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Description of key information

Skin irritation/corrosion: not irritating
Eye irritation/corrosion: not irritating

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:
11 Spet - 17 Sept 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
(adopted 22 July 2010)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
23 July 2009
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
The Department of Health of the Government of the United Kingdom
Species:
human
Strain:
other: EpiSkin; reconstructed three-dimensional human epidermis
Details on test animals or test system and environmental conditions:
TEST SKIN MODEL
- Source: SkinEthic Laboratories, Nice, France

TEST METHOD
The EPISKIN model is a three-dimensional reconstructed human epidermis model consisting of adult human-derived epidermal keratinocytes seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen (EPISKIN Model Kit, SkinEthic Laboratories, Nice, France). A highly differentiated and stratified epidermis model is obtained after a 13-Day culture period comprising the main basal, spinous and granular layers and a functional stratum corneum. The test item is applied topically to the straturm corneum surface, at the air interface, so that undiluted and/or end use dilutions can be tested directly. The test is based on the experience that irritant chemicals are cytotoxic after exposure to the EPISKIN model. Irritant chemicals are able to penetrate the stratum corneum and are sufficiently cytotoxic to cause cell death in the underlying cell layers after a longer incubation period. Toxicity is determined by the metabolic conversion of the vital dye MTT to formazan by viable cells in the test item treated cultures relative to the negative control. Additionally, IL-1alpha in the culture medium is measured to for test items which are borderline non-irritant based on MTT reduction. This complementary endpoint will be used to either confirm a non-irritant result or to override the non-irritant result.

ADAPTATION TO CELL CULTURE CONDITIONS
Tissues were transferred into 12-well plates containing 2 mL of prewarmed maintenance medium and incubated for 1 day at 5% CO2.
Type of coverage:
open
Preparation of test site:
other: intact reconstructed human epidermis
Vehicle:
unchanged (no vehicle)
Controls:
other: 106concurrent control tissues treated with 10 µL of DPBS served as negative controls, positive controls were exposed to 5% (w/v) SDS
Amount / concentration applied:
TEST MATERIAL: 10 µL

POSITIVE CONTROL SUBSTANCE: 5% (w/v) SDS; 10 µL; to ensure uniform contact SDS was spread over the whole surface with a pipette tip, after 7 minutes, SDS was re-spread to ensure distribution
Duration of treatment / exposure:
15 minutes followed by 42 hours post-exposure in medium
Observation period:
not applicable
Number of animals:
not applicable
The test was performed in duplicates for each test or control group and treatment period
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing: The test item was washed from the skin surface with DPBS.
- Time after start of exposure: 15 min

CELL VIABILITY MEASUREMENTS & IL-1alpha
Following the 42-Hour post-exposure incubation period each 12-well plate was placed onto a plate shaker for 15 minutes to homogenise the released mediators in the maintenance medium. 1.6 ml of the maintenance medium from beneath each tissue was transferred to pre-labelled micro tubes and stored in a freezer at -14 to -30ºC for possible inflammatory mediator determination.
The tissues were transferred to 2 ml of a 0.3 mg/ml MTT solution, freshly prepared in assay medium and incubated for 3 hours at 37°C, 5% CO 2 in air. At the end of the 3-Hour incubation period each tissue was placed onto absorbent paper to dry. A total biopsy of the epidermis was made using the EPISKINTM biopsy punch. The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) placed into labelled 1.5 ml micro tubes containing 500 μl of acidified isopropanol, ensuring that both the epidermis and collagen matrix were fully immersed. Each tube was plugged to prevent evaporation and mixed thoroughly on a vortex mixer. The tubes were refrigerated at 1 to 10°C until Day 6 of the experiment, allowing the extraction of formazan crystals out of the MTT-loaded tissues. At the end of the formazan extraction period each tube was mixed thoroughly on a vortex mixer to produce a homogenous coloured solution. The optical density of 200 µL (duplicate measurements) was measured (quantitative viability analysis) at 540 nm (without a reference filter) using the Anthos 2001 microplate reader.
Irritation / corrosion parameter:
other: other: cell viability (%)
Value:
100
Remarks on result:
other:
Remarks:
Basis: other: mean value of negative controls. Time point: 15 min + 42 hours. Reversibility: other: not applicable. (migrated information)
Irritation / corrosion parameter:
other: other: cell viability (%)
Value:
106.1
Remarks on result:
other:
Remarks:
Basis: other: mean value of the test item. Time point: 15 min + 42 hours. Reversibility: other: not applicable. (migrated information)
Irritation / corrosion parameter:
other: other: cell viability (%)
Value:
9.9
Remarks on result:
other:
Remarks:
Basis: other: mean value of the positive control. Time point: 15 min + 42 hours. Reversibility: other: not applicable. (migrated information)
Irritant / corrosive response data:
The relative mean viability of the test item treated tissues was 106.1% after a 15-Minute exposure period and is therefore in range with the negative control and no irritating effect was observed. The positive control showed irritation. Additionally, the acceptance criteria are fulfilled.
Other effects:
The MTT solution containing the test item (in a pretest) did not turn blue which indicated that the test item did not directly reduce MTT.

Table 1: Mean OD540Values and Percentage Viabilities for the Negative Control Item, Positive Control Item and Test Item

Item

OD540 of tissues

Mean OD 540 of triplicate tissues

±SD of OD540

Relative individual tissue viability (%)

Relative mean viability (%)

± SD of Relative mean viability (%)

 

Negative Control Item

0.920

0.854

0.060

107.7

100

7.0

0.839

98.2

0.804

94.1

Positive Control Item

0.047

0.085

0.051

5.5

9.9

6.0

0.064

7.5

0.143

16.7

Test Item

0.816

0.906

0.078

95.6

106.1

9.1

0.955

111.8

0.947

110.9

 

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
CLP: not classified
DSD: not classified
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Version / remarks:
(adopted 24 April 2002)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2400 (Acute Eye Irritation)
Version / remarks:
(Aug 1998)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Japanese Ministry of Agriculture, Forestry and Fisheries, Testing Guidelines for Toxicology Studies, 12 NohSan No. 8147 (adopted 2002)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
The Department of Health of the Government of the United Kingdom, London, UK
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 57033
- Expiration date of the lot/batch: 25 November 2014

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature in the dark.
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Details on strain: Hsdlf:NZW
- Source: Harlan Laboratories UK Ltd. Leicestershire, UK
- Age at study initiation: 12-20 weeks
- Weight at study initiation: 2.33-2.66 kg
- Housing: individually in suspended cages
- Diet: 2930C Teklad Global Rabbit diet (Harlan Laboratories UK Ltd., Oxon, UK), ad libitum
- Water: tap water, ad libitum
- Acclimation period:at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17-23
- Humidity (%): 30-70
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: To:
Vehicle:
unchanged (no vehicle)
Controls:
other: the adjacent eye of the same animal served as control
Amount / concentration applied:
0.1 mL
Duration of treatment / exposure:
1 single application
Observation period (in vivo):
7 days (reading time points: directly after application, 1, 24, 48, and 72 hours and 7 days thereafter)
Number of animals or in vitro replicates:
3
Details on study design:
SCORING SYSTEM: Draize Scoring System

Additionally, a modified version of the system (Kay and Calandra (1962), J. Soc. Chem. 13: 281-289) was used to classify the ocular irritancy potential of the test item. This was achieved by adding together the scores for the cornea, iris and conjunctivae for each time point for each rabbit. The scores for cornea, iris and conjunctivae were calculated as follows:
Conjunctivae total score = (redness score + chemosis score + discharge score) x 2
Iris score = iris score x 5
Cornea score = (degree of opacity x area of cornea involved) x 5

TOOL USED TO ASSESS SCORE: standard ophthalmoscope

OTHER
Body weights were recorded on day 0 and at the end of the observation period.
Irritation parameter:
cornea opacity score
Basis:
mean
Remarks:
out of all 3 animals
Time point:
other: mean over 24, 48, and 72 h
Score:
0
Max. score:
4
Reversibility:
other: reversibility: not applicable
Irritation parameter:
iris score
Basis:
mean
Remarks:
out of all 3 animals
Time point:
other: mean over 24, 48, and 72 h
Score:
0
Max. score:
2
Reversibility:
other: revrsibility: not applicable
Irritation parameter:
conjunctivae score
Basis:
mean
Remarks:
out of animal #1 and #2
Time point:
other: mean over 24, 48, and 72 h
Score:
1
Max. score:
3
Reversibility:
fully reversible within: 7 days
Remarks on result:
other: 1 h after treatment: score = 2 in both animals
Irritation parameter:
conjunctivae score
Basis:
animal #3
Time point:
other: mean over 24, 48, and 72 h
Score:
1.67
Max. score:
3
Reversibility:
fully reversible within: 7 days
Remarks on result:
other: 1 h after treatment: score = 2
Irritation parameter:
chemosis score
Basis:
mean
Remarks:
out of animal #1 and #2
Time point:
other: mean over 24, 48, and 72 h
Score:
1
Max. score:
4
Reversibility:
fully reversible within: 7 days
Remarks on result:
other: 1 h after treatment: score = 2 in both animals
Irritation parameter:
chemosis score
Basis:
animal #3
Time point:
other: mean over 24, 48, and 72 h
Score:
1.33
Max. score:
4
Reversibility:
fully reversible within: 7 days
Remarks on result:
other: 1 h after treatment: score = 2
Irritant / corrosive response data:
No effects on cornea and iris were observed during the study. All animals showed conjunctival redness and swelling (score = 2) at the 1 h reading. The effects were attenuated within 24 to 72 h and were fully reversible within 72 h in animal #1 and within 7 days in animal #2 and #3.
Other effects:
All animals showed expected gain in bodyweight during the study.

Table 1: Eye irritation – Details on result

Animal #

Time after treatment

Cornea

Iris

Conjunctival redness

Chemosis

Individual total score

#1

1 h

0

0

2

2

17

24 h

0

0

2

2

10

48 h

0

0

1

1

4

72 h

0

0

0

0

0

7 days

-

-

-

-

-

Mean (24-72 h)

0.00

0.00

1.00

1.00

-

#2

1 h

0

0

2

2

17

24 h

0

0

1

1

4

48 h

0

0

1

1

4

72 h

0

0

1

1

4

7 days

0

0

0

0

0

Mean (24-72 h)

0.00

0.00

1.00

1.00

-

#3

1 h

0

0

2

2

10

24 h

0

0

2

2

8

48 h

0

0

2

1

6

72 h

0

0

1

1

4

7 days

0

0

0

0

0

Mean (24-72 h)

0.00

0.00

1.67

1.33

-

Group mean score(24-72 h)

0.00

0.00

1.22

1.11

 

 

 

Group mean total score

1 h

14.7

24 h

7.3

48 h

4.7

72 h

2.7

7 d

0.0
Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
CLP: not classified
DSD: not classified
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

An in-vitro study, investigating the skin corrosion properties of 2-(1-methylethoxy)ethyl acetate (CAS 19234-20-9) is available (Warren, 2012). The study was performed according to OECD guideline 431 under GLP conditions. In the study, the undiluted test material was applied onto reconstructed three-dimensional human epidermis for 3, 60 and 240 min. The corrosivity potential of the test item was predicted from the relative mean tissue viabilities obtained after the different exposure periods and compared to the mean of the controls. The positive control substance glacial acetic acid and the negative control 0.9% sodium chloride solution were used in the study and showed the expected results. The cell viability measurements were performed using the MTT reduction assay.

The mean cell viability of the test item treated tissues after 3, 60 and 240 min was > 100% compared to the negative control. Thus, the test item was considered not corrosive according to the evaluation criteria of the test.

In addition, an in vitro-study, investigating the skin irritation properties of 2-(1-methylethoxy)ethyl acetate (CAS 19234-20-9) is available (Warren, 2012). The study was performed according to OECD guideline 439 under GLP conditions. In the study, the undiluted test material was applied onto reconstructed three-dimensional human epidermis for 15 min followed by 42 h post-exposure treatment in medium. The irritation potential of the test item was predicted from the relative mean tissue viabilities obtained after the different exposure periods and compared to the mean of the controls. The positive control substance 5% SDS and the negative control DPBS were used in the study and showed the expected results. The cell viability measurements were performed using the MTT reduction assay. The mean cell viability of the test item treated tissues after 15 min exposure and 42 h post-exposure treatment was 106.1% compared to the negative control. Thus, the test item was considered not irritating according to the evaluation criteria of the test.

An in-vitro study, investigating ocular corrosives and severe irritants with 2-(1-methylethoxy)ethyl acetate (CAS 19234-20-9) is available (Warren, 2012). The study was performed according to OECD guideline 437 under GLP conditions. The cornea form bovine eyes were exposed to the unchanged test substance for 10 min and thereafter incubated in MEM for 120 ± 10 min. The ocular corrosion potential of the test item was predicted from the in-vitro irritancy score and permeability measurements and compared to the mean of the controls. A test item that induces an in-vitro irritancy score ≥55.1 is defined as an ocular corrosive or severe irritant. The positive control substance ethanol and the negative control 0.9% sodium chloride solution were used in the study and showed the expected results. The in vitro irritancy score of the test item treated corneas after15 + 120 min was 15.9 compared to 1.1 of the negative control. Thus, the test item was considered not to be n ocular corrosive or severe irritant according to the evaluation criteria of the test.

The eye irritation potential of 2-(1-methylethoxy)ethyl acetate (CAS 19234-20-9) was investigated according to OECD guideline 405 and in compliance with GLP (Pooles, 2012). The undiluted test material (0.1 mL) was placed into the conjunctival sac of one eye of 3 New Zealand White rabbits. The other eye remained untreated and served as control. The eyes were examined and scored 1, 24, 48, 72 h and 7 days after application. No effects on cornea and iris were observed during the study. All animals showed conjunctival redness and swelling (score = 2) at the 1 h reading. The effects were attenuated within 24 to 72 h and were fully reversible within 72 h in animal #1 and within 7 days in animal #2 and #3. The mean conjunctivae scores after 24, 48, and 72 h were 1, 1 and 1.67 for the individual animals. The mean chemosis scores after 24, 48, and 72 h were 1, 1 and 1.33 for the individual animals. All animals showed expected gain in bodyweight during the study. Thus, the test substance was not considered as eye irritant.

 

Conclusion for irritation/corrosion

In conclusion, no evidence of skin corrosion or irritation properties were seen after treatment with 2-(1-methylethoxy)ethyl acetate (CAS 19234-20-9). In addition, there is no evidence of irreversible effects on the eye and the substance is not considered to be an eye irritant and thus no hazard for skin and eye irritation / corrosion was identified for 2-(1-methylethoxy)ethyl acetate (CAS 19234-20-9).


Justification for selection of skin irritation / corrosion endpoint:
The selected study is the most adequate and reliable study.

Justification for selection of eye irritation endpoint:
The selected study is the most adequate and reliable study.

Justification for classification or non-classification

Based on the available in-vitro data on skin corrosion and irritation properties, 2-(1-methylethoxy)ethyl acetate (CAS 19234-20-9) do not meet the criteria for classification according to Regulation (EC) 1272/2008 or Directive 67/548/EEC and the data are therefore conclusive but not sufficient for classification.

Based on the available data on eye irritation properties, 2-(1-methylethoxy)ethyl acetate (CAS 19234-20-9) do not meet the criteria for classification according to Regulation (EC) 1272/2008 or Directive 67/548/EEC and the data are therefore conclusive but not sufficient for classification.