Registration Dossier

Administrative data

Description of key information

Skin irritation: irritating (OECD 439, GLP, K, rel. 1)
Eye irritation: not irritating (similar to OECD 405, read-across, 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 August 13 to 18, 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study conducted according to OECD test Guideline No. 439 without any deviation.
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.
EpiSkinTM Tissues (0.38cm2) lot number: 14-EKIN-030.
Maintenance Medium lot number: 14-MAIN3-033.
Assay Medium lot number: 14-ESSC-030.
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 14.8% 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

0.976

0.923

0.071

105.7

100*

7.7

0.951

103.0

0.842

91.2

Positive Control Item

0.252

0.289

0.071

27.3

31.3

7.7

0.244

26.4

0.371

40.2

Test Item

0.135

0.137

0.005

14.6

14.8

0.6

0.143

15.5

0.133

14.4

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 3.1% relative to the negative control treated tissues and the standard deviation value of the percentage viability was 7.7%. The positive control acceptance criterion was therefore satisfied.

 

The mean OD562 for the negative control treated tissues was 0.923 and the standard deviation value of the percentage viability was 7.7%. 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 0.6%. The test item acceptance criterion was therefore satisfied.

 

Historical Control Data

 

For the previous 22 experiments conducted from April 2014 to August 2014 using this test method, the mean OD of the positive control was 0.084 ± 0.046 and the mean percentage viability was 9.2 ± 5.3. In this same period the mean OD of the negative control was 0.938 ± 0.102.

Although the positive control parameters for this study were greater than ranges based on the previous 22 experiments the positive control was considered to have reflected the ability to respond to an irritant chemical under the conditions of the test ie the recommended acceptance criterion for the positive control was achieved for this test (≤40% relative to the concurrent negative control). The negative control in this study was comparable to the negative control achieved in the previous 22 experiments conducted with this test method.

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 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 14.8% 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 3.1% relative to the negative control treated tissues and the standard deviation value of the percentage viability was 7.7%. The positive control acceptance criterion was therefore satisfied.

The mean OD562 for the negative control treated tissues was 0.923 and the standard deviation value of the percentage viability was 7.7%. 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 0.6%. 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:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From July 09 to 16, 1979
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Basic data given: comparable to standard guideline. No details on test animals and environmental conditions. The supporting substance is considered adequate for read-across purpose (see Iuclid section 13 for justification).
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
other: Section 1500.42 - Hazardous Substances and Articles, Administration and Enforcement Regulations, Federal Register, Vol. 38. No. 187, p. 27019, 27 September 1973.
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
no
Remarks:
pre-GLP
Species:
rabbit
Strain:
not specified
Details on test animals or tissues and environmental conditions:
TEST ANIMALS: Healthy young adult albino rabbits were used.

IN-LIFE DATES: From: July 09, 1979 To: July 16, 1979
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 g
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: According to “Illustrated guide for grading eye irritation by hazardous substances”.

TOOL USED TO ASSESS SCORE: No data
Irritation parameter:
cornea opacity score
Basis:
mean
Remarks:
(6 animals)
Time point:
other: 24, 48 and 72 h
Score:
0
Max. score:
4
Irritation parameter:
iris score
Basis:
mean
Remarks:
(6 animals)
Time point:
other: 24, 48 and 72 h
Score:
0
Max. score:
2
Irritation parameter:
conjunctivae score
Basis:
mean
Remarks:
(6 animals)
Time point:
other: 24, 48 and 72 h
Score:
0.39
Max. score:
3
Reversibility:
fully reversible within: 3 days
Irritation parameter:
chemosis score
Basis:
mean
Remarks:
(6 animals)
Time point:
other: 24, 48 and 72 h
Score:
0.33
Max. score:
4
Reversibility:
fully reversible within: 5 days
Irritant / corrosive response data:
- Animals showed conjunctival reactions (redness, chemosis and discharge) which were reversible within 5 days.
Other effects:
None

Table 7.3.2/1: Mean eye irritation response data of 6 animals at each observation time

Score at time point / Reversibility

Cornea

Iris

(/2)

Conjunctivae

Opacity

(/4)

Area

(/4)

Redness

(/3)

Chemosis

(/4)

Discharge

(/3)

24 h

0

0

0

0.83

0.67

1

48 h

0

0

0

0.33

0.17

0.33

72 h

0

0

0

0

0.17

0.33

Mean

0

0

0

0.39

0.33

0.55

Reversibility

-

-

-

Completely

reversible

Completely reversible

Completely reversible

Average time for reversion

-

-

-

3 days

5 days

5 days

Table 7.3.2/2: 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 (Day 1)

0 / 0 / 0 / 0 / 0 / 0

0 / 0 / 0 / 0 / 0 / 0

0 / 0 / 0 / 0 / 0 / 0

1 / 2 / 1 / 0 / 0 / 1

1 / 1 / 1 / 0 / 0 / 1

1 / 3 / 2 / 0 / 0 / 0

48 h (Day 2)

0 / 0 / 0 / 0 / 0 / 0

0 / 0 / 0 / 0 / 0 / 0

0 / 0 / 0 / 0 / 0 / 0

0 / 2 / 0 / 0 / 0 / 0

0 / 1 / 0 / 0 / 0 / 0

0 / 2 / 0 / 0 / 0 / 0

72 h (Day 3)

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 / 1 / 0 / 0 / 0 / 0

0 / 2 / 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 24, 48 and 72 h

0 / 0 / 0 / 0 / 0 / 0

0 / 0 / 0 / 0 / 0 / 0

0 / 0 / 0 / 0 / 0 / 0

0.33 / 1.33 / 0.33 / 0 / 0 / 0.33

0.33 / 1 / 0.33 / 0 / 0 / 0.33 

0.33 / 2.33 / 0.67 / 0 / 0 / 0

Reversibility

 -

 -

-

Completely

reversible

Completely reversible

Completely reversible

Average time (unit) for reversion

-

-

-

3 days

5 days

5 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 5 days. Therefore, the test material is not classified as irritant to the eyes.
Executive summary:

In an eye irritation study, 0.1 g 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 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 “Illustrated guide for grading eye irritation by hazardous substances”.

The calculated mean score for each animal within 3 scoring times (24, 48 and 72 h) were 0.39 for redness, 0.33 for chemosis, 0.0 for iris lesions and 0.0 for corneal opacity. The observed conjunctival reactions were fully reversible within 5 days.

Under the test conditions, the test material is not classified as irritating to the eyes according to the annex I of 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.

The supporting substance is considered adequate for read-across purpose (see Iuclid section 13 for justification).

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation / corrosion: A study was available on the substance itself but was not reliable (Biosearch, 1979, Rel. 3). Indeed, the protocol deviates from standard method : no data on test animals, 24-hour exposure instead of 4 -hour, occlusive dressing instead of semi-occlusive dressing, observation at 24- and 72-hour only (no 48-hour scoring), reversibility not assessed although more severe effects - not sufficient for classification - were observed at the end of the study period (72-hour). In addition, desquamation was observed during an acute dermal toxicity study (Borriston, 1979). Therefore a new study, an in vitro skin irritation test, was performed (Harlan, 2015, Rel.1). This study was performed according to the OECD Guideline 439 and in compliance with GLP, using the EPISKIN TM reconstructed human epidermis model. The relative mean viability of the test item treated tissues was 14.8% 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 3.1% relative to the negative control treated tissues and the standard deviation value of the percentage viability was 7.7%. The positive control acceptance criterion was therefore satisfied. The mean OD562 for the negative control treated tissues was 0.923 and the standard deviation value of the percentage viability was 7.7%. 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 0.6%. The test item acceptance criterion was therefore satisfied. Under the test conditions, test item is a skin irritant. In accordance with the integrated testing strategy, the bottom-up approach normally includes an additional in vitro test for skin corrosion when the result of the in vitro skin irritation test is positive. However, taking into account: 1/ the absence of corrosion in the acute dermal toxicity study on the supporting substance, 2/ the absence of corrosion and irritation in the in vivo eye irritation study on the supporting substance, it can be safely concluded that the registered substance is also not corrosive to the skin without performing a new test. Eye irritation: A study was available on the substance itself, however it was performed on a dilution (Biosearch, 1979, Rel.3). Therefore a read-across approach was used. The supporting substance, (2E)-1-(2,6,6-trimethyl-2-cyclohexen-1-yl)-2-buten-1-one is considered adequate for read-across purposes (see Iuclid section 13 for additional justification). The key study (Biosearch, 1979) is of good quality (Klimisch score = 2). The key study was performed similarly to the OECD Guideline No. 405. Undiluted test material was instilled into the eye of 6 rabbits. The eyes were not rinsed after instillation of the test item. Animals were observed at 24, 48 and 72 h after instillation of test material into eyes and then on Days 5 and 7. The calculated mean score for each animal within 3 scoring times (24, 48 and 72 h) were 0.39 for redness, 0.33 for chemosis, 0.0 for iris lesions and 0.0 for corneal opacity. The observed conjunctival reactions were fully reversible within 5 days. The supporting substance does not require classification for eye irritation. Even if only tested at 50%, the study on the registered substance supports this conclusion. The registered substance is therefore not classified for eye irritation.


Justification for selection of skin irritation / corrosion endpoint:
A key study was available on the substance itself and is fully reliable (Klimisch score = 1).

Justification for selection of eye irritation endpoint:
A study was available on the substance itself, however it was performed on a dilution. Therefore a read-across approach was used. The supporting substance is considered adequate for read-across purposes (see Iuclid section 13 for additional justification). The key study is of good quality (Klimisch 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 including ATP6.

Self-classification:

Based on the available information, the registered substance should be classified as Skin irr. 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 information was available regarding respiratory irritation.