diethyl sulphate, compound with 2-(heptadec-8-enyl)-4,5-dihydro-1H-imidazole-1-ethanol (1:1)

Cas Number:

68527-63-9

Molecular formula:

C22H42N2O.C4H10O4S

IUPAC Name:

2-{2-[(8E)-heptadec-8-en-1-yl]-4,5-dihydro-1H-imidazol-1-yl}ethan-1-ol diethyl sulfate

Test material form:

liquid

Details on test material:

Test item formulations were found to be homogeneous and stable up to 24 hour in vehicle corn oil.

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: R-127., Ltd. / Batch 170825
- Date of manufacture: 2017-06-19
- Expiration date of the lot/batch: 2019-06-19

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Solubility and stability of the test substance in the solvent/vehicle: soluble in water, acetone, alcohol

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Details on animal used as source of test system:

The tissue consisted of normal, human-derived epidermal keratinocytes which have be en cultured to form a multilayered, highly differentiated model of the human epidermis. It consisted of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo.

Justification for test system used:

Investigation of skin corrosive properties

Vehicle:

unchanged (no vehicle)

Details on test system:

Test system preparation
After receipt of the EpiSkinTM kit, all of the epidermis units used in the study were pre-incubated in maintenance medium (provided by kit supplier) at 37 oC, 5% CO2 for at least 24 hours before dosing.

Dose of test and control substances
Before topical application, the six epidermis units for test substance were transferred to new maintenance medium:

a) Two epidermis units were dosed with 50 µl test item and incubated at room temperature for 3 minutes

b) Two epidermis units were dosed with 50 µl test item and incubated at room temperature for 60 minutes

c) Two epidermis units were dosed with 50 µl test item and incubated at room temperature for 240 minutes

Before topical application, the six epidermis units for negative control substance (0.9% NaCl saline) were transferred to new maintenance medium:

a) Two epidermis units were dosed with 50 µl negative control substance and incubated at room temperature for 3 minutes

b) Two epidermis units were dosed with 50 µl negative control substance and incubated at room temperature for 60 minutes

c) Two epidermis units were dosed with 50 µl negative control substance and incubated at room temperature for 240 minutes

Before topical application, the two epidermis units for positive control substance (glacial acetic acid) were transferred to new maintenance medium. The two epidermis units were dosed with 50 µl positive control substance and incubated at room temperature for 240 minutes

Removal of test chemicals

After treatment, each treated epidermis unit was rinsed with 25ml sterile 0.01M PBS pH 7.4 to remove the residue of test chemicals from the epidermal surface. After rinsing, the unit was placed on absorbent paper to remove the PBS solution from epidermal surface. Cotton-bud was used to sweep the surface carefully when necessary.

MTT assay
The epidermis units were transferred into assay medium with 0.3 mg/ml MTT (protected from light) and incubated at 37 oC, 5% CO2 for 3 hours protected from light.
The epidermis units were dried on absorbent paper at the end of incubation. Each epidermis tissue was then gently separated using forceps from the collagen matrix. Both epidermis tissue and collagen matrix were placed into a micro tube with 500 µl of acidic isopropanol and mixed thoroughly. The micro tubes were stored at room temperature overnight.
The solution in the micro tubes were vortexed to homogenize the solution colour. Two 200 µl of solution from each tube was transferred into a 96-well plate. The absorbance (OD) at 570 nm was read using microplate reader.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µl

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µl
- Concentration (if solution): 0.9 % NaCl saline

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µl
- Concentration (if solution): glacial acetic acid

Duration of treatment / exposure:

3 min, 60 min, 240 min

Number of replicates:

2

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 min exposure

Value:

94

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 min exposure

Value:

82.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

240 min exposure

Value:

74

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

Refers to corrosion, only.

Conclusions:

Based on the prediction model for the EpiSkinTM skin corrosion test method and associated with UN GHS classification system, the test item- Diethyl sulphate, compound with 2-(heptadec-8-enyl)-4,5-dihydro-1H-imidazole-1-ethanol (1:1) is considered as non-corrosive.

Executive summary:

The potential of corrosive propetries to the skin were eaxamined ina guideline study according to OECD 431 with the reconstructed human epidermis test method.

Based on the prediction model for the EpiSkinTM skin corrosion test method and associated with UN GHS classification system, the test item- Diethyl sulphate, compound with 2-(heptadec-8-enyl)-4,5-dihydro-1H-imidazole-1-ethanol (1:1) is considered as non-corrosive.