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Administrative data

Description of key information

Skin irritation: No adverse skin irritation effects were observed in all tests.
Eye irritation: No acute eye irritation potential was determined in a study according to OECD guideline No. 405.

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:
weight of evidence
Study period:
2015-01-23 to 2015-04-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
2013
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
2009
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Species:
other: Human skin model EpiDerm
Strain:
other: Human skin model EpiDerm
Details on test animals and environmental conditions:
Epi-200- SIT Kit Lot No.: 21627
MTT-100 Assay Kit Components

Epi-200 SIT kits and MTT-100 assays diluent were purchased from MatTek Corporation (82105 Bratislava, Slovakia). The EpiDerm™ tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDerm™ tissues (surface 0.6 cm²) are cultured on specially prepared cell culture inserts (MILLICELLs, 10 mm ∅).

EpiDerm™ tissues were shipped with cool packs on medium-supplemented agarose gels in a 24-well plate and reached Harlan CCR on 17 February 2015. On day of receipt the pre-incubation phase of the EpiDerm™ tissues started.
Type of coverage:
other: In-vitro human skin model Epiderm
Preparation of test site:
other: In-vitro human skin model Epiderm
Vehicle:
unchanged (no vehicle)
Controls:
other: No control animals because Human skin model. Negative and positive control were used in this test.
Amount / concentration applied:
30 µL (47µL/cm^2) of the undiluted test item was dispensed directly atop the EpiDerm tissue and spread to match the surface of the tissue.
Duration of treatment / exposure:
60 min
Observation period:
Not applicable
Number of animals:
Human in vitro skin model, 3 tissues per dose
Details on study design:
Treatment
Time: 60 min
Incubator: Within this period the 6-well plates were put into the incubator for 35 min at 37 +/- °C, 5+/- 0.5 % CO2.
Rinsing: After the end of the treatment interval the inserts were removed immediately with DPBS at least 15 times in order to remove any residual test material.
Incubation time: 42 hours

MTT Assay
A volume of 300 µL of the MTT solution was added to each well and the plates were kept in an incubator (37 +/- 1 °C, 5 +/- % CO2) until further use.
Incubation period: 3 hours
Time: After the 42 hours incubation period was completed for all tissues and exposure groups, culture inserts were transferred from the holding plates to the MTT plates.
Rinsing: After 3 h incubation, wells were rinsed three times with DPBS and were transferred onto new 24-well plates.
Extraction time: About 68 hours at room temperature the formazan salt was extracted.
Extractant solution: Isopropanol

OD reading: OD was read in a microplate reader (Versamax Molecular Devices, Softmax Pro, version 4.7.1) with 570 +/- nm filter. Mean values were calculated from the 3 wells per tissue.


Irritation / corrosion parameter:
other: Tissue viability after exposure compared to negative control in %
Value:
89.1
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 60 min treatment. Max. score: 100.0. Remarks: Not irritant.
Other effects / acceptance of results:
After treatment with the test item the mean relative absorbance value decreased to 89.1% compared to the relative absorbance value of the negative control. This value is above the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritant potential.

Dose group

Treatment Inveral

Absorbance
570 nm
Tissue 1 *

Absorbance
570 nm
Tissue 2 *

Absorbance
570 nm
Tissue 3 *

Mean
Absorbance
of 3 Tissues

Rel. Absorbance
[%] Tissue
1, 2 +3 **

Relative
Standard
Deviation [%]

Mean Rel.
Absorbance
[% of
Negative
Control] ***

Negative Control

60 min

1.618

1.628

1.564

1.603

100.9
101.6
97.5

2.2

100

Positive Control

60 min

0.068

0.069

0.062

0.067

4.3
'4.3
'3.9

5.8

4.2

Test item

60 min

1.491

1.461

1.335

1.429

93
91.1
83.3

5.8

89.1

* Mean of three replicate wells after blank correction

** relative absorbance per tissue (rounded values): 100 x (absorbance tissue)/mean absorbance negative control)

*** relative absorbance per treatment group (rounded values): 100 x (mean absorbance test/positive control)/(mean absorbance negative control)

The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water did not led to a change in colour and the colour of the test item was not intensive => additional test with viable tissues without MTT addition was not necessary

Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did not show blue colour => additional test with freeze-killed tissues without MTT addition was not necessary

The mean relative absorbance value of the test item, corresponding to the cell viability, was not reduced (89.1%; threshold for irritancy: ≤ 50%), consequently the test item was not irritant to skin.

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item is not irritant to skin
Executive summary:

This in vitro study was performed to assess the irritation potential of the test item by means of the Human Skin Model Test. The test item did not prove to be a MTT reducer (test for direct MTT reduction), and it did not change colour when mixed with deionised water (test for colour interference). Also its intrinsic colour was not intensive. Consequently, additional tests with freeze-killed or viable tissues were not necessary. Each three tissues of the human skin model EpiDerm™ were treated with the test item, the negative or the positive control for 60 minutes. Each 30 μL of the test item, of the negative control (DPBS), and of the positive control (5% SLS) were applied to each tissue and spread to match the surface of the tissue.

After treatment with the negative control the absorbance values were well in the required range of the acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for the 60 minutes treatment interval, thus assuring the quality of the tissues. Treatment with the positive control induced a sufficient decrease in the relative absorbance as compared to the negative control for the 60 minutes treatment interval, and thus assuring the validity of the test system. After treatment with the test item the mean relative absorbance value decreased to 89.1% compared to the relative absorbance value of the negative control. This value is above the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritant potential.

In conclusion, it can be stated that in this study and under the experimental conditions reported, the tes titem is not irritant to skin.

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
Study period:
2000-03-13 to 2000-03-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
4 animals tested instead of 3
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Version / remarks:
1987
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
Version / remarks:
1997
Deviations:
no
GLP compliance:
yes
Test material information:
Composition 1
Species:
rabbit
Strain:
other: Chbb:HM(SPF)-Littlerussian
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: BI Pharma KG, 88397 Biberach
- Housing: The study took place in animal room No. 5. During a pre-period of at least one week and throughout the experiment the rabbits were caged individually in PPO cages (floor area: 2576 sq.cm) with perforated floor.
- Diet: "Altromin 2123" from Altromin, 32791 Lage, Lippe, ad libitum.
- Water: Free access to bottles with domestic quality drinking water acidified with hydrochloric acid to pH 2.5 in order to prevent microbial growth.

ENVIRONMENTAL CONDITIONS
- Temperature: 20°C +/- 3 °C
- Humidity: 55 % +/- 15 %
- Air changes (per hr): 10 times
- Photoperiod (hrs dark / hrs light): 12/12
Vehicle:
unchanged (no vehicle)
Controls:
other: The right eye served as control
Amount / concentration applied:
0.1 mL
Duration of treatment / exposure:
The test article was placed in the left eye and the lids were then held together for 1 second.
Observation period (in vivo):
72 hours
Number of animals or in vitro replicates:
4 female rabbits
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing: With 0.9 % sodium chloride solution.
- Time after start of exposure: Washing after 24 hours

SCORING SYSTEM: According to Draize et al.

TOOL USED TO ASSESS SCORE: Fluorescein
Irritation parameter:
cornea opacity score
Basis:
mean
Remarks:
all animals
Time point:
other: 24, 48, 72 h
Score:
0
Max. score:
4
Irritation parameter:
iris score
Basis:
mean
Remarks:
all animals
Time point:
other: 24, 48, 72 h
Score:
0
Max. score:
2
Irritation parameter:
conjunctivae score
Basis:
mean
Remarks:
animal 1
Time point:
other: 24, 48, 72 h
Score:
0.33
Max. score:
3
Reversibility:
fully reversible within: 48 h
Irritation parameter:
conjunctivae score
Basis:
mean
Remarks:
animal 2
Time point:
other: 24, 48, 72 h
Score:
0.33
Max. score:
3
Reversibility:
fully reversible within: 48 h
Irritation parameter:
conjunctivae score
Basis:
mean
Remarks:
animal 3
Time point:
other: 24, 48, 72 h
Score:
0.33
Max. score:
3
Reversibility:
fully reversible within: 48 h
Irritation parameter:
conjunctivae score
Basis:
mean
Remarks:
animal 4
Time point:
other: 24, 48, 72 h
Score:
0.33
Max. score:
3
Reversibility:
fully reversible within: 48 h
Irritation parameter:
chemosis score
Basis:
mean
Remarks:
animal 1
Time point:
other: 24, 48, 72 h
Score:
0
Max. score:
4
Irritation parameter:
chemosis score
Basis:
mean
Remarks:
animal 2
Time point:
other: 24, 48, 72 h
Score:
0.33
Max. score:
4
Reversibility:
fully reversible within: 48 h
Irritation parameter:
chemosis score
Basis:
mean
Remarks:
animal 3
Time point:
other: 24, 48, 72 h
Score:
0.33
Max. score:
4
Reversibility:
fully reversible within: 48 h
Irritation parameter:
chemosis score
Basis:
mean
Remarks:
animal 4
Time point:
other: 24, 48, 72 h
Score:
0
Max. score:
4
Irritant / corrosive response data:
not irritating
Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
According to the Directive (EC) No 1272/2008 Ethyl Phenyl Acetate shall not be classified as eye irritating.
Executive summary:

The local effect of the test item was investigated according to the method recommended in the OECD guideline No. 405, Feb. 1987 and EEC guideline B.5, Jan. 1997. Four female albino rabbits were exposed to 0.1 mL of the test article in the left eye. The eyes were examined and the changes were graded according to a numerical scale one hour, 24, 48 and 72 hours after dosing. Slight signs of irritation were observed on the treated eyes. All effects were fully reversible within 72 hours. The following mean values were calculated.

Animal 1: Cornea, Iris, Conjunctiva = 0, Redness = 0.33

Animal 2: Cornea, Iris = 0, Redness and Conjunctiva = 0.33

Animal 3: Cornea, Iris = 0, Redness and Conjunctiva = 0.33

Animal 4: Cornea, Iris, Conjunctiva = 0, Redness = 0.33

Conclusion

According to the Directive (EC) No 1272/2008 Ethyl Phenyl Acetate shall not be classified as eye irritating.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation

One in vitro skin irritation study and 2 human patch tests were available. All studies showed no irritating effects. The results of the human patch test are described in section 7.10.5.

WoE

In-vitro skin irritation test

This in vitro study was performed to assess the irritation potential of the test item by means of the Human Skin Model Test. The test item did not prove to be a MTT reducer (test for direct MTT reduction), and it did not change colour when mixed with deionised water (test for colour interference). Also its intrinsic colour was not intensive. Consequently, additional tests with freeze-killed or viable tissues were not necessary. Each three tissues of the human skin model EpiDerm™ were treated with the test item, the negative or the positive control for 60 minutes. Each 30 μL of the test item, of the negative control (DPBS), and of the positive control (5% SLS) were applied to each tissue and spread to match the surface of the tissue.

After treatment with the negative control the absorbance values were well in the required range of the acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for the 60 minutes treatment interval, thus assuring the quality of the tissues. Treatment with the positive control induced a sufficient decrease in the relative absorbance as compared to the negative control for the 60 minutes treatment interval, and thus assuring the validity of the test system. After treatment with the test item the mean relative absorbance value decreased to 89.1% compared to the relative absorbance value of the negative control. This value is above the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritant potential.

In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item is not irritant to skin.

Human patch study of T. Fujii, S. Furukawa and S. Suzuki, 1972 (Yukugaku 21 (12) pg 904 -908).

In a closed human patch study the skin irritation potential of the test item was tested. 1763 healthy male and female subjects were tested in condition 1 and 1830 subjects in condition 2. The following application way was chosen: In condition 1 the site of application was the small of back in condition 2 the upper inside of arm. Vaselinum Aldum, Unguentum Hydrophilicum were used as vehicle in condition 1 and Unguentum Simplex, Unguentum Hydrophlicum were used as vehicle in condition 2. The test item was applied in a concentration of 3 -20 % and in a concentration of 0.2 -2 %. No irritation reactions were observed in 30 subjects.

Human patch study of A.E. Katz, 1946 (The Spice Mill 69 pg 46-47)

A 24 hour closed patch test was conducted on male and female volunteers. Test material was applied undiluted to an area on the inner arm of the subjects measuring 1 cm in diameter. Immediately following application, the area was covered with an adhesive bandage for 24 hours. Reactions were read daily for 5 days. No effects, no positive irritating reactions were observed in 23 tests.

Conclusion

No positive skin irritating reactions with the test item were found in two human patch tests and one in-vitro skin irritation test. Thus the substance was judged to be not irritating to the skin.

Eye irritation

Key study

Acute Eye Irritation/Corrosion study in the rabbit (Frey-Tox, 2000, Report No: 01723)

The acute eye irritation potential of the test item was investigated according to the method recommended in the OECD guideline No. 405, Feb. 1987 and EEC guideline B.5, Jan. 1997. Four female albino rabbits were exposed to 0.1 mL of the test article in the left eye. The eyes were examined and the changes were graded according to a numerical scale one hour, 24, 48 and 72 hours after dosing. Slight signs of irritation were observed on the treated eyes. All effects were fully reversible within 72 hours. The following mean values were calculated.

Animal 1: Cornea, Iris, Conjunctiva = 0, Redness = 0.33

Animal 2: Cornea, Iris = 0, Redness and Conjunctiva = 0.33

Animal 3: Cornea, Iris = 0, Redness and Conjunctiva = 0.33

Animal 4: Cornea, Iris, Redness = 0, Conjunctiva = 0.33

Conclusion

According to the Directive (EC) No 1272/2008 the test item shall not be classified as eye irritating.


Justification for selection of skin irritation / corrosion endpoint:
GLP and guideline study

Justification for selection of eye irritation endpoint:
Guideline and GLP study

Justification for classification or non-classification

Skin Irritation

 

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. As a result the substance is not considered to be classified for skin irritation under Regulation (EC) No 1272/2008

Eye irritation

 

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. As a result the substance is not considered to be classified for eye irritation under Regulation (EC) No 1272/2008