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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 May 2020 - 28 August 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Reaction mass of 2- (palmitoylamino)ethyl acrylate and 2-(stearoylamino)ethyl acrylate
EC Number:
950-492-4
Cas Number:
2361378-62-1
Molecular formula:
C21-23H39-43NO3
IUPAC Name:
Reaction mass of 2- (palmitoylamino)ethyl acrylate and 2-(stearoylamino)ethyl acrylate
impurity 1
Reference substance name:
Amides, C16-18, N-(hydroxyethyl)
EC Number:
309-819-0
EC Name:
Amides, C16-18, N-(hydroxyethyl)
Cas Number:
101226-97-5
IUPAC Name:
Amides, C16-18, N-(hydroxyethyl)
impurity 2
Reference substance name:
Zirconium compounds
IUPAC Name:
Zirconium compounds
Test material form:
solid: granular
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: sample supplied by sponsor :91112Y
- Expiration date of the lot/batch: 12 March 2021
- Purity test date: 92.4%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Controlled room temperature (15-25°C, ≤70% relative humidity)

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
The test item was applied in this original form; although it was ground to fine powder in a mortar and pestle.

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
other: epidermal keratinocytes (NHEK)
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ Model
- Tissue batch number(s): 30865
- Date of initiation of testing:13 May 2020

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C(first 35 min), then 25 minutes at room temperature (23.9-24.7°C)
- Temperature of post-treatment incubation (if applicable): 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: After the incubation time the EpiDerm™ units were removed and rinsed thoroughly with DPBS solution(= insert was filled and emptied 20 times in a constant soft stream of DPBS in a glass beaker filled with at least 100 mL DPBS solution) to remove all of the test material from the epidermal surface


MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg MTT / ml medium
- Incubation time: 3 hours
- Spectrophotometer: Thermo Fisher Scientific
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3 per test item, negative and positive control

CONTROL TISSUES USED IN CASE OF MTT DIRECT/COLOUR INTERFERENCE
1. MTT: 25 mg of test item was added to 1 mL MTT medium and mixed. The mixture was incubated at 37°C in an incubator with 5 % CO2, in a >95% humidified atmosphere for 1 hour protected from light, and then any colour change was recorded:
-Test items which do not react with MTT: other colours
-Test items reacting with MTT: blue or purple
After one hour incubation, red colour of the mixture was detected in the test tube. Thus, the test item did not react with MTT and therefore the use of additional controls was not necessary.

2. Colour: Prior to treatment, the test item was evaluated for its intrinsic colour or ability to become coloured in contact with water (simulating a tissue humid environment).
The test item had a light yellow colour, therefore, in addition to the normal procedure, two additional test item-treated living tissues were used for the non-specific OD evaluation. These tissues followed the same test item application and all steps as for the other tissues, except for the MTT step: MTT incubation was replaced by incubation with fresh Assay Medium to mimic the amount of colour from the test item that may be present in the test disks. OD reading was conducted following the same conditions as for the other tissues.

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be non-irritant to skin if the mean cell viability was >50% compared to the negative control.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg

VEHICLE
- Amount(s) applied (volume or weight with unit):25 µL DPBS
- Lot: RNBH7035 (Sigma-Aldrich)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL DPBS

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution): 5% (w/v) SDS solution
Duration of treatment / exposure:
35 minutes at 37°C; 25 minutes at room temperature (23.9-24.7°C)
Duration of post-treatment incubation (if applicable):
42 hrs (± 2hrs)
Number of replicates:
3

Test system

Vehicle:
other: DPBS

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
test item
Value:
92.7
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: 92.7±5.9
Other effects / acceptance of results:
- OTHER EFFECTS:
- Direct-MTT reduction: As no colour change was observed after one hour of incubation of the test item in MTT working solution, the test material did not interact with MTT. Therefore, additional controls and data calculations were not necessary. The false estimation of viability can be excluded.
- Colour interference with MTT:
As the test item was coloured (light yellow), two additional test item-treated living tissues were used for the non-specific OD evaluation. The mean optical density (measured at 570 nm) of these tissues was determined as 0.003, Non-Specific Colour % (NSCliving%) was calculated as 0.2% compared to negative control (see Table 3). This value was below 5%, therefore additional data calculation was not necessary

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD value of the three negative control tissues was in the recommended range (1.956). Standard deviation of the viability results for negative control samples was 3.2%.
- Acceptance criteria met for positive control: The positive control treated tissues showed 1.9% viability demonstrating the proper performance of the assay.
- Acceptance criteria met for variability between replicate measurements: The standard deviation of viability values of the three test item-treated tissue samples in the MTT assay was 5.9%. The standard deviation of the viability results for positive control samples was 0.1%.
- Range of historical values if different from the ones specified in the test guideline: OD data of two replicate tissues at the negative control in this study were higher (OD: 2.014 and 1.966) than the maximum OD of the historical control range (OD: 1.941). OD data of one replicate tissue at the positive control in this study was lower (OD: 0.035) than the minimum OD of the historical control range (OD: 0.038). This fact has no impact on the results or integrity of the study since the positive control material showed clear positive result and the acceptable mean percentage viability range for the positive controls is 0-20% according to the OECD No. 439.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Remarks:
Not classified according to CLP.
Conclusions:
In an in vitro skin irritation assay in the human epidermal model EpiDerm, the test item is not a skin irritant.
Executive summary:

In an in vitro skin irritation assay in the human epidermal model EpiDerm (20/071-051B), reconstructed human keratinocytes (moistened with 25µL DPBS) was exposed to 25 mg of the test item (92.4%) for 35 minutes at 37°C and then 25 minutes at room temperature (23.9- 24.7°C). DPBS was used for the negative control and 5% SDS was used for the positive control. After removal of the test substance via washing, tissues were post-incubated for 42 ± 2hrs. Tissues were then incubated with MTT for 3 hours. The OD570 of isopropyl alcohol extracts was measured on a spectrophotometer. Relative cell viability was calculated for each tissue as % of the mean viability of the negative control tissues.

The controls confirmed the validity of the study. The mean OD570 for the negative control treated tissues was between ≥0.8 and ≤ 2.8 (1.956). The mean relative tissue viability of the positive control was ≤ 20% (1.9%). Standard deviation of viability of replicate tissues of all dose/negative control/positive control groups was ≤ 18% (5.9%/3.2%/0.1%).

The test item was not directly MTT reducing. The NSCliving % value for two additional test item-treated living tissues was below 5% so additional data calculation was not necessary to consider colour interference. The average viability of tissues treated by the test item was 92.7 ±5.9% of the negative control average value i.e. viability was > 50 %. According to these results, the test item is not irritating.