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Administrative data

Description of key information

Skin irritation: irritating  (OECD 439, GLP, K, Rel.1)
Eye irritation: not irritating (similar to OECD 405, K, Rel. 2)
Respiratory irritation: no data available

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:
From February 25 to March 02, 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed according to OECD test guideline No. 439 and in compliance with GLP.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
UK GLP Compliance Program (inspected on March 12 to 14, 2014 / Signed on May 12, 2014)
Species:
other: EPISKIN™ Reconstructed Human Epidermis Model Kit
Details on test animals and environmental conditions:
EPISKIN™ Reconstructed Human Epidermis Model Kit
- Supplier: SkinEthic Laboratories, Lyon, France
- Date received: 25 February 2015
- Expiry date : 02 March 2015
- EpiSkinTM Tissues (0.38cm2) lot number : 15-EKIN-008
- Maintenance Medium lot number : 15-MAIN3-008
- Assay Medium lot number : 15-ESSC-008
Type of coverage:
other: not applicable
Preparation of test site:
other: not applicable
Vehicle:
other: not applicable
Controls:
yes
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 µL of the test item was applied to the epidermis surface
- Concentration: The test item was used as supplied.
Duration of treatment / exposure:
Triplicate tissues were treated with the test item for an exposure period of 15 minutes at room temperature in 12 well plate containing 2 mL of maintenance medium.
At the end of the exposure period, tissues were rinsed and incubated at 37 °C, 5% CO2 in air for 42 hours
Observation period:
- On Day 3, at the end of the 42 h post-treatment incubation period: MTT test (MTT Loading/Formazan Extraction) was performed and tissues were incubated for 3 hours at 37 °C, 5 % CO2 in air.
- On Day 6, at the end of the formazan extraction period: The optical density was measured (quantitative viability analysis) at 562 nm (without a reference filter) using the Anthos 2001 microplate reader.
Number of animals:
Triplicate tissues for test item, negative and positive controls
Details on study design:
TEST SITE
- Test item was applied to the epidermis surface

REMOVAL OF TEST SUBSTANCE
- Washing (if done): At the end of the exposure period, each tissue was removed from the well and rinsed using DPBS with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test item.

Negative and positive controls (reference substances).
- Negative control: 10 µL of DPBS
- Positive control: 10 µL of 5 % w/v aqueous solution of Sodium Dodecyl Sulfate (SDS)
Irritant / corrosive response data:
The relative mean viability of the test item treated tissues was 9.2% after a 15-minute exposure period and 42 hours post exposure incubation period.
Other effects:
None

Direct MTT Reduction: The MTT solution containing the test item remained yellow which indicated that the test item did not directly reduce MTT.

 

Main test:

Table 7.3.1/1: 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 (%)

± SD of Relative mean viability (%)

Negative Control Item

1.016

1.044

0.02

97.3

100*

2.3

1.056

101.1

1.059

101.4

Positive Control Item

0.154

0.129

0.03

14.8

12.4

3.3

0.090

8.6

0.144

13.8

Test Item

0.074

0.096

0.02

7.1

9.2

1.9

0.109

10.4

0.106

10.2

SD=        Standard deviation

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

 

Quality Criteria

The relative mean tissue viability for the positive control treated tissues was 12.4% relative to the negative control treated tissues and the standard deviation value of the percentage viability was 3.3%. The positive control acceptance criterion was therefore satisfied.

 

The mean OD562 for the negative control treated tissues was 1.044 and the standard deviation value of the percentage viability was 2.3%. The negative control acceptance criterion was therefore satisfied.

 

The standard deviation calculated from individual percentage tissue viabilities of the three identically treated test item tissues was 1.9%. The test item acceptance criterion was therefore satisfied.

 

Historical Control Data

For the previous 19 experiments up to the start of this study (approximately 6 months data) using this test method, the mean OD of the positive control was 0.119 ± 0.053 and the mean percentage viability was 14.3 ± 6.3. In this same period the mean OD of the negative control was 0.866 ± 0.128. Although the negative control parameter for this study was greater than range based on the previous 19 experiments the negative control was within the defined acceptance criterion for this test method using the EpiSkinTM Reconstructed Human Epidermis Model ie ≥0.6 and ≤ 1.5.
Interpretation of results:
irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the test conditions, test item was classified as irritant according to the annex VI of the Regulation (EC) No. 1272/2008 (CLP).
Executive summary:

An in vitro skin irritation study was performed according to the OECD Guideline 439 and in compliance with GLP, using the EPISKINTM reconstructed human epidermis model. Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. At the end of the post‑exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre‑labelled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT-loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT‑loaded tissues. At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 µL samples were transferred to the appropriate wells of a pre‑labelled 96‑well plate. The optical density was measured at 562 nm.

Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues). The relative mean viability of the test item treated tissues was 9.2% after the 15‑minute exposure period and 42 hours post‑exposure incubation period.

The relative mean tissue viability for the positive control treated tissues was 12.4% relative to the negative control treated tissues and the standard deviation value of the percentage viability was 3.3%. The positive control acceptance criterion was therefore satisfied.

The mean OD562for the negative control treated tissues was 1.044 and the standard deviation value of the percentage viability was 2.3%. The negative control acceptance criterion was therefore satisfied.

The standard deviation calculated from individual percentage tissue viabilities of the three identically treated test item tissues was 1.9%. The test item acceptance criterion was therefore satisfied.

 

Under the test conditions, test item was classified as irritant according to the annex VI of the Regulation (EC) No. 1272/2008 (CLP).

This study is considered as acceptable and satisfies the requirement for skin irritation endpoint.

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:
From February 16 to 23, 1981
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Basic data given: GLP study comparable to OECD test guideline No 405. No details on test animals and environmental conditions.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
equivalent or similar to
Guideline:
other: Section 1500.42 - Federal Hazardous Substances Act Regulations CFR 16 P.125
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Deviations:
yes
Remarks:
No details on test animals and environmental conditions.
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Species:
rabbit
Strain:
not specified
Details on test animals or tissues and environmental conditions:
Healthy young albino rabbits were used.
Vehicle:
unchanged (no vehicle)
Controls:
other: untreated eye served as control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 mL
Duration of treatment / exposure:
Eyes of the animals were not rinsed.
Observation period (in vivo):
7 days
Number of animals or in vitro replicates:
6
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing (if done): No

SCORING SYSTEM: Draize Scale.

TOOL USED TO ASSESS SCORE: No data.
Irritation parameter:
cornea opacity score
Basis:
animal: #1, #2, #3, #4, #5 & #6
Time point:
other: 24, 48 and 72 h
Score:
0
Max. score:
4
Irritation parameter:
iris score
Basis:
animal: #1, #2, #3, #4, #5 & #6
Time point:
other: 24, 48 and 72 h
Score:
0
Max. score:
2
Irritation parameter:
conjunctivae score
Basis:
animal: #1, #2, #3, #4 & #6
Time point:
other: 24, 48 and 72 h
Score:
0.33
Max. score:
3
Reversibility:
fully reversible within: 2 days
Irritation parameter:
conjunctivae score
Basis:
animal #5
Time point:
other: 24, 48 and 72 h
Score:
0
Max. score:
3
Irritation parameter:
chemosis score
Basis:
animal: #1 & #3
Time point:
other: 24, 48 and 72 h
Score:
0.33
Max. score:
4
Reversibility:
fully reversible within: 2 days
Irritation parameter:
chemosis score
Basis:
animal #2
Time point:
other: 24, 48 and 72 h
Score:
0.67
Max. score:
3
Reversibility:
not fully reversible within: 2 days
Irritation parameter:
chemosis score
Basis:
animal: #4, #5 & #6
Time point:
other: 24, 48 and 72 h
Score:
0
Max. score:
3
Irritant / corrosive response data:
- Animals showed conjunctival reactions (redness, chemosis and discharge) which were reversible within 2 days
Other effects:
None

Table 7.3.2/1: Eye irritation response data for each animal at each observation time

Score at time point

Cornea

Iris

(/2)

Conjunctivae

Opacity

(/4)

Area

(/4)

Redness

(/3)

Chemosis

(/4)

Discharge

(/3)

24 h

0 / 0 / 0 / 0 / 0 / 0

0 / 0 / 0 / 0 / 0 / 0

0 / 0 / 0 / 0 / 0 / 0

1 / 1 / 1 / 1 / 0 / 1

1 / 1 / 1 / 0 / 0 / 0

1 / 2 / 1 / 0 / 0 / 0

48 h

0 / 0 / 0 / 0 / 0 / 0

0 / 0 / 0 / 0 / 0 / 0

0 / 0 / 0 / 0 / 0 / 0

0 / 0 / 0 / 0 / 0 / 0

0 / 0 / 0 / 0 / 0 / 0

0 / 0 / 0 / 0 / 0 / 0

72 h

0 / 0 / 0 / 0 / 0 / 0

0 / 0 / 0 / 0 / 0 / 0

0 / 0 / 0 / 0 / 0 / 0

0 / 0 / 0 / 0 / 0 / 0

0 / 0 / 0 / 0 / 0 / 0

0 / 0 / 0 / 0 / 0 / 0

Day 5

0 / 0 / 0 / 0 / 0 / 0

0 / 0 / 0 / 0 / 0 / 0

0 / 0 / 0 / 0 / 0 / 0

0 / 0 / 0 / 0 / 0 / 0

0 / 0 / 0 / 0 / 0 / 0

0 / 0 / 0 / 0 / 0 / 0

Day 7

0 / 0 / 0 / 0 / 0 / 0

0 / 0 / 0 / 0 / 0 / 0

0 / 0 / 0 / 0 / 0 / 0

0 / 0 / 0 / 0 / 0 / 0

0 / 0 / 0 / 0 / 0 / 0

0 / 0 / 0 / 0 / 0 / 0

Average 24h, 48h, and 72h

0 / 0 / 0 / 0 / 0 / 0

0 / 0 / 0 / 0 / 0 / 0

0 / 0 / 0 / 0 / 0 / 0

0.33 / 0.33 / 0.33 / 0.33 / 0 / 0.33

0.33 / 0.33 / 0.33 / 0 / 0 / 0

0.33 / 0.67 / 0.33 / 0 / 0 / 0

Reversibility

 -

 -

-

Completely

reversible

Completely reversible

Completely reversible

Average time (unit) for reversion

-

-

-

2 days

2 days

2 days

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the test conditions, the test material induced mild irritation being reversible within 2 days. Therefore, the test material is not classified as irritant to the eyes.
Executive summary:

In an eye irritation study, 0.1 mL of undiluted test material was instilled into the right eye of 6 albino rabbits while the left eye remained untreated and served as control. The eyes were not rinsed after the instillation of test material. Animals were observed at 24, 48 and 72 after instillation of test material into eyes and then on Days 5 and 7. The reactions in the conjunctivae (redness, chemosis and discharge), the iris and the cornea (opacity and area involved) were scored according to the Draize scale of scoring ocular lesions.

The calculated mean score for each animal within 3 scoring times (24, 48 and 72 h) were 0.33 / 0.33 / 0.33 / 0.33 / 0 / 0.33 for redness, 0.33 / 0.33 / 0.33 / 0.33 / 0 / 0.33 for chemosis, 0.33 / 0.33 / 0.33 / 0.33 / 0 / 0.33 for iris lesions and corneal opacity. The observed conjunctival reactions were disappeared completely within 2 days. 

Under the test conditions, the test material is not classified as irritating to the eyes according to the annex VI of the Regulation EC No. 1272/2008 (CLP).

Although some details on test animals and experimental conditions were missing, this study is considered as acceptable and satisfies the requirement for eye irritation endpoint.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation / corrosion:

An in vivo study was available on the substance itself but was not sufficiently robust to be the key study (Biosearch, 1981, Rel.4). Indeed, deviations from OECD Test Guideline No. 404 were noted: the exposure period was 24 hours instead of 4 hours, occlusive dressing was used instead of semi-occlusive dressing, and the animals were observed for 3 days instead of 14 days although complete reversibility was not achieved. This study was thus not sufficient on its own to conclude on the skin irritation potential.

Therefore, a new in vitro study was conducted on the substance (Harlan, 2015, rel. 1). The skin irritation potential of the test material was evaluated using the EPISKINTM reconstructed human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours. This test was designed to be compatible with the OECD Test Guideline No. 439 and was performed in compliance with GLP. The quality criteria required for acceptance of results in the test were satisfied. The relative mean viability of the test item treated tissues was 9.2 %, after the 15‑minute exposure period. With a tissue viability < 50%, the test material was considered to be irritant to skin.

Eye irritation:

A key study was identified (Biosearch, 1981, Rel.2). In this eye irritation study performed similarly to the OECD Test Guideline No 405, 0.1 mL of undiluted test material was instilled into the conjunctival sac of one eye of 6 New Zealand White rabbits while the remained untreated eye served as control. The eyes were not rinsed after the instillation of test material. Animals were observed at 24, 48 and 72 h after instillation of test material into eyes and then on Days 5 and 7. The reactions in the conjunctivae (redness, chemosis and discharge), the iris and the cornea (opacity and area involved) were scored according to the Draize scale.

The calculated mean score for each animal within 3 scoring times (24, 48 and 72 h) were 0.33 / 0.33 / 0.33 / 0.33 / 0 / 0.33 for redness, 0.33 / 0.33 / 0.33 / 0.33 / 0 / 0.33 for chemosis, 0.33 / 0.33 / 0.33 / 0.33 / 0 / 0.33 for iris lesions and corneal opacity. The observed conjunctival reactions were disappeared completely within 2 days. 

Under the test conditions, the test material is not irritating to the eyes.


Justification for selection of skin irritation / corrosion endpoint:
A key study is available. The study is GLP-compliant and of high quality (Klimisch score = 1).

Justification for selection of eye irritation endpoint:
A key study is available (similar to OECD 405, GLP, Klimish score=2)

Effects on skin irritation/corrosion: irritating

Justification for classification or non-classification

Harmonized classification:

The substance has no harmonized classification according to the Regulation (EC) No. 1272/2008.

 

Self-classification:

Based on the available information, the substance should be classified as Skin irritant Category 2 (H315: Causes skin irritation) according to the Annex VI of the Regulation (EC) No. 1272/2008 (CLP).

No additional self-classification is proposed regarding eye irritation according to the Annex VI of the Regulation (EC) No. 1272/2008 (CLP).

No data was available regarding respiratory irritation.