1,2-Heptanediol

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2020-08-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: MatTek Corporation Protocol: In vitro EpiDermTM Skin Irritation Test (EPI-200-SIT) For use with MatTek Corporation’s Reconstructed Human Epidermal Model EpiDerm (EPI-200-SIT)

Version / remarks:

2019-02-10

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:

2012-07-06

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2019-06-18

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

heptane-1,2-diol

EC Number:

855-780-2

Cas Number:

3710-31-4

Molecular formula:

C7H16O2

IUPAC Name:

heptane-1,2-diol

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: All cells used to produce EpiDerm are derived from tissue obtained by MatTek Corporation from accredited institutions.

Source strain:

other: not applicable, cells from human donors are used

Details on animal used as source of test system:

Not applicable, cells from human donors are used

Justification for test system used:

In an international prevalidation study performed by ECVAM, the in vitro skin irritation test using the human skin model EpiDerm™ and EpiSkin™ and measurement of cell viability by dehydrogenase conversion of MTT into a blue formazan salt have turned out as a sufficiently promising predictor for skin irritancy potential.

Vehicle:

unchanged (no vehicle)

Remarks:

The test item was tested neat.

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Epi-200-SIT (MatTek Corporation, 82105 Bratislava, Slovakia) is a three-dimensional human epidermis model which develops a highly differentiated and stratified epidermis analogous to those found in vivo.
- Tissue batch number: 30880
- Date of initiation of testing: 12th May 2020

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: At 37 ± 1.5°C (first 35 min), room temperature (last 25 min)
- Temperature of post-treatment incubation: 37 ± 1.5°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: The tissues were gently rinsed with PBS several times in order to remove any residual test material

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL in DMEM
- Incubation time: 3 hours in MTT solution, 3.5 hours in isorpopanol
- Spectrophotometer: Versamax® (Molecular Devices)
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: OD 0.03 to OD 0.11 ( positive control, corresponds to 2.24% - 6-19% viability according to HCD of laboratory); ≥ OD 0.8 and ≤ OD 2.8 (negative control, according to OECD439/MatTek protocol); ≥ OD 1.28 and ≤ OD 2.0 (according to HCD of laboratory)
- Barrier function: The barrrier function has been tested by ET-50 assay using 100 µL Triton X-100, 4 time points, n=3 using MTT assay. The acceptance criterium was fulfilled when the ET-50 was reached between 4.77 and 8.72 hours. The test result of the tissue batch used was 5.51 hours.
- Contamination: The cells used to produce EpiDerm tissue were screened for potential biological contaminants (HIV-1 virus, Hepatitis B and C virus by oligonucleotide direct amplification, bacteria, yeast and other funghi were ensured by long-term antibiotic, antimycotic-free culture). None of the listed contaminants were detected in the tissue batch used.

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues: Since test item/ water and test item/ isopropanol solutions did not change color significantly and the test item did not interfere with MTT, no additional tissues were necessary in the main experiment.

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

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive or irritant to skin if the tissue viability after exposure and post-incubation is ≤50%.
- The test substance is considered to be non-irritant to skin if the tissue viability after exposure and post-incubation is ≥50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 30 µL
- Concentration: The item was applied undiluted

NEGATIVE CONTROL
- Amount applied: Not indicated
- Concentration: The negative control (DPBS) was applied undiluted

POSITIVE CONTROL
- Amount applied: Not indicated
- Concentration: 5% SDS in deionised water

Duration of treatment / exposure:

1 hour (35 min at 37°C and 25 min at room temperature)

Duration of post-treatment incubation (if applicable):

45.5 hours (including a medium change after 25.5 hours)

Number of replicates:

3 tissue replicates, one experiment

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean of 3 tissue replicates

Value:

2.84

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: Not indicated
- Direct-MTT reduction: The test item did not interfere with MTT.
- Colour interference with MTT: The test item/ water and test item/ isopropanol solutions did not change color significantly.

DEMONSTRATION OF TECHNICAL PROFICIENCY:
See "Attached background material"

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: Yes, the standard deviations between the three tissue percentage viability values of each group (test item, positive and negative controls) in the main test were below 18
- Range of historical values if different from the ones specified in the test guideline: See "Details on test system"

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Remarks:

The test item is identified as requiring classification and labelling according to UN GHS/ EU CLP “Category 2” or “Category 1”. Further testing is required to resolve between UN GHS Categories 1 and 2 and decide on the final classification of the test item.

Conclusions:

In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item is irritant to skin.

Executive summary:

This GLP compliant in vitro study was performed to assess the irritation potential of the test item by means of the Human Skin Model Test according to OECD guideline 439. The test item did not prove to be a MTT reducer in the MTT interference pre-experiment. Also, its intrinsic color was not intensive and it did not change color when mixed with deionised water or isopropanol. Therefore, additional tests with freeze-killed tissues and/or viable tissues (without MTT addition) did not have to be performed. Each three tissues of the human skin model EpiDerm™ were treated with the test item, the negative control (DPBS) or the positive control (5% SDS) for 60 minutes. After treatment with the negative control the absorbance values were well within the required range of the acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for the 60 minutes treatment interval, thus assuring the quality of the tissues. Treatment with the positive control induced a sufficient decrease in the viability as compared to the negative control for the 60 minutes treatment interval, thus assuring the validity of the test system. After treatment with the test item, the mean relative viability value was 2.84% compared to the relative absorbance value of the negative control. This value is below the threshold for irritancy of ≤ 50%. Therefore, the test item is considered to possess an irritant potential. In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item is irritant to skin. The test item is identified as requiring classification and labelling according to UN GHS/ EU CLP “Category 2” or “Category 1”, since the tissue viability after exposure and post-incubation is less or equal to 50%. Further testing is required to resolve between UN GHS Categories 1 and 2 and decide on the final classification of the test item.