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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 August 2017 - 01 September 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
29 July 2016
Deviations:
no
Qualifier:
according to
Guideline:
other: EC Guideline No. 440/2008. Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.40 BIS: "In Vitro Skin Corrosion: Human Skin Model Test". Official Journal of the European Union No. L142
Version / remarks:
31 May 2008
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Physical appearance: white powder
Storage conditions: at room temperature

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
skin obtained from plastic surgery from multiple donors
Justification for test system used:
Recommended test system in international guidelines (OECD and EC).
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm Skin Model
- Tissue batch number(s): 26787
- Surface: 0.6 cm^2
- On the day of receipt the tissues were kept on agarose and stored in the refrigerator.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 3 minute exposure: room temperature, 1 hour exposure: 36.3 - 37.1°C.
- Temperature of post-treatment incubation: 37°C.

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: the tissues were washed with phosphate buffered saline (one washing step)
- Observable damage in the tissue due to washing: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm
- The test item was checked for interference with the MTT endpoint before the start of the study.

NUMBER OF REPLICATE TISSUES: 4 (2 tissues for the 3 minute exposure period, 2 tissues for the 1 hour exposure period) + 2 tissues for the negative and the positive control for each exposure period

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

PREDICTION MODEL / DECISION CRITERIA (see table 1 in ''any other informatin on methods'')
A test item is considered corrosive in the in vitro skin corrosion test if:
a) The relative mean tissue viability obtained after 3-minute treatment compared to the negative control tissues is decreased below 50%.
b) In addition, a test item considered non-corrosive (viability ≥ 50%) after the 3-minute treatment is considered corrosive if the relative tissue viability after 1-hour treatment with the test item is decreased below 15%.

A test item is considered non corrosive in the in vitro skin corrosion test if:
a) The relative mean tissue viability obtained after the 3-minute treatment compared to the negative control tissues is not decreased below 50%.
b) In addition, the relative tissue viability after the 1-hour treatment is not decreased below 15%.

ACCEPTABILITY CRITERIA:
a) The absolute mean OD570 of the two tissues of the negative control should reasonably be within the acceptance limits of OECD 431 (lower acceptance limit ≥0.8 and upper acceptance limit ≤2.8)
b) The mean relative tissue viability following 1-hour exposure to the positive control should b e <15 %.
c) In the range 20 - 100% viability, the Coefficient of Variation (CV) between tissue replicates should be ≤ 30%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount applied: 38.6-45.6 mg on tissues moistened with 25 μL Milli-Q water.

NEGATIVE CONTROL
- Amount applied: 50 μL Milli-Q water

POSITIVE CONTROL
- Amount: 50 μL 8N KNOH
Duration of treatment / exposure:
- 3 minutes
- 1 hour
Duration of post-treatment incubation (if applicable):
3 hours
Number of replicates:
4 in total; 2 per exposure period

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1 hour exposure (mean of 2 replicates)
Value:
101
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks:
Mean tissue viability: 8.4%
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 minute exposure (mean of 2 replicates)
Value:
109
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks:
Mean tissue viability: 10%
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no
- Colour interference with MTT: no

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, the absolute mean OD570 was between the acceptance limits (i.e. 1.644 for the 3-minute exposure and 1.779 for the 1-hour exposure).
- Acceptance criteria met for positive control: yes, the mean relative tissue viability following 1 hour exposure was <15% (i.e., 8.4%).
- Acceptance criteria met for variability between replicate measurements: yes, CV between tissue replicates was ≤30% (i.e., 7.2% for the 3 minute exposure period and 5.7% for the 1 hour exposure period).

- Because the mean relative tissue viability for HYDROPIP-NR was not below 50% after 3 minutes treatment and not below 15% after 1 hour treatment, the test item is considered to be not corrosive.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Remarks:
Not classified according to Regulation (EC) No. 1272/2008
Conclusions:
The results of a skin corrosion test, performed according to OECD guideline 431, showed that HYDROPIP-NR was not corrosive to the skin. The test substance is therefore not classified according to GHS and according to Regulation (EC) 1272/2008.
Executive summary:

A skin corrosion test was performed, according to OECD guideline 431, to assess the potential of HYDROPIP-NR to induce skin corrosion on a human three dimensional epidermal model. The test item was applied at an amount of >25 mg directly on top of an EpiDerm model for a 3 -minute and a 1 -hour exposure period. For each exposure period two tissues were treated with the test item, two tissues were treated with a positive control (potassium hydroxide) and two tissues were treated with Milli-Q water as a negative control. After exposure, the tissues were incubated for 3 hours in MTT-medium and subsequently the cell viability was measured at 570 nm using a spectrophotometer. The mean cell viability for the tissues treated with test item compared to the negative control were 109 and 101% for the 3 -minute and the 1 -hour exposure period, respectively. The mean tissue viability for the positive control after 1 -hour exposure was 8.4%. All acceptability criteria were met and the study was considered to be valid. Since the mean tissue viability was >50% after the 3 -minute exposure and >15% after the 1 -hour exposure, HYDROPIP-NR is considered not corrosive to the skin. The test substance is therefore not classified according to GHS and according to Regulation (EC) 1272/2008.