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Description of key information

Skin Irritation/Corrosion: not irritating (OECD 439, GLP)

Serious eye damage/eye irritation: not irritating (equivalent/similar to OECD 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:
key study
Study period:
11-10-12 to 12-11-12
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD Guideline and GLP compliant
Qualifier:
according to guideline
Guideline:
other: OECD 439 Guideline For The Testing of Chemicals: In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method
Version / remarks:
2010
GLP compliance:
yes
Details on test animals or test system and environmental conditions:
EPISKIN™ Skin Irritation Test 15 Min-42 Hours using human epidermis skin constructs with a functional multi-layered stratum corneum was used. The epidermis surface area supplied is 0.38cm2. The kit was supplied by SkinEthic Laboratories, Lyon, France.

The Technical Data, Safety Sheet and Certificate of Analysis for the EPISKIN construct kit that provides information on performance (viability, barrier function and morphology) is attached to the study record as Annex I.

Batch number: 12-EKIN-041; Passage number: Second. The kit expiry date was: 12/11/12
On receipt of the kit, the inserts with tissues on agarose were stored at room temperature until use.

Reduction of MTT by test substance: The MTT reducing capability of the test substance was investigated by mixing approximately 10 µL ofthe test substance with2 mL of 0.3 mg/mL MTT solution in duplicate. A control of 10 µL ofdistilled water, mixed with 2 mL of 0.3 mg/mL MTT solution was also included in duplicate.

Check for colouring potential of test substance: The test substance was evaluated for its colour or ability to become coloured in contact with water (simulating a tissue humid environment). The test substance (10 µL) was mixed with 90 µL of distilled water in a transparent container. 100 µL of distilled water was included as a control. The solution was mixed for 15 minutes on a shaker. At the end of the shaking period the
colour of the solution was assessed by eye.

Test substance pH: An estimate of the pH of the test substance as a 10% (v/v) solution in distilled water was determined using pH indicator paper and recorded.
Type of coverage:
other: Not applicable
Preparation of test site:
other: Not applicable
Vehicle:
unchanged (no vehicle)
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 µL of the test substance were applied to each of triplicate tissues.

Positive controls: 10 µL to each of triplicate tissues (5% Sodium Dodecyl Sulphate (SDS) in distilled water)
Negative controls: 10 µL to each of triplicate tissues (Sterile Dulbecco’s Phosphate Buffered Saline (DPBS) with magnesium and calcium.
Duration of treatment / exposure:
15 ± 0.5 minutes
Details on study design:
Replicates: Triplicate tissues of human epidermis skin were dosed with the test substance, negative and positive control.

Positive controls: 5% Sodium Dodecyl Sulphate (SDS) in distilled water
Negative controls: Sterile Dulbecco’s Phosphate Buffered Saline (DPBS) with magnesium and calcium

REMOVAL OF TEST SUBSTANCE
- Washing (if done): Each tissue was rinsed with 25 mL sterile Dulbeccos Phosphate Buffered Saline (DPBS) to remove residual test substance. Inserts were then blotted on absorbent paper to remove remaining DPBS
- Time after start of exposure: 15 ± 0.5 minutes

After incubation for 42 ± 1 hr at 37 ± 2°C in a humidified atmosphere of 5% CO2 in air, each tissue was incubated with 2 mL of 0.3 mg/mL MTT (3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) for 3 hours ± 5 minutes at 37 ± 2°C in a humidified atmosphere of 5% CO2 in air. The
epidermis was removed from the insert using a biopsy punch, the epidermis separated from the collagen matrix using forceps and both parts placed in a micro tube and vortexed with 500 µL of acidic isopropanol (0.04 N HCl final concentration). The tissues were extracted by storing at 2-8 ºC, protected fromlight, for 48 to 70 hours. The absorbance of the extractant was read at 540 nm, with six wells containing acidified isopropanol (0.04 N HCl final concentration) as a blank.

SCORING SYSTEM: The mean Optical Density (OD) for the six replicate blanks was subtracted from the individual substance and control tissues OD. The mean absorbance of the tri plicate negative control values should be ≥0.6 and ≤1.5. The Standard Deviation (SD) value ofthe % viability should be <18. The OD of the positive control is an indicator of the sensit ivity of the tissues. The mean viability shoul d be ≤40% ofthe negative control and the SD <18%. If the mean tissue viabilit y was equal to or less than 50% of the negative control value, the sample was classed as Irritant R38 (EU classification) or Category2 (GHS classification).
Irritation / corrosion parameter:
other: other: Tissue viability
Value:
100
Remarks on result:
other:
Remarks:
Basis: mean negative control (Tissue 1,2,3). Remarks: Tissue viability as percentage of mean OD negative control . (migrated information)
Irritation / corrosion parameter:
other: other: Tissue viability
Value:
24.3
Remarks on result:
other:
Remarks:
Basis: mean positive control (Tissue 1,2,3). Remarks: Tissue viability as percentage of mean OD negative control . (migrated information)
Irritation / corrosion parameter:
other: other: Tissue viability
Value:
76.2
Remarks on result:
other:
Remarks:
Basis: mean Methyl Cedryl Ketone (Tissue 1,2,3). Remarks: Tissue viability as percentage of mean OD negative control . (migrated information)
Other effects / acceptance of results:
Individual tissue data (a,b,c) is presented below for negative and positive control and test substance (Table 1).

There was no change in the test substance, Methyl Cedryl Ketone/MTT solution or the water control/MTT solution after three hours incubation in the dark 37 ± 2°C in a humidified atmosphere of 5% CO2 in air. The test substance had not interacted with the MTT.

The test substance, Methyl Cedryl Ketone/water solution and water control were colourless after the 15 minute shaking period. The test substance, Methyl Cedryl Ketone, had not shown any potential for colouring water.

The pH of the test substance, Methyl Cedryl Ketone, as a 10% v/v solution in purified water, measured using pH indicator paper, was approximately 7.0.

Table 1: EPISKIN study data

Sample Tissue Replicate Optical Density (OD) OD - Blank % Negative Control
Negative Control a 0.885 0.753 100.7
0.845 0.712
b 0.869 0.737 96
0.793 0.661
c 0.907 0.775 103.4
0.862 0.73
Replicates a,b,c Average 0.728 100
sd 0.039 3.7
Positive Control a 0.434 0.302 40.3
0.418 0.285
b 0.233 0.1 12.5
0.214 0.082
c 0.288 0.156 20.2
0.27 0.138
Replicates a,b,c Average 0.177 24.3
sd 0.094 14.4
Methyl Cedryl Ketone a 0.708 0.576 77.6
0.685 0.553
b 0.672 0.539 71
0.627 0.494
c 0.728 0.596 79.9
0.7 0.568
Replicates a,b,c Average 0.554 76.2
sd 0.035 4.6
Blank 0.128
0.134
0.134
0.131
0.133
0.134
Average 0.132
sd 0.003

sd = Standard Deviation

Table 2:Summary of EPISKIN assay results

Sample Tissue viability as percentage of mean OD negative control
Replicate Tissues Mean ± Prediction
MTT endpoint
a b c SD
Negative Control 100.7 96 103.4 100.0 ± 3.7 Not applicable
Positive Control 40.3 12.5 20.2 24.3 ± 14.4 Irritant
Methyl Cedryl Ketone 77.6 71 79.9 76.2 ± 4.6 Non-irritant
Interpretation of results:
GHS criteria not met
Conclusions:
It was concluded that the test substance, Methyl Cedryl Ketone, with a mean tissue viability of 76.2 ± 4.6%, was predicted as non-irritant to the skin.
Executive summary:

In an In Vitro Skin Irritation test Reconstructed Human Epidermis Test Method (MBB0004), reconstituted human epidermis skin cultured in vitro was exposed to 10 µL undiluted Methyl Cedryl Ketone for 15 ± 0.5 minutes.

Both the negative and positive control results met the assay acceptance criteria. The percentage mean viability of the Methyl Cedryl Ketone was 76.2 ± 4.6 of the negative control. As the mean tissue viability is greater than 50% of the negative control value, Methyl Cedryl Ketone is classed as non-irritant.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 439 in vitro skin irritation (Reconstructed Human Epidermis Test Method; EPISKIN™ Skin Irritation Test).

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:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Equivalent or similar to OECD 405. There is no GLP compliance statement in the report but it is indicated that the study was designed to ensure compliance with the Code of GLP published in the Federal Register, December 1978.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
GLP compliance:
no
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Age at study initiation: young adult
- Weight at study initiation: between 2.0 and 3.5 kg
- Housing: Housed individually under standard lab conditions
- Diet (e.g. ad libitum): 100g purina rabbit chow supplemented with whole oats
- Water (e.g. ad libitum): ad libitum
The criteria for housing and maintenance were those specified in the 'Guide for the Care and Use of Laboratory Animals' (DHEW Publication No. (NIH) 78-23, Revised 1978).

Vehicle:
unchanged (no vehicle)
Controls:
not required
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1mL

Duration of treatment / exposure:
24 hrs
Observation period (in vivo):
24, 48 and 72 hrs and at 4 and 7 days after dosing.
Number of animals or in vitro replicates:
6 animals
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing (if done): The eye was not washed after instillation of the test article.

SCORING SYSTEM: Method of Draize/Table: Grading of Ocular Lesions in OECD 405 with additions noted below in Tables 1 and 2.

TOOL USED TO ASSESS SCORE: Both eyes examined with fluorescein under UV light (24 hrs and 7 days)
Irritation parameter:
cornea opacity score
Remarks:
Opacity
Basis:
animal: 1-6
Time point:
24/48/72 h
Score:
0
Irritation parameter:
iris score
Basis:
animal: 1-6
Time point:
24/48/72 h
Score:
0
Irritation parameter:
conjunctivae score
Remarks:
Redness
Basis:
animal: 1-6
Time point:
24/48/72 h
Score:
0
Irritation parameter:
chemosis score
Basis:
animal: 1-6
Time point:
24/48/72 h
Score:
0
Irritant / corrosive response data:
None of the dosed eyes had any reaction during the 7 day observation period (Table 3) so no animal was retained for examination on day 10. All eyes were negative for fluorescein retention. Individual animal data is presented below.

The mean ocular irritation scores were:

0 at 24 hours
0 at 48 hours
0 at 72 hours
0 at 4 days
0 at 7 days

Table 3: Score chart for primary eye irritation (all timepoints)

  Cornea (Max. 4) Iris (Max. 2) Conjunctivae (Max. 3/3 and 4)
  Opacity and Area   Redness/Discharge and Chemosis
Animal 24h 48h 72h Day 4 Day 7 24h 48h 72h Day 4 Day 7 24h 48h 72h Day 4 Day 7
A-1132 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
A-1131 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
A-1130 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
A-1134 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
A-1133 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
A-1135 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
Control 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
Weighted score  0 0 0
Total 0 0 0
Primary Occular Irritation Score 0 0 0
Interpretation of results:
GHS criteria not met
Conclusions:
The substance methyl cedryl ketone was not irritating in the eyes of New Zealand White rabbits in this study.
Executive summary:

In a primary eye irritation study (0267), 0.1 mL of  Methyl Cedryl Ketone was instilled into the conjunctival sac of the right eye of 6 young female rabbits (New Zealand white) for 24 hours.  The eye was not washed after instillation of the test article.  Animals were then observed for 24, 48 and 72 hour and at 4 and 7 days after dosing.  Irritation was scored by the method of Draize/Table: Grading of Ocular lesions (OECD 405) with additions noted in Tables 1 and 2.

 

None of the dosed eyes had any reaction during the 7 day observation period.

In this study, Methyl Cedryl Ketone is not an eye irritant.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation/corrosion:

There is one in vitro skin irritation study available.

In an In Vitro Skin Irritation test Reconstructed Human Epidermis Test Method (OECD 439/GLP), reconstituted human epidermis skin cultured in vitro was exposed to 10 µL undiluted Methyl Cedryl Ketone for 15 ± 0.5 minutes. Both the negative and positive control results met the assay acceptance criteria. The percentage mean viability of the Methyl Cedryl Ketone was 76.2 ± 4.6 of the negative control. As the mean tissue viability is greater than 50% of the negative control value, Methyl Cedryl Ketone is classed as non-irritant.

Serious eye damage/eye irritation:

There is no in vitro eye irritation study available. There is one in vivo eye irritation study in rabbits available.

In a primary eye irritation study (equivalent/similar to OECD 405), 0.1 mL of Methyl Cedryl Ketone was instilled into the conjunctival sac of the right eye of 6 young female rabbits (New Zealand white) for 24 hours.  The eye was not washed after instillation of the test article.  Animals were then observed for 24, 48 and 72 hour and at 4 and 7 days after dosing. None of the dosed eyes had any reaction during the 7 day observation period. In this study, Methyl Cedryl Ketone is not an eye irritant.

The results from these studies are acceptable to use in the human health risk assessment.


Justification for classification or non-classification

Based on the available information in the dossier, the substance Methyl Cedryl Ketone (CAS No. 32388-55-9) does not need to be classified for skin irritation/corrosion or serious eye damage/eye irritation when considering the criteria outlined in Annex I of 1272/2008/EC.

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