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Administrative data

Description of key information

Skin irritation/corrosion:

One in-vitro skin corrosion study is available. It is concluded that the test substance is not corrosive.

Eye irritation:

A BCOP study is available. The test substance did not induce ocular irritation.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 25 to 29 January 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
Batch number: E010016621
Purity: Not indicated
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Details on animal used as source of test system:
Source: MatTek Corporation, Ashland MA, U.S.A.
Justification for test system used:
Recommended test system in international guidelines (OECD and EC).
Vehicle:
unchanged (no vehicle)
Details on test system:
Application/Treatment of the test item
The skin tissues were kept in the refrigerator the day they were received. The next day, at least 1 hour before the assay was started the tissues were transferred to 6-well plates containing 0.9 ml DMEM medium per well. The level of the DMEM medium was just beneath the tissue. The plates were incubated for 1 – 2.5 hours at 37.0 ± 1.0ºC. The medium was replaced with fresh DMEM medium just before the test substance was applied. The test was performed on a total of 4 tissues per test item together with a negative control and positive control. Two tissues were used for a 3-minute exposure to the test substance and two for a 1-hour exposure. The skin was moistened with 25 μl Milli-Q water to ensure close contact of the test item to the tissue and 38.0 to 50.0 mg of the solid test item was added 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. After the exposure period, the tissues were washed with phosphate buffered saline to remove residual test item. Rinsed tissues were kept in 24 well plates on 300 μl DMEM medium until 6 tissues (= one application time) were dosed and rinsed.
Cell viability measurement
The DMEM medium 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. The amount of extracted formazan was determined spectrophotometrically at 570 nm in triplicate with the TECAN Infinite® M200 Pro Plate Reader.
Cell viability was calculated for each tissue as percentage of the mean of the negative control tissues. Skin corrosion potential of the test item was classified according to remaining cell viability following exposure of the test item with either of the two exposure times.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 38.0 to 50.0 mg

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL
Duration of treatment / exposure:
3 minutes, 1 hour
Number of replicates:
Two tissues each group
Irritation / corrosion parameter:
% tissue viability
Value:
80
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Remarks:
The exposure time is 3 minutes
Irritation / corrosion parameter:
% tissue viability
Value:
90
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Remarks:
The exposure time is 1 hour
Other effects / acceptance of results:
The test substance was checked for colour interference in aqueous conditions and possible direct MTT reduction by adding the test item to MTT medium. Because the solutions did not turn blue / purple nor a blue / purple precipitate was observed it was concluded that the test substance did not interfere with the MTT endpoint.
The maximum inter-tissue variability in viability between two tissues treated identically was less than 17% and the maximum difference in percentage between the mean viability of two tissues and one of the two tissues was less than 9% for the negative control and test item. For the positive control, the maximum inter-tissue variability in viability between two tissues treated identically was less than 31% and the maximum difference in percentage between the mean viability of two tissues and one of the two tissues was less than 18%, however since the viabilities were below 20% the acceptability criteria were met. It was therefore concluded that the test system functioned properly.
Interpretation of results:
GHS criteria not met
Conclusions:
Finally, it is concluded that this test is valid and that the test substance is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 18 to 19 January 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
Batch number: E010016621
Purity: Not indicated
Species:
cattle
Details on test animals or tissues and environmental conditions:
Source: Bovine eyes from young cattle were obtained from the slaughterhouse (Vitelco's Hertogenbosch, The Netherlands), where the eyes were excised by a slaughterhouse employee as soon as possible after slaughter.
Transport: Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 311.9 to 329.7 mg
Duration of treatment / exposure:
240 ± 10 minutes
Number of animals or in vitro replicates:
Three corneas each group
Details on study design:
Treatment of corneas and opacity measurements
The test substance was weighed in a bottle and applied directly on the corneas in such a way that the cornea was completely covered (311.9 to 329.7 mg).The holder was slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the solutions over the entire cornea. Corneas were incubated in a horizontal position for 240 ± 10 minutes at 32 ± 1°C. After the incubation the solutions and the test compound were removed and the epithelium was washed at least three times with MEM with phenol red. Possible pH effects of the test item on the corneas were recorded. Each cornea was inspected visually for dissimilar opacity patterns. The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM and the opacity determinations were performed.
Permeability determinations
After the incubation period, the medium in the posterior compartment of each holder was removed and placed into a sampling tube labelled according to holder number. 360 μl of the medium from each sampling tube was transferred to a 96-well plate. The optical density at 490 nm (OD490) of each sampling tube was measured in triplicate using a microplate reader. Any OD490 that was 1.500 or higher was diluted to bring the OD490 into the acceptable range (linearity up to OD490 of 1.500 was verified before the start of the experiment). OD490 values of less than 1.500 were used in the permeability calculation.
The mean OD490 for each treatment was calculated using cMEM corrected OD490 values. If a dilution has been performed, the OD490 of each reading of the positive control and the test item was corrected for the mean negative control OD490 before the dilution factor was applied to the reading.
Irritation parameter:
in vitro irritation score
Value:
-0.3
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
The individual in vitro irritancy scores for the negative controls ranged from -0.1 to 0.9. The individual positive control in vitro irritancy scores ranged from 101 to 154. The corneas treated with the positive control were turbid after the 240 minutes of treatment.
The corneas treated with the test substance showed opacity values ranging from -0.8 to 0.0 and permeability values ranging from 0.000 to 0.013. The corneas were clear after the 240 minutes of treatment with the test substance. No pH effect of the test item was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from -0.8 to 0.2 after 240 minutes of treatment with the test substance.
Interpretation of results:
GHS criteria not met
Conclusions:
Since the test substance induced an IVIS≤3, no classification is required for eye irritation or serious eye damage.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Additional information

Skin irritation/corrosion:

One in-vitro skin corrosion study is available which was performed according to OECD guideline 431 under GLP (Eurlings, 2016). It is concluded that the test substance is not corrosive.

Eye irritation:

A BCOP study is available which was performed according to OECD guideline 437 under GLP (Eurlings, 2016). The test substance did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of -0.3 after 240 minutes of treatment.

Justification for classification or non-classification

Skin irritation/corrosion:

Because the mean relative tissue viability for the test substance was not below 50% after 3 minutes treatment and not below 15% after 1 hour treatment it is considered to be not corrosive. In the absence of in-vivo data no definitive conclusion can be drawn.

Eye irritation:

Since the test substance induced an IVIS≤3 (actual value -0.3), no classification is required for eye irritation or serious eye damage based on GHS criteria. In the absence of in-vivo data no definitive conclusion can be drawn.