Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
skin irritation / corrosion
Remarks:
in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04-12-2017 to 12-03-2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Guideline study performed under GLP. All relevant validity criteria were met.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Deviations:
yes
Remarks:
Minor deviation in environmental conditions (humidity minimum 47%) which does not affect the reliability of the study
Qualifier:
according to guideline
Guideline:
other: EU Method B.40 BIS : In Vitro Skin Corrosion: Human Skin Model Test
Deviations:
yes
Remarks:
Minor deviation in environmental conditions (humidity minimum 47%) which does not affect the reliability of the study
GLP compliance:
yes

Test material

Constituent 1
Reference substance name:
Reaction products of 2-hydroxyethyl methacrylate and diphosphorous pentoxide and water
Molecular formula:
Not applicable (a generic molecular formula cannot be provided for this specific UVCB substance)
IUPAC Name:
Reaction products of 2-hydroxyethyl methacrylate and diphosphorous pentoxide and water
Test material form:
liquid
Details on test material:
- Physical state: Liquid
- Storage condition of test material: In refrigerator (2-8°C) container protected from light
- Other: Yellow liquid

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
foreskin from a single donor
Vehicle:
unchanged (no vehicle)
Details on test system:
EpiDerm Skin Model (EPI-200, Lot no.: 27636 kit L and M and 27667 Kit H and G). The test consists of topical application of the test item on the skin tissue for 3-minute and 1-hour. After exposure the skin tissue is thoroughly rinsed to remove the test item followed by immediate determination of the cytotoxic (corrosive) effect.
Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) at the end of the treatment. The EpiDerm Skin Model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists 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. The EpiDerm tissues (surface 0.6 cm²) were cultured on polycarbonate membranes of 10 mm cell culture inserts.

Preincubation:
On the day of receipt the tissues were kept on agarose and stored in the refrigerator. On the next day, at least one hour before starting the assay the tissues were transferred to 6-well plates with 0.9 ml DMEM.

Application of test item and rinsing:
The plates were incubated for approximately 1 to 1.5 hours at 37 ± 1.0°C. The medium was replaced with fresh DMEM just before the test item was applied. Two tissues were used for a 3-minute exposure and two for a 1-hour exposure. Test item: 50 µl of the undiluted test item was added into the 6-well plates on top of the skin tissues. Negative Control: similarly treated with 50 µl Milli-Q water only. Positive control: 50 µl 8N KOH (potassium hydroxide 8 normal solution). After the exposure periods (3 minutes and 1 hour, respectively), the tissues were washed with phosphate buffered saline to remove residual test item. The skin inserts were carefully dried. Rinsed tissues were kept in 24 well plates on 300 µl DMEM until 6 tissues (= one application time) were dosed and rinsed.

The repeat experiment 2 (3-minutes repeated exposure only), was similarly conducted.

All incubations, with the exception of the test item incubation of 3 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 47- 84%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.0 - 37.3°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Based on testing laboratory historical data these deviations are not considered significant.
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
- Concentration (if solution): undiluted

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 μl
- Concentration (if solution): Not applicable

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 μl
- Concentration (if solution): 8N KOH(aq) (pre-diluted)
Duration of treatment / exposure:
Observations are made 3-minutes and 1-hour post-test item application
Duration of post-treatment incubation (if applicable):
The DMEM was replaced by 300 µl MTT-medium and tissues were incubated for 3 hours at 37°C in air containing 5% CO2. After incubation the tissues were washed with PBS and formazan was extracted with 2 ml isopropanol (MatTek corporation) over night at room temperature.
Number of replicates:
1. Experiment 1: Duplicate; two tissues used for a 3-minute exposure to the test item and two tissues for a 1-hour exposure, and equivalent NC and PC tissue exposures.
2. Experiment 2 (3-minute exposure only): Duplicate; two tissues used for a 3-minute exposure to the test item, and equivalent NC and PC tissue exposures.

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean (n = 2)
Value:
35
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Basis: EXPERIMENT 1: mean. Time point: 3 minute exposure. Remarks: n = 2 ; CV = 41 % within range of 20 - 100% viability; Score in terms of percentage of negative control. Discarded and test repeated in EXPERIMENT 2.
Irritation / corrosion parameter:
% tissue viability
Remarks:
3 minute exposure
Run / experiment:
mean (n = 2)
Value:
8.6
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Basis: EXPERIMENT 2: mean. Time point: 3 minute exposure. Remarks: n = 2 ; CV = 45 % ; Score in terms of percentage of negative control.
Irritation / corrosion parameter:
% tissue viability
Remarks:
1 hour exposure
Run / experiment:
mean (n = 2)
Value:
12
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Basis: EXPERIMENT 1: mean. Time point: 1 hour exposure. Remarks: n = 2 ; CV = 85 % ; Score in terms of percentage of negative control.
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: None reported.
- Direct-MTT reduction: Screened prior to test. Not applicable.
- Colour interference with MTT: Screened prior to test. Not applicable.

DEMONSTRATION OF TECHNICAL PROFICIENCY: The test laboratory has validated the specified assay.

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. Experiment 2 was conducted due to excessive variability in Experiment 1 in the 3-minute exposure, and a Coefficient of Variation between 20 and 100% viability of > 30% (35% and 12% in each replicate). In the repeated 3-minute exposure: the Coefficient of Variation between 20 and 100% viability was inapplicable as the viability was < 20%. The validity criterion was met.
- Range of historical values if different from the ones specified in the test guideline: Historic Control Data: Negative Control OD570: 3-minutes (n=111): 1.258 – 2.615 ; 1-hour (n=110): 1.371 – 2.371. Positive Control HCD data are presented within the full study report and the concurrent PC was within the specified ranges.

Applicant's summary and conclusion

Interpretation of results:
Category 1A (corrosive) based on GHS criteria
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of this in vitro study, the test item is considered to be corrosive to the skin. The test item caused a mean tissue viability of < 50% after 3 minute exposure and < 15% after 1 hour exposure and would be classified under Regulation (EC) 1272/2008: skin corrosive - category 1A.
Executive summary:

The study was performed to OECD TG 431 and EU Method B.40 BIS to assess the skin corrosion potential of the test item in accordance with GLP using a human three-dimensional epidermal model (EpiDerm (EPI-200)). The test was performed on a total of 4 tissues per test item per experiment, together with a negative control and positive control. Four tissues were used for a 3-minute exposure to the test item (two in experiment 1 and two in experiment 2) and two for a 1-hour exposure (in experiment 1). 50 μl of the undiluted test item was added (with a pipette) into the 6-well plates on top of the skin tissues. The remaining tissues were treated with 50 μl Milli-Q water (negative control) and with 50 μl 8N KOH (positive control), respectively. The positive control had a mean relative tissue viability of 13% after 1 hour exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. In the range of viability 20 - 100% the Coefficient of Variation between tissue replicates was < 8% for the negative control and 41% for the test item.The relative mean tissue viability obtained after 3-minute and 1-hour treatments with the test item compared to the negative control tissues was 35% and 12%, respectively. However, since the acceptability criteria were not met for the 3-minute exposure, this part of the test was repeated. In the repeat experiment 2, the positive control had a mean relative tissue viability of 15% after the 3-minute exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The relative mean tissue viability obtained after 3-minute treatment with the test item compared to the negative control tissues was 8.6%. Skin corrosion is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with the test item compared to the negative control tissues was 8.6% and 12%, respectively. Because the mean relative tissue viability for the test item was below 50% after the 3-minute treatment and below 15% after the 1-hour treatment the test item is considered to be corrosive. Under the conditions of this study, the test item is skin corrosive in the in vitro skin corrosion test and classified under Regulation (EC) 1272/2008: skin corrosive - category 1A.