Reaction mass of 1-Hydroxyoctane, 2-methanoate and 1,2-Dihydroxyoctane, dimethanoate and 2-Hydroxyoctane 1-methanoate and 1,2-Dihydroxyoctane

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2 - 27 May 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Version / remarks:

adopted 29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of 1-Hydroxyoctane, 2-methanoate and 1,2-Dihydroxyoctane, dimethanoate and 2-Hydroxyoctane 1-methanoate and 1,2-Dihydroxyoctane

EC Number:

937-960-3

Molecular formula:

not applicabe (reaction mass)

IUPAC Name:

Reaction mass of 1-Hydroxyoctane, 2-methanoate and 1,2-Dihydroxyoctane, dimethanoate and 2-Hydroxyoctane 1-methanoate and 1,2-Dihydroxyoctane

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: EpiDerm™ (EPI-200)

Source strain:

not specified

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE)
- Model used: EpiDerm™ (EPI-200) (MatTek Corporation, Bratislava, Slovakia)
- Tissue batch number: 23338
- Delivery date: 24 May 2016
- Date of initiation of testing: 24 May 2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature (3 minutes exposure), 37 ± 1.5 °C (60 minutes exposure)

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Tissues were gently rinsed with DPBS (20 times) in order to remove any residual test material.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h
- Spectrophotometer: microplate reader (Versamax, Molecular Devices, SoftMax Pro Enterprise v.4.7.1)
- Wavelength: 570 nm
- Filter: without reference filter

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: The quality of the EpiDerm™ tissue was assessed by an MTT cell viability test.
- Barrier function: The barrier function was assessed by determination of the exposure time required to reduce tissue viability by 50% (ET-50) upon application of 100 µL of 1% Triton X-100. The ET-50 value was determined to be 5.64 h.
- Contamination: The cells used to produce the EpiDerm™ tissue were screened for the presence of viruses, bacteria, yeast and other fungi.

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
Since the test substance did not directly reduce the MTT solution, an additional functional check was not performed.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 50 µL

NEGATIVE CONTROL
- Amount applied: 50 µL

POSITIVE CONTROL
- Amount applied: 50 µL
- Concentration: 8 N

Duration of treatment / exposure:

3 ± 0.5 and 60 ± 5 min

Number of replicates:

duplicates for each treatment and control group

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean value of 2 tissues

Run / experiment:

3 minutes exposure

Value:

98.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean value of 2 tissues

Run / experiment:

60 minutes exposure

Value:

10.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Direct-MTT reduction: The test substance did not interfere with the MTT assay (no reducing capacity).

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean negative control OD, both for the 3 and 60 min exposure period, was in the range of ≥ 0.8 and ≤ 2.8.
- Acceptance criteria met for positive control: Exposure to the positive control induced a decrease in the relative absorbance as compared to the negative control, both for the 3 min exposure period (16.2%) and for the 60 min exposure period (10.7%) thus confirming the validity of the test system and the specific batch of tissue models.
- Acceptance criteria met for variability between replicate measurements: The coefficient of variation in the range 20 – 100% viability between tissue replicates was < 30%.

Any other information on results incl. tables

Table 2. Results after treatment with the test substance and controls

Test group	Absorbance at 570 nm*		Mean absorbance of 2 tissues	Coefficient of variation (%)	Rel. absorbance (% of negative control)**
	Tissue 1	Tissue 2
3 minutes treatment
Negative control	1.701	1.598	1.649	4.4	100.0
Positive control	0.239	0.297	0.268	15.4	16.2
Test substance	1.656	1.605	1.630	2.2	98.8
60 minutes treatment
Negative control	1.619	1.625	1.622	0.2	100.0
Positive control	0.198	0.148	0.173	20.2	10.7
Test substance	0.169	0.170	0.170	0.1	10.5

* Mean of three replicate wells after blank correction

** Relative absorbance (rounded values): 100 × (absorbance test substance/positive control) / (absorbance negative control)

Applicant's summary and conclusion

Interpretation of results:

other: Skin Corr. Cat. 1

Remarks:

according to Regulation (EC) No 1272/2008

Conclusions:

Under the conditions of the conducted test, the test substance possessed corrosive properties towards reconstructed human epidermis tissue.

Executive summary:

After exposure of the tissues to the test item the relative absorbance value decreased to 98.8% after 3 minutes exposure. After 1 hour exposure the relative absorbance value was reduced to 10.5%. The value for the 3 minutes exposure did not exceed the threshold for corrosivity which is defined to be 50% after the 3 minutes exposure, but the value for the 1 hour exposure was below the threshold of 15%. Therefore, the test item was considered to be corrosive.