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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:
2017-03-06
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study

Data source

Reference
Reference Type:
other company data
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
2015
Deviations:
no
GLP compliance:
no
Remarks:
The report does not meet the definition of a GLP study

Test material

Constituent 1
Chemical structure
Reference substance name:
Methyl 2-hydroxy-2-methylpropionate
EC Number:
218-301-2
EC Name:
Methyl 2-hydroxy-2-methylpropionate
Cas Number:
2110-78-3
Molecular formula:
C5H10O3
IUPAC Name:
methyl 2-hydroxy-2-methylpropionate
Test material form:
liquid

In vitro test system

Test system:
human skin model
Remarks:
EpiDerm tissue model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: not specified
Source strain:
not specified
Justification for test system used:
The potentials for chemical-induced skin irritation and corrosion are important considerations in establishing procedures for the safe handling, packaging, and transport of chemicals (UN, 2007). The EpiDerm three-dimensional human skin model has been extensively characterized (EC-ECVAM, 2009) and is approved (OECD, 2013) for identification and classification of skin irritation hazard according to the UN GHS and EU classification system (Category 2/H315 or No label). The EpiDerm model uses human-derived epidermal keratinocytes as the cell source, which are cultured to form a multi-layered, highly differentiated model that closely mimics human epidermis at biochemical and physiological levels.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm tissue model (model number EPI-200 EpiDerm cultures)
- Supplier: MatTek Corporation; Ashland, MA.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Following the 60 ± 1min treatment period, the tissues were rinsed with sterile DPBS
- Modifications to validated SOP: none

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 300 µL MTT (1 mg/mL)
- Incubation time: 3 ± 0.1 hrs

NUMBER OF REPLICATE TISSUES: 3

CALCULATION METHOD:
The mean OD570 value of the blank wells was calculated. Individual blank corrected OD570 values for each test chemical or control tissue were determined by subtracting the mean OD570 value of the blank wells from their individual OD570 values. The mean of the corrected OD570 values for the negative control was calculated. Corrected Individual Tissue OD570 = individual tissue OD570 – mean blank OD570 For each individual tissue, % viability relative to negative control was calculated using the following formula:

% Viability = Corrected Individual OD570 of Test Chemical (or Control) / Corrected Mean OD570 of Negative Control X 100

The individual relative viabilities were tabulated for each tissue and the mean and standard deviations for viability values were calculated for the test chemical and control

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be an irritant to skin if the mean relative tissue viability of the 3 individual tissues exposed to the test chemical is ≤ 50% of mean viability of the negative control
- The test substance is considered to be non-irritant to skin if the mean relative tissue viability of the 3 individual tissues exposed to the test chemical is >= 50% of mean viability of the negative control
Control samples:
yes, concurrent negative control
yes, concurrent positive control
yes, concurrent MTT non-specific colour control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 µL
- Concentration (if solution): undiluted

VEHICLE
- none
Duration of treatment / exposure:
total of 60 ± 1 min
Duration of post-treatment incubation (if applicable):
total post-treatment incubation period of 42 ± 2 hrs
Number of replicates:
3

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Replicate 1, Methyl 2- hydroxyisobutyrate
Value:
87
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Replicate 2, Methyl 2- hydroxyisobutyrate
Value:
101
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Replicate 3, Methyl 2- hydroxyisobutyrate
Value:
106.2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
- Colour interference with MTT: no


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
The mean relative tissue viability for EpiDerm tissue models treated with methyl 2-hydroxyisobutyrate and positive control (1% TRITON™ X-100) were 98.1% and 3.2%, respectively

Any other information on results incl. tables

Table 1. Culture Viability in EpiDerm Skin Irritation Model

Chemical Name

1 Hr Treatment plus 42 Hr Recovery

Mean
Viability (%)

Replicate 1

Replicate 2

Replicate 3

Methyl 2-hydroxyisobutyrate

87.0

101.0

106.2

98.1

Negative Control*

101.6

101.7

96.7

100.0

Positive Control*

4.6

1.9

3.2

3.2

*Negative Control: DPBS; Positive Control: 1% TRITON™ X-100

Table 2. In Vitro Classification in EpiDerm Skin Irritation Model

Chemical Name

EpiDerm Irritancy Classification
Prediction (I/NI)

Methyl 2-hydroxyisobutyrate

Non-Irritant (UN GHS Cat NC)

Negative Control*

Non-Irritant (UN GHS Cat NC)

Positive Control*

Irritant (I) (UN GHS Cat 1 or 2)

*Negative Control: DPBS; Positive Control: 1% TRITON™ X-100

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The mean relative tissue viability for EpiDerm tissue models treated with methyl 2-hydroxyisobutyrate and positive control (1% TRITON™ X-100) were 98.1% and 3.2%, respectively. Methyl 2-hydroxyisobutyrate was considered to be a non-irritant (UN GHS Cat NC) to skin.
Executive summary:

The skin irritatin potential of the test substance methyl 2-hydroxyisobutyrate was evaluated in a non-GLP in vitro EpiDerm skin irritation assay (MatTek Corporation; Ashland, MA) according to OECD Guideline 439. The EpiDerm tissue model consists of normal, human-derived epidermal keratinocytes that are cultured to form a multilayered, differentiated model of human epidermis. In this assay, methyl 2-hydroxyisobutyrate was topically applied to the EpiDerm tissue models for 60 minutes, followed by a 42-hour post-exposure recovery. Following recovery, the cell viability was measured in treated and control tissues using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay and the data reported as a percentage of the mean of negative control. A test chemical was considered to possess skin irritation potential (UN GHS Cat 1 or 2) if the relative cell viability was less than or equal to 50%. In this study, Dulbecco’s Phosphate Buffered Saline (DPBS) and 1% TRITON™ X-100 served as the negative and positive controls, respectively.

The mean relative cell viability of methyl 2-hydroxyisobutyrate- and positive control-treated tissues were 98.1% and 3.2% (i.e. ≤ 50%), respectively, therefore, methyl 2-hydroxyisobutyrate was considered as a non-irritant (UN GHS Cat NC) in the EpiDerm irritation assay in accordance to Regulation (EC) No 1272/2008 (CLP Regulation).

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