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Description of key information

Skin corrosion: the substance is not considered corrosive based on absence of corrosive effects and in the presence of some irritation effects in the acute dermal toxicity test in rabbits (OECD TG 402).

Skin irritation (OECDTG439): irritating

Eye irritation (OECDTG405): 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:
24 May 2016 - 30 May 2016
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:
28 July 2015
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
2009
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Test system:
human skin model
Source species:
human
Cell type:
other: epidermal keratinocytes
Cell source:
other: SkinEthic Laboratories, Lyon, France.
Source strain:
other: Not applicable
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Small ModelTM, 0.38 cm^2
- Tissue batch number: 16-EKIN-021
- Twenty five μL of the undiluted test substance was added into 12-well plates on top of the skin tissues.
- The test item was applied topically to the corresponding tissues ensuring uniform covering.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37.0°C

PRE-TEST PROCEDURE:
Assessment of Direct Test Item Reduction of MTT
MTT Salt Metabolism, Cell Viability Assay
The MTT assay, a colorimetric method of determining cell viability, is based on reduction of the yellow tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt by mitochondrial succinate dehydrogenase in viable cells. One limitation of the assay is possible interference of the test item with MTT. A test item may directly reduce MTT, thus mimicking dehydrogenase activity of the cellular mitochondria. This property of the test item is only a problem, if at the time of the MTT test (after rinsing) there are still sufficient amounts of the test item present on or in the tissues. In this case, the true metabolic MTT reduction and the false direct MTT reduction can be differentiated and quantified.
Test for Direct MTT Reduction + colour interference:
Fleuramone was checked for possible direct MTT reduction and colour interference before the study was started. Some non-coloured test items may change into coloured items in aqueous conditions and thus stain the skin tissues during the exposure. To assess the colour interference, 10 μL of Fleuramone was added to 90 μL Milli-Q water. The mixture was mixed for approximately 15 minutes. A negative control, 10 μL Milli-Q water was tested concurrently. At the end of the shaking period a colour check was performed. To assess the ability of the test item to reduce MTT, 10 μL of the test item was added to 2 mL MTT solution (0.3 mg/mL in PBS). The mixture was incubated for 3 hours at 37°C. A negative control, sterile Milli-Q water was tested concurrently. At the end of the incubation period a colour check was performed.

PRE-INCUBATION:
On the day of receipt the tissues were transferred to 12-well plates and preincubated with prewarmed Maintenance Medium overnight at 37°C. Maintenance medium and Assay medium were supplied by Skinethic Laboratories, Lyon, France.

APPLICATION/TREATMENT OF TEST SUBSTANCE:
The test was performed on a total of 3 tissues per test item together with negative and positive controls. Ten μL (26.3 μL/cm^2) of the undiluted test item was added into 12-well plates on top of the skin tissues. Three tissues were treated with 10 μL PBS (negative control) and 3 tissues with 10 μL 5% SDS (positive control) respectively. The positive control was re-spread after 7 minutes contact time. After the exposure period of 15 ± 0.5 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test item. After rinsing, the cell culture inserts were each dried carefully and moved to a new well on 2 mL pre-warmed maintenance medium until all tissues were dosed and rinsed. Subsequently the skin tissues were incubated for 42 hours at 37°C.

CELL VIABILITY MEASUREMENT:
After incubation, cell culture inserts were dried carefully to remove excess medium and were transferred into a 12-wells plate prefilled with 2 mL MTT-solution (0.3 mg/mL in PBS). The tissues were incubated for 3 h at 37°C. After incubation the tissues were placed on blotting paper to dry the tissues. Total biopsy was made by using a biopsy punch. Epidermis was separated from the collagen matrix and both parts were placed in prelabeled microtubes and extracted with 500 μL acidified isopropanol. Tubes were stored refrigerated until Day 6 op the experiment. The amount of extracted formazan was determined spectrophotometrically at 562 nm with the Anthos 2001 microplate reader.
Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues. Skin irritation potential of the test item was classified according to remaining cell viability following exposure of the test item.

DECISION CRITERIA
- A test substance is considered irritant in the skin irritation test if: The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test substance and 42 hours of post incubation is ≤ 50% of the mean viability of the negative controls.
- A test substance is considered non-irritant in the in vitro skin irritation test if: The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test substance and 42 hours of post incubation is > 50% of the mean viability of the negative controls.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
Test material
- Applied volume: 10 μL
Duration of treatment / exposure:
15-Minute exposure period and 42 hours post-exposure incubation period.
Number of replicates:
A total of 9 tissues were used: Triplicate tissues were treated with: test substance, positive control or negative control.
Irritation / corrosion parameter:
other: relative mean viability
Value:
21.2
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: The relative mean tissue viability compared to the negative control tissues (100%).
Other effects / acceptance of results:
Direct MTT Reduction:
The MTT solution containing the test item did not turn blue or purple which indicated that the test item did not directly reduce MTT.

Quality Criteria:
The relative mean tissue viability for the positive control treated tissues was 4.9% relative to the negative control treated tissues and the standard deviation value of the viability was 0.9%. The positive control acceptance criteria were therefore satisfied.
The mean OD562 for the negative control treated tissues was 1.293 and the standard deviation value of the viability was 3.5%. The negative control acceptance criteria were therefore satisfied.
The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 1.1%. The test item acceptance criterion was therefore satisfied.

Mean OD562 Values and Percentage Viabilities for the Negative Control Item, Positive Control Item and Test Item:

Item

OD562 of

tissues

Mean OD562

of triplicate

tissues

± SD of

OD562

Relative

individual

tissue

viability (%)

Relative

mean

viability (%)

Negative

Control Item

1.265

1.293

0.045

97.8

1.345

104.0

1.269

98.1

Positive Control Item

0.076

0.063

0.011

5.9

4.9

0.058

4.5

0.055

4.3

Test Item

0.262

0.274

0.014

20.3

21.2

0.290

22.4

0.270

20.9

SD = Standard deviation

*The mean viability of the negative control tissues is set at 100 %

Interpretation of results:
other: Skin irritant Category 2
Remarks:
According to Regulation (EC) No. 1272/2008 and its updates.
Conclusions:
The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the substance compared to the negative control tissues was 21.2%. Since the mean relative tissue viability for the substance was below 50% the substance is considered to be a skin irritant.
Executive summary:

The possible skin irritation potential of the substance was tested in vitro using a human skin model through topical application for 15 minutes. The study procedures described in this report were according to OECD TG 439 guideline and GLP principles. Skin tissue was treated by topical application of 10 μL undiluted test substance. After 42 hours incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) MTT at the end of treatment. Skin irritation is expressed as the remaining cell viability after exposure to the test substance. Reliable negative and positive controls were included. The positive control had a mean cell viability of 4.9% after 15 minutes exposure. The standard deviation value of the percentage viability of three tissues treated identically was less than 4%, indicating that the test system functioned properly. The relative mean tissue viability obtained after 15 minutes treatment with the substance compared to the negative control tissue was 21.2%. Since the mean relative tissue viability for the substance was below 50% after 15 minutes treatment the substance is considered to be a skin irritant category 2.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (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:
21 July, 1980 - 01 September, 1980
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Reliability has been presented as 2 because similar to OECD Guideline protocol has been followed but not GLP.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Version / remarks:
1981
Deviations:
yes
Remarks:
no details on purity, no details on animal housing and environmental conditions.
Principles of method if other than guideline:
Modified Federal Hazardous Substances Labelling Act Method.
GLP compliance:
no
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Unilever Environmental Safety Division, Colworth Laboratory
- Age at study initiation: 7 weeks
- Weight at study initiation: 1.9 - 2.0 kg

ENVIRONMENTAL CONDITIONS
No data.
Vehicle:
unchanged (no vehicle)
Controls:
other: One eye of each animal remained untreated and served as the reference control.
Amount / concentration applied:
Amount applied: 0.1 mL
Duration of treatment / exposure:
Single instillation on Day 1
Observation period (in vivo):
5 days
Number of animals or in vitro replicates:
2 females and 1 male.
Details on study design:
STUDY DESIGN
The test material was applied undiluted in 3 animals.

TREATMENT
The substance was applied to one eye of the rabbits by gently pulling the lower lid away from the eye ball and placing 0.1 mL in the sac so formed.

REMOVAL OF TEST SUBSTANCE
-Washing: No

OBSERVATIONS
- Irritation:
The eyes were examined 24 hours after treatment and thereafter at daily intervals and graded for corneal, conjunctival and iridial damage. Eyes were examined before application of materials and at daily intervals afterwards with a slit lamp and corneal swelling is measured.
The irritation scores and a description of all other (local) effects were recorded. The irritation was assessed according to OECD 405 (1981).
Irritation parameter:
cornea opacity score
Basis:
mean
Remarks:
animal #1 and #3
Time point:
24/48/72 h
Score:
0.17
Max. score:
4
Reversibility:
fully reversible within: 72 hours
Irritation parameter:
cornea opacity score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0.5
Max. score:
4
Reversibility:
fully reversible within: 4 days
Irritation parameter:
iris score
Basis:
mean
Remarks:
animal #1, #2 and #3
Time point:
24/48/72 h
Score:
0
Max. score:
2
Irritation parameter:
conjunctivae score
Remarks:
redness
Basis:
mean
Remarks:
animal #1 and #3
Time point:
24/48/72 h
Score:
0.33
Max. score:
3
Reversibility:
fully reversible within: 72 hours
Irritation parameter:
conjunctivae score
Remarks:
redness
Basis:
animal #2
Time point:
24/48/72 h
Score:
0.67
Max. score:
3
Reversibility:
fully reversible within: 4 days
Irritation parameter:
chemosis score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0.33
Max. score:
4
Reversibility:
fully reversible within: 72 hours
Irritation parameter:
chemosis score
Basis:
mean
Remarks:
animal #2 and #3
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritant / corrosive response data:
The substance caused slight to moderate corneal opacities in all three animals, with conjunctival swelling in two animals. All three animals had slight conjunctivitis. The observed effects were fully reversible within 4 days.

Ocular Irritant response data (for the undiluted test substance)

 Animal #1

 Time after administration         

 

 15 minutes

 48 hours  72 hours  4 days
 Cornea score (opacity)  -  0.5  N  
 Iris score  -  0  0  
 Conjunctivae score (redness)  1  0  
 Chemosis score  1  1  0  

 Animal #2

 Time after administration         

 

 15 minutes

 48 hours  72 hours  4 days
 Cornea score (opacity)  -  1  0.5  N
 Iris score  -  0  0  0
 Conjunctivae score (redness)  1  1  0
 Chemosis score  0  0  0  0

 Animal #3  Time after administration         
   15 minutes  48 hours  72 hours  4 days
 Cornea score (opacity)  -  0.5  N  
 Iris score  -  0  0  
 Conjunctivae score (redness)  1  0  
 Chemosis score  0  0  0  

N = Normal

Interpretation of results:
other: Not an eye irritant.
Remarks:
According to Regulation (EC) No 1272/2008 and its updates.
Conclusions:
In an eye irritation study with two female and one male rabbit, performed equivalent to OECD 405 guideline, limited irritation was observed which was fully reversible within 4 days. Based on the results of this study, the substance is not considered an eye irritant.
Executive summary:

The substance was tested in an eye irritation test in two female and one male rabbit according a method/protocol equivalent to OECD 405 guideline. The substance caused slight to moderate corneal opacities in all three animals, with conjunctival swelling in two animals. All three animals had slight conjunctivitis. The observed effects were fully reversible within 4 days. The results showed that the substance is not considered an eye irritant.

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

Respiratory irritation

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

Additional information

Skin corrosion: Skin corrosion: the substance is not considered corrosive based on absence of corrosive effects and in the presence of some irritation effects in the acute dermal toxicity test in rabbits (OECD TG 402).

Skin irritation:

The possible skin irritation potential of the substance was tested in vitro using a human skin model through topical application for 15 minutes. The study procedures described in this report were according to OECD TG 439 guideline and GLP principles. Skin tissue was treated by topical application of 10 μL undiluted test substance. After 42 hours incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) MTT at the end of treatment. Skin irritation is expressed as the remaining cell viability after exposure to the test substance. Reliable negative and positive controls were included. The positive control had a mean cell viability of 4.9% after 15 minutes exposure. The standard deviation value of the percentage viability of three tissues treated identically was less than 4%, indicating that the test system functioned properly. The relative mean tissue viability obtained after 15 minutes treatment with the substance compared to the negative control tissue was 21.2%. Since the mean relative tissue viability for the substance was below 50% after 15 minutes treatment the substance is considered to be a skin irritant.

Eye irritation:

The substance was tested in an eye irritation test in two female and one male rabbit according a method/protocol equivalent to OECD 405 guideline. The substance caused slight to moderate corneal opacities in all three animals, with conjunctival swelling in two animals. All three animals had slight conjunctivitis. The observed effects were fully reversible within 4 days. The results showed that the substance is not considered an eye irritant.

Respiratory irritation: the substance is not considered a respiratory irritant in absence of human data indicating such effects. In addition, the substance is not corrosive to skin or eye irritating with irreversible effects. Therefore the substance is not expected to be a respiratory irritant.

Justification for classification or non-classification

The substance is not considered corrosive based on the absence of such effects in the acute dermal toxicity test. The substance is a skin irritant in the OECD TG 439 and needs to be classified according toEU CLP ((EC) No. 1272/2008 and its updates), which results in skin irritation, Category 2, H315: Causes skin irritation.

Based on the negative results in the eye irritation test, the substance does not need to be classified for this endpoint according to EU CLP ((EC) No. 1272/2008 and its updates).

The substance is not considered a respiratory irritant in absence of human data indicating such effects. Therefore the substance does not need to be classified for this endpoint in accordance with EU CLP ((EC) No. 1272/2008 and its updates).

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