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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:
30th October 2017 - 27th November 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 guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Reaction mass of allyl (2-methylbutoxy)acetate and allyl (3-methylbutoxy)acetate
EC Number:
916-328-0
Molecular formula:
C10H18O3
IUPAC Name:
Reaction mass of allyl (2-methylbutoxy)acetate and allyl (3-methylbutoxy)acetate
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: O’Laughlin (Nantong) Fine Chemicals Co., Ltd.; NTA375
- Expiration date of the lot/batch: Sep 25, 2020
- Purity: CAS No. 67634-00-8: 79.45 %; CAS No.: 67634-01-9 20.28 %

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Store in cool place. Keep container tightly closed in a dry and well-ventilated place.

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
The reconstructed human epidermal model EpiDerm™ (EPI-200, MatTek, Bratislava, SR) consists of normal human-derived epidermal keratinocytes, which have been cultured to form a multilayered highly differentiated model of the human epidermis. The EpiDerm™ tissues (surface 0.63 cm²) are cultured on specially prepared cell culture inserts and shipped as kits, containing 24 tissues on shipping agarose together with the necessary amount of culture media. The certificate of analysis from the EpiDerm™ model (Lot No. 25851, kit B) is presented in Annex 1.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 3 mins at room temperature and 60 minutes 37±1°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: After exposition, tissues were thoroughly rinsed and blotted to remove the test item/controls.
- Observable damage in the tissue due to washing: None
- Modifications to validated SOP:None

DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- Spectrophotometer: Libra S22
- Wavelength:OD570

NUMBER OF REPLICATE TISSUES: In each time interval three tissues were used per test item, three tissues for the positive control (PC) and three tissues for negative control (NC).

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE

Direct MTT reduction - functional check in tubes
Functional check for this possibility was performed as follows: 50 µL of the test item was added to 1 mL MTT medium (red) and it was incubated in the incubator (37±1 °C, 5±1 % CO2, humidified) for 1 hour. At the end of
the exposure time, the presence and intensity of the staining (if any) was observed.

Concurrent MTT non-specific colour control
For this purpose 50 µL of the test item were added to 1.0 mL of water and it was kept at culture conditions (37±1 °C, 5±1 % CO2, humidified) for 1 hour. Another 50 µL of the test item were added to 2 mL of isopropyl
alcohol and it was left at 2 hours at room temperature.

PREDICTION MODEL / DECISION CRITERIA
Corrosivity potential of the test item is predicted from the relative mean tissue viabilities obtained after 3 minutes and 60 minutes of treatment compared to the negative control tissues concurrently treated with H2O.

According to the OECD TG 431 as well as to the EU Method B.40, the test item is considered to be corrosive to skin:
i) if the viability after 3-minute exposure is less than 50 %, or
ii) if the viability after 3-minute exposure is greater than or equal to 50 % and the viability after 1-hour exposure is less than 15 %.
The test item is considered to be non-corrosive to skin:
i) if the viability after 3 minute exposure is greater than or equal to 50 % and the viability after 1-hour exposure is greater than or equal to 15 %.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
yes, concurrent MTT non-specific colour control
other: Direct MTT reduction
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µL

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

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL
- Concentration (if solution): 8N KOH
Duration of treatment / exposure:
3 mins at room temperature
60 minutes at 37±1°C
Number of replicates:
3

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
AAG (3 mins)
Value:
95.2
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
AAG (60 mins)
Value:
104.4
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Positive Control (3 mins)
Value:
7.5
Vehicle controls validity:
not applicable
Negative controls validity:
not applicable
Positive controls validity:
not applicable
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Positive Control (60 mins)
Value:
4.6
Vehicle controls validity:
not examined
Negative controls validity:
not applicable
Positive controls validity:
not applicable
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: None
- Direct-MTT reduction: The test item did not change colour of MTT medium from red to blue (see Figure 1). The test item does not reduce MTT directly.
- Colour interference with MTT: The colour of water/isopropyl alkohol did not change, so colour of the test item did not interfere with evaluation (see Figures 2 a, b).

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The assay meets the acceptance criterion - OD570 of the NC tissues was 1.790 (3 min) and 1.747 (60 min) which is ≥ 0.8 and ≤ 2.8.
- Acceptance criteria met for positive control: Viability of tissues treated with 8N KOH after 60 minutes of treatment was 4.6 % which is < 15 %.
- Acceptance criteria met for variability between replicate measurements: CV values in all triplets of tissues were ≤ 0.3.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In the in vitro skin corrosion test using the reconstructed human epidermal model EpiDerm, Allyl Amyl Glycolate was not corrosive.
Executive summary:

In an in vitro skin corrosion assay using a human epidermal model EpiDerm (17 -698), reconstructed human epidermis tissue was exposed to 50 µL mg of Allyl Amyl Glycolate for 3 mins at room temperature and 60 minutes at 37±1°C. Water was used for the negative control and 8N KOH was used for the positive control. After removal of the test substance, tissues were incubated with MTT for 3 hours incubation. 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 colour of the test substance did not interfere with the endpoint. The test substance is not directly MTT reducing. The average viability of Allyl Amyl Glycolate-treated tissues was 95.2 % of the negative control average value after 3 minutes of treatment and 104.4 % after 1 hour of treatment. The average viability of 8N KOH-treated tissues was 7.5 % of the negative control average value after 3 minutes of treatment and 4.6 % after 1 hour of treatment According to these results, Allyl Amyl Glycolate is not corrosive.

This in vitro skin corrosion assay in the human epidermal model EpiDerm is acceptable and satisfies the guideline requirement for an OECD 431 study.