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Description of key information

Skin irritation/corrosion: Key study: Test method OECD 439. GLP study. EAC3 was determined to be non-irritant to the skin.
Eye irritation/corrosion: Key study: Test method OECD 437. GLP study. EAC3 does not require a classification as a severe eye irritant.
Key study: Test method OECD 405. GLP study. EAC3 was considered to be non irritant to the eye.

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:
July 11, 2012 - July 13, 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Test method according to OECD Guideline 439. GLP study.
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Species:
other: In vitro: Human skin
Details on test animals or test system and environmental conditions:
The test system used was EPISKIN-SM, a three-dimensional human epidermis model.
Type of coverage:
other: In-vitro test
Preparation of test site:
other: In-vitro test
Vehicle:
unchanged (no vehicle)
Controls:
other: In-vitro test. Negative and positive controls were conducted.
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µL
Duration of treatment / exposure:
15 minutes.
Observation period:
After approx. 42 hours.
Number of animals:
Not applicable: In-vitro test.
3 replicate wells for the test item and 3 negative controls + 3 positive controls were used
Details on study design:
PRE-INCUBATION (Day -1):
The assay platewells were filled with a pre-warmed medium (2 mL per well, 37 ºC). The epidermis units were placed, with the media below in contact with the epidermis, into each prepared well and then incubated overnight at 37°C in an incubator with 5% CO2.

APPLICATION AND RINSING (Day 0):
- Test skin units: 50 μL of test item (3 replicates)
- Negative control skin units: 50 μL Phosphate Buffered Saline (PBS) (3 replicates)
- Positive control skin units: 50 μL Sodium Dodecyl Sulphate (SDS) 5% aq. solution (3 replicates)
- For additional control for staining effects of the test item, 50 μL of the test item was applied evenly to the epidermal surface.
The treated plates were incubated for 15 min. exposure time at room temperature (21.7-22.3 ºC). Then, the EPISKIN-SM units were removed and rinsed with PBS 1x solution (0.9%). The units were place into the plate well with fresh pre-warmed maintenance medium (2mL/well) and incubated for 42 h at 37 ºC with 5% CO2.

MTT TEST (Day 2):
The EPISKIN-SM units were transferred into the MTT solution filled wells (2 mL of 0.3 mg/mL MTT per well). The staining control well instead, was transferred to well filled with fresh assay medium. The units were incubated for 3 h at 37 ºC with 5% CO2, protected from light.

FORMAZAN EXTRACTION (Day 2):
The epidermises from each disk were separated with the aid of forceps and both parts (epidermis and collagen matrix) and were placed into a tube of 500 µL acidified isopropanol. The capped tubes were thoroughly mixed by using a vortex mixer and then incubated for 2 hours at room temperature protected from light with gentle agitation (~150 rpm) for formazan extraction.

CELL VIABILITY MEASUREMENT (Day 2):
2×200 μL samples from each tube were placed into the wells of a 96-well plate. The OD (Absorbance / Optical Density) of the samples in a 96-well plate spectrophotometer was read at a wavelength 540 nm, using acidified isopropanol solution blank (6×200 μL). The viability was expressed as a % relative to negative control.
Irritant / corrosive response data:
The mean relative viability of test item EAC3 after 15 minutes exposure and 42 hours post incubation period (based on the optical activity measured at 540 nm) was 115% (SD± 8.89). According to EU classification, the substance was determined to be non-irritant since the mean tissue viability % was >50.

CELL VIABILITY:

The results of the optical density (OD) measured at 540 nm of each extract and the calculated % viability of the cells:

Substance

Optical density (OD)

Viability (%)

Negative control (PBS)

1

0.797

104

2

0.744

97

3

0.753

98

Mean

0.765

100

Stand. dev. (SD)

3.79

Positive control

(SDS 5%)

1

0.246

32

2

0.207

27

3

0.083

11

Mean

0.179

23

Stand. dev. (SD)

10.97

Test ítem

(EAC3)

1

0.856

112

2

0.959

125

3

0.823

108

Mean

0.879

115

Stand. dev. (SD)

8.89

VALIDITY OF THE TEST:

The mean OD value of the three negative control tissues was 0.765.

The positive control result showed a mean of 23% viability.

Each standard deviation value (SD) of the % viability was below 18.

Control OD values were below historically established boundaries.

The SD calculated from individual % tissues viabilities of the three identically test item treated replicates was 8.89.

All validity criteria were within acceptable limits and therefore the study can be considered as valid.

INDICATOR FOR POTENTIAL FALSE VIABILITY:

The test material did not interact with the MTT (based on no colour change observed).

The non specific colour % was calculated as 3.5% (below the threshold of 5.0) and therefore additional data calculation was not necessary.

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The mean relative viability of test item EAC3 after 15 minutes exposure and 42 hours post incubation period was 115% (SD± 8.89). According to EU classification, the substance is considered to be non-irritant since the mean tissue viability % was >50.
Executive summary:

An in-vitro skin irritation test in the EPISKIN model was performed on the substance EAC3 in accordance with OECD Guideline 439 and GLP. Disks of EPISKIN (three units / chemical) were treated with EAC3 and incubated for 15 minutes at room temperature. Exposure of test material was terminated by rinsing with PBS. Epidermis units were then incubated at 37°C for 42 hours in an incubator with 5% CO2. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37°C in 5% CO2 protected from light. The formazan extract in acidified isopropanol was then spectrophotometrically evaluated for optical density (OD) and quantified. SDS 5% and PBS treated epidermis were used as positive and negative controls respectively. For each treated tissue, optical density (OD) was calculated and the tissue viability was expressed as a % relative to negative control. Following exposure with EAC3, the mean treated skin value was 115% and therefore non-irritant to the skin (>50%). All validity criteria were within acceptable limits and therefore the study can be considered as valid.

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:
other: Test method according to OECD 405. GLP study.
Qualifier:
according to guideline
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: S&K-LAP Kft. 2173 Kartal, Császár út 135, Hungary
- Age at study initiation: ~13 weeks old (adult)
- Weight at study initiation: 3294 – 3525 g
- Housing: Individually in AAALAC approved metal wire rabbit cages
- Diet (e.g. ad libitum): Ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: 21 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 3°C
- Humidity (%): 49 – 89 %
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent no treatment
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 mL
Duration of treatment / exposure:
Single exposure
Observation period (in vivo):
72 hours.
Number of animals or in vitro replicates:
3 male animals.
Details on study design:
OBSERVATIONS: The eyes were examined at 1, 24, 48 and 72 hours after treatment. Any clinical signs of toxicity or signs of ill-health during the study were recorded. Individual body weight was recorded at the beginning of the experiment and before euthanasia
SCORING SYSTEM: The eye irritation scores were evaluated according to the scoring system by Draize (1977) and OECD 405 (24 April 2002).
Irritation parameter:
chemosis score
Basis:
animal #1
Time point:
other: mean 24-72 h
Score:
0
Max. score:
4
Irritation parameter:
chemosis score
Basis:
animal #2
Time point:
other: mean 24-72 h
Score:
0
Max. score:
4
Irritation parameter:
chemosis score
Basis:
animal #3
Time point:
other: mean 24-72 h
Score:
0
Max. score:
4
Irritation parameter:
conjunctivae score
Remarks:
Discharge
Basis:
animal #1
Time point:
other: mean 24-72 h
Score:
0
Max. score:
3
Irritation parameter:
conjunctivae score
Remarks:
Discharge
Basis:
animal #2
Time point:
other: mean 24-72 h
Score:
0.33
Max. score:
3
Reversibility:
fully reversible within: 48 h
Irritation parameter:
conjunctivae score
Remarks:
Discharge
Basis:
animal #3
Time point:
other: mean 24-72 h
Score:
0
Max. score:
3
Irritation parameter:
conjunctivae score
Remarks:
Redness
Basis:
animal #1
Time point:
other: mean 24-72 h
Score:
0.33
Max. score:
3
Reversibility:
fully reversible within: 48 h
Irritation parameter:
conjunctivae score
Remarks:
Redness
Basis:
animal #2
Time point:
other: mean 24-72 h
Score:
0.33
Max. score:
3
Reversibility:
fully reversible within: 48 h
Irritation parameter:
conjunctivae score
Remarks:
Redness
Basis:
animal #3
Time point:
other: mean 24-72 h
Score:
0.33
Max. score:
3
Reversibility:
fully reversible within: 48 h
Irritation parameter:
cornea opacity score
Remarks:
Opacity
Basis:
animal #1
Time point:
other: mean 24-72 h
Score:
0
Max. score:
4
Irritation parameter:
cornea opacity score
Remarks:
Opacity
Basis:
animal #2
Time point:
other: mean 24-72 h
Score:
0
Max. score:
4
Irritation parameter:
cornea opacity score
Remarks:
Opacity
Basis:
animal #3
Time point:
other: mean 24-72 h
Score:
0
Max. score:
4
Irritation parameter:
iris score
Basis:
animal #1
Time point:
other: mean 24-72 h
Score:
0
Max. score:
2
Irritation parameter:
iris score
Basis:
animal #2
Time point:
other: mean 24-72 h
Score:
0
Max. score:
2
Irritation parameter:
iris score
Basis:
animal #3
Time point:
other: mean 24-72 h
Score:
0
Max. score:
2
Irritant / corrosive response data:
Initial Pain Reaction (IPR) (score 2) was observed in all animals. One hour after the application: Conjunctival redness (score 1 or 2), conjunctival chemosis (score 1 or 2) and conjunctival discharge (score 2 or 3) were found in all animals. At 24 hours after the application: Conjunctival redness (score 1) was found in all animals. In addition conjunctival discharge (score 1) was noted in one animal. At 48 and 72 hours after the application no signs of eye irritation or other clinical signs were observed. As all signs of eye irritation had fully reversed the study was terminated after a period of 72 hours observation.

No mortality was observed.

The body weight and body weight change were considered to be normal with no indication of a treatment related effect.

There were no clinical signs observed that could be related to treatment.

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Instillation of 0.1 mL of EAC3 into one eye of each of three rabbits resulted in effects conjunctival redness (score 1) in all animals and conjunctival discharge (score 1) in one animal att 24 hours after the application. Based on these results, EAC3 was considered to be non irritant to the eye according to Regulation (EC) No 1272/2008.
Executive summary:

An acute eye irritation study of the test item EAC3 was performed in New Zealand White rabbits according to OECD Guideline 405 and GLP. 0.1 mL of the test item was placed as a single dose into the conjunctival sac of one eye of each animal (3 male rabbits). The untreated right eye served as control. Individual body weight was recorded at the beginning of the experiment and before euthanasia. Morbidity and clinical signs of toxicity were checked daily. No adverse effects were observed. The eyes were examined at 1, 24, 48 and 72 hours after the application. Initial Pain Reaction (IPR) (score 2) was observed in all animals. At 24 hours after the application conjunctival redness (score 1) was found in all animals and conjunctival discharge (score 1) was noted in one animal. The effects were fully reversible within 48 hours. Based on these results, EAC3 was considered to be non irritant to the eye according to Regulation (EC) No 1272/2008.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation/corrosion:

Key study: An in-vitro skin irritation test in the EPISKIN model was performed on the substance EAC3 in accordance with OECD Guideline 439 and GLP. Disks of EPISKIN (three units / chemical) were treated with EAC3 for a 15 minutes exposure time and 42 hours post incubation, and incubated for 15 minutes at room temperature. SDS 5% and PBS treated epidermis were used as positive and negative controls respectively. For each treated tissue, optical density (OD) was calculated and the tissue viability was expressed as a % relative to negative control. Following exposure with EAC3, the mean treated skin value was 115% and therefore, it was concluded that the test item was non-irritant to the skin (mean tissue viability % >50).

Data waiving (other justification): In accordance with REACH Regulation Annex XI, the in-vivo skin irritation study does not need to be conducted since the following conditions are met:

- results are derived from an in-vitro method whose scientific validity has been established by a validation study, according to internationally agreed validation principles;

- results are adequate for the purpose of classification and labelling and risk assessment; and

- adequate and reliable documentation of the applied method is provided.

Eye irritation/corrosion:

Key study: An in vitro eye irritation study of the test item EAC3 was performed in isolated chicken’s eyes in accordance with the OECD Guideline 438 and GLP. The corneas were exposed to 30 μL of EAC3 for 10 seconds and then, the surface was rinsed with saline. The positive and negative control eyes were treated in a similar way with 30 μL Trichloroacetic acid 30 (w/v) %. and with 30 μL of Sodium chloride (Salsol solution 0.9%) respectively. The maximum corneal thickness change, corneal opacity change and fluorescein retention where calculated. The results suggest that the test item is not irritating. According to the guideline OECD 438, EAC3 does not require a classification as a severe eye irritant and an in vivo study is required for classification.

Key study: An acute eye irritation study of the test item EAC3 was performed in New Zealand White rabbits according to OECD Guideline 405 and GLP. 0.1 mL of the test item was placed as a single dose into the conjunctival sac of one eye of each animal (3 male rabbits). Initial Pain Reaction (IPR) (score 2) was observed in all animals. At 24 hours after the application conjunctival redness (score 1) was found in all animals and conjunctival discharge (score 1) was noted in one animal. The effects were fully reversible within 48 hours. Based on these results, EAC3 was considered to be non irritant to the eye according to Regulation (EC) No 1272/2008.


Justification for selection of skin irritation / corrosion endpoint:
Only one study available.

Justification for selection of eye irritation endpoint:
Only one in-vivo study is available.

Justification for classification or non-classification

Skin irritation/corrosion: Based on the available data, the substance EAC3 is not classified for skin irritation in accordance with CLP Regulation (EC) No 1272/2008.

Eye irritation/corrosion: Based on the available data, the substance EAC3 is not classified for eye irritation in accordance with CLP Regulation (EC) No 1272/2008 .