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Administrative data

Description of key information

Skin irritation: non-corrosive, in vitro EpiDerm OECD 431, Wil Research 2014
Skin irritation: irritating, in vitro EpiSkin OECD 431, Wil Research 2014
Eye irritation: non-irritating, eq or similar to OECD 405, Consumer Product Testing Inc. 1979

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP.
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
yes
Remarks:
Minor deviation in environmental conditions (humidity minimum 72%) which does not affect the reliability of the study
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
yes
Remarks:
Minor deviation in environmental conditions (humidity minimum 72%) which does not affect the reliability of the study
GLP compliance:
yes
Species:
other: EPISKIN Small ModelTM
Strain:
not specified
Details on test animals and environmental conditions:
EPISKIN Small Model is a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. The keratinocytes were cultured for 13 days, which results in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

All incubations, with the exception of the test substance incubation of 15 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 72 - 89%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.0 - 36.9°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the humidity (with a maximum of 11%) occurred which were caused by opening and closing of the incubator door, but the time of these deviations did not exceed 1 hour. Based on laboratory historical data these deviations are considered not to affect the study integrity.
Controls:
other: negative and positive controls were used
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 microlitres
- Concentration (if solution): undiluted

Duration of treatment / exposure:
Tissues were treated with the test material for 15 minutes and then washed with phosphate buffered saline to remove residual test material. Subsequently the skin tissues were incubated for 42 hours at 37°C.
Details on study design:
TEST SITE
- Area of exposure: Twenty five μl of the undiluted test substance was added (with a pipette) into 12-well plates on top of the skin tissues.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): yes
- Time after start of exposure: 15 minutes

SCORING SYSTEM: Skin irritation potential of the test substance was classified according to remaining cell viability following exposure of the test substance.
Irritation / corrosion parameter:
other:
Value:
6
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 15 minute exposure. Reversibility: no data. Remarks: Score in terms of percentage of negative control. (migrated information)
Other effects:
The test substance was checked for possible direct MTT reduction by adding the test substance to MTT medium. Because no colour change was observed it was concluded that the test substance did not interact with MTT.

Table 1. Mean absorption and tissue viability in the in vitro skin irritation test with the test substance

  OD570 Mean  SD Mean tissue viability (% of control)
A B C
Negative control 0.966 1.118 1.135 1.073 ±0.093 100
Test Material 0.062 0.071 0.063 0.065 ±0.005 6
Positive control 0.084 0.108 0.135 0.109 ±0.025 10

OD = optical density SD = Standard deviation Triplicate exposures are indicated by A, B and C.

Values are corrected for background adsoption (0.041). Isopropanol was used to measure background adsorption.

Interpretation of results:
irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of this in vitro study, the test material is considered to be irritating to skin.
Executive summary:

The study was performed to OECD 439 and EU Method B.46 to assess the skin irritation potential of the test substance in accordance with GLP using a human three dimensional epidermal model (EPISKIN Small Model). The test was performed on a total of 3 tissues per test substance together with negative and positive controls. Twenty five μl of the undiluted test substance was added (with a pipette) into 12-well plates on top of the skin tissues. Three tissues were treated with 25 μl PBS (negative control) and 3 tissues with 25 μl 5% SDS (positive control) respectively. The positive control was re-spread after 7 minutes contact time. After the exposure period of 15 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test substance. After rinsing the cell culture inserts were each dried carefully and moved to a new well on 2 ml pre-warmed maintenance medium until all tissues were dosed and rinsed. Subsequently the skin tissues were incubated for 42 hours at 37°C. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate. Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues. The positive control had a mean cell viability of 10% after 15 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was less than 9%, indicating that the test system functioned properly. The relative mean tissue viability obtained after 15 minutes treatment with the test substance compared to the negative control tissues was 6%. Since the mean relative tissue viability for the test substance was below or equal to 50% after 15 minutes treatment it is considered to be irritant.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Eye irritation

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation:

In vitro, OECD 439 2014 - The study was performed to OECD 439 and EU Method B.46 to assess the skin irritation potential of the test substance in accordance with GLP using a human three dimensional epidermal model (EPISKIN Small Model). The test was performed on a total of 3 tissues per test substance together with negative and positive controls. Twenty five μl of the undiluted test substance was added (with a pipette) into 12-well plates on top of the skin tissues. Three tissues were treated with 25 μl PBS (negative control) and 3 tissues with 25 μl 5% SDS (positive control) respectively. The positive control was re-spread after 7 minutes contact time. After the exposure period of 15 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test substance. After rinsing the cell culture inserts were each dried carefully and moved to a new well on 2 ml pre-warmed maintenance medium until all tissues were dosed and rinsed. Subsequently the skin tissues were incubated for 42 hours at 37°C. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate. Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues. The positive control had a mean cell viability of 10% after 15 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was less than 9%, indicating that the test system functioned properly. The relative mean tissue viability obtained after 15 minutes treatment with the test substance compared to the negative control tissues was 6%. Since the mean relative tissue viability for the test substance was below or equal to 50% after 15 minutes treatment it is considered to be irritant.

 

Skin corrosion:

In vitro, OECD 431 2014 - The study was performed to OECD 431 and EU Method B.40 BIS to assess the skin corrosion potential of the test substance 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 substance 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. Fifty μl of the undiluted test substance 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 9% after 3 minutes exposure. The absolute mean OD540 (optical density at 540 nm) of the negative control tissues was within the laboratory historical control data range. The maximum inter-tissue variability in viability between two tissues treated identically was less than 12% and the maximum difference in percentage between the mean viability of two tissues and one of the two tissues was less than 7%, indicating that the test system functioned properly. Skin corrosion is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with the test material compared to the negative control tissues was 92% and 42%, respectively. Because the mean relative tissue viability for the test substance was not below 50% after the 3-minute treatment and not below 15% after the 1-hour treatment the test substance is considered to be not corrosive. Under the conditions of this study the test material is not corrosive in the in vitro skin corrosion test.

Eye irritation/corrosion:

In vivo eq. or similar to OECD 405 1979 - The study was performed to assess the irritancy potential of the test material to the eye following a single application in the New Zealand White rabbit. The non-GLP study was completed under a method similar to OECD 405 and following definitions similar to the US 16 CFR 1500.42. A volume of 0.1 ml of the test material was placed into the conjunctival sac of one eye of 6 animals. The other eye remained untreated and was used for control purposes. Assessment of ocular damage/irritation was made approximately 24, 48 and 72 hours following treatment, and further assessments made at 4 and 7 days using scoring based on Draize et al. Corneal, iritis and conjunctival irritation was observed in the majority of test animals on all 3 days. Under the conditions of this study the test material is considered to be a mild eye irritant. Based on the applicants recalculation of the mean scores following grading at 24, 48 and 72h, 4 out of 6 organisms mean scores did not meet the EU classification criteria. The mean scores indicated that not more than three organisms met a positive scoring criteria for corneal opacity which in all cases fully reversed within 7 days. Furthermore there was an absence of significant variation in effects. Mean iritis and conjunctival scores in all organisms were insufficient for classification purposes. The substance has the potential to produce mild transient eye irritation but is insufficient for classification. Therefore the substance cannot be considered as an eye irritant under Regulation (EC) 1272/2008.


Justification for selection of skin irritation / corrosion endpoint:
two in vitro GLP compliant Klimisch 1 studies; selected study in accordance with sequential testing strategy of Regulation (EC) 440/2008 and its subsequent ammendments.

Justification for selection of eye irritation endpoint:
one non-GLP in vivo study assigned Klimisch score 2; following a method equivalent to recognised guideline and is well documented

Effects on skin irritation/corrosion: irritating

Justification for classification or non-classification

The substance meets the classification criteria under EU Directive 67/548/EEC for skin irritation, R38 irritating to the skin

The substance meets the classification criteria under Regulation (EC) No 1272/2008 for skin irritation category 2, H315

The substance does not meet classification criteria under EU Directive 67/548/EEC for eye irritation.

The substance does not meet classification criteria under Regulation (EC) No 1272/2008 for eye irritation.