reaction mass of 2-[[1-(chloromethyl)-2-[[4-(oxiran-2-ylmethoxymethyl)cyclohexyl]methoxy]ethoxy]methyl]oxirane and cis-1,4-bis[(2,3-epoxypropoxy)methyl]cyclohexane and trans-1,4-bis[(2,3-epoxypropoxy)methyl]cyclohexane

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

2015

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

2-[[1-(chloromethyl)-2-[[4-(oxiran-2-ylmethoxymethyl)cyclohexyl]methoxy]ethoxy]methyl]oxirane

Molecular formula:

C17H29ClO5

IUPAC Name:

2-[[1-(chloromethyl)-2-[[4-(oxiran-2-ylmethoxymethyl)cyclohexyl]methoxy]ethoxy]methyl]oxirane

Constituent 2

Reference substance name:

cis-1,4-bis[(2,3-epoxypropoxy)methyl]cyclohexane

Cas Number:

1197197-64-0

Molecular formula:

C14H26O4

IUPAC Name:

cis-1,4-bis[(2,3-epoxypropoxy)methyl]cyclohexane

Constituent 3

Reference substance name:

trans-1,4-bis[(2,3-epoxypropoxy)methyl]cyclohexane

Cas Number:

158307-92-7

Molecular formula:

C14H26O4

IUPAC Name:

trans-1,4-bis[(2,3-epoxypropoxy)methyl]cyclohexane

impurity 1

Reference substance name:

[4-(oxiran-2-ylmethoxymethyl)cyclohexyl]methanol

Molecular formula:

C11H20O3

IUPAC Name:

[4-(oxiran-2-ylmethoxymethyl)cyclohexyl]methanol

impurity 2

Reference substance name:

[4-[[3-chloro-2-(oxiran-2-ylmethoxy)propoxy]methyl]cyclohexyl]methanol

Molecular formula:

C14H25ClO4

IUPAC Name:

[4-[[3-chloro-2-(oxiran-2-ylmethoxy)propoxy]methyl]cyclohexyl]methanol

impurity 3

Reference substance name:

2-[[3-chloro-2-[[4-[[3-chloro-2-(oxiran-2-ylmethoxy)propoxy]methyl]cyclohexyl]methoxy]propoxy]methyl]oxirane

Molecular formula:

C20H34Cl2O6

IUPAC Name:

2-[[3-chloro-2-[[4-[[3-chloro-2-(oxiran-2-ylmethoxy)propoxy]methyl]cyclohexyl]methoxy]propoxy]methyl]oxirane

impurity 4

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 3M Company
- Expiration date of the lot/batch: 26 May 2022
- Purity test date:

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity:
- Specific activity:
- Locations of the label:
- Expiration date of radiochemical substance:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
- Stability under test conditions:
- Solubility and stability of the test substance in the solvent/vehicle:
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
- Preliminary purification step (if any):
- Final dilution of a dissolved solid, stock liquid or gel:
- Final preparation of a solid:

FORM AS APPLIED IN THE TEST (if different from that of starting material)

OTHER SPECIFICS:

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Multiple adult donors.

Details on animal used as source of test system:

SOURCE ANIMAL
- Source: Adult human donors
- Sex: No data

Justification for test system used:

Recommended by OECD Guideline 439.

Vehicle:

unchanged (no vehicle)

Details on test system:

SKIN DISC PREPARATION
- Procedure used:
- Quality control for skin discs: Electrical resistance obtained with two of the isolated skin discs was [complete, e.g. 10 kΩ]
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
- Number of washing steps: Not specified
- Observable damage in the tissue due to washing: Not specified
DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- Spectrophotometer: TECAN Infinite M200 Pro Plate Reader
- Wavelength: 570 nm
NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION: 3 per test article or control

PREDICTION MODEL / DECISION CRITERIA
A test item is considered irritant in the skin irritation test if:
The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is ≤ 50% of the mean viability of the negativecontrols.
A test item is considered non-irritant in the in vitro skin irritation test if:
The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is > 50% of the mean viability of the negative controls.

The in vitro skin irritation test is considered acceptable if it meets the following criteria:
a) The absolute mean OD570 (optical density at 570 nm) of the three tissues of the negative control should reasonably be within the laboratory historical control data range and the Standard Deviation value (SD) of the % viability should be less than or equal to 18.
b) The mean relative tissue viability of the positive control should be less than or equal to 40% relative to the negative control and the Standard Deviation value (SD) of the % viability should be less than or equal to 18.
c) The SD calculated from individual % tissue viabilities of the three identically treated replicates should be less than or equal to 18.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 uL

VEHICLE: None

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): Phosphate buffered saline

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 5% (aq) Sodium dodecyl sulfate (SDS, Sigma-Aldrich Chemie GmbH, Steinheim, Germany) [CAS Number 151-21-3] in PBS
- Concentration (if solution): 5%

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3 per test article or control

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Assay 1

Value:

ca. 46

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: See 'Remarks'

Remarks:

The standard deviation value of the percentage viability was 44% and the results of the individual tissues were in 2 different categories (96%, 26% and 15%). To gain more information the assay was repeated.

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Assay 2

Value:

ca. 73

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: See 'Remarks'

Remarks:

The standard deviation was 41% and the results of the individual tissues were in 2 different categories (31%, 113% and 75%)

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: None
- Direct-MTT reduction: None
- Colour interference with MTT: None

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: No, the standard deviation was greater than 40% for both assays.

Applicant's summary and conclusion

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

Based on the results of the study, test article is equivocal in the in vitro skin irritation test under the experimental conditions described. The test was concluded to be valid, however the test item is not suitable for the assay. In the absence of a conclusive finding for skin irritation, a Skin 2 classification is assigned.

Executive summary:

The dermal irritation potential of the test article was evaluated in an in vitro reconstructed epidermal model. The study was conducted in compliance with OECD GLP (1997) according to OECD Test Guideline 439 (2015). The undiluted test article (25 uL) was applied to the tissues (n=3) and the tissues were incubated for 15 minutes at room temperature. Positive (5% Sodium dodecyl sulfate (SDS) in phosphate buffered saline (PBS)) and negative (PBS) controls were tested in parallel. Following exposure, the tissues were washed to removed the test article and controls and incubated for 42 hours at 37 C. Following the post-exposure incubation, tissue viability was determined using the MTT assay. Test article tissue viability was compared to negative control tissues. In the fist assay, the mean test article-treated tissue viability was 46%. The standard deviation value of the percentage viability was 44% and the results of the individual tissues were in 2 different categories (96%, 26% and 15%).  To gain more information the assay was repeated. In the second assay, the mean test article-treated tissue viability was 73%. However, the standard deviation was 41% and the individual results were again divided over two categories (31%, 113% and 75%). Based on the results of the study, test article is equivocal in the in vitro skin irritation test under the experimental conditions described.  The test was concluded to be valid, however the test item is not suitable for the assay.  In the absence of a conclusive finding for skin irritation, a Skin 2 classification is assigned.