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Description of key information

Skin irritation (OECD TG 439): not irritating
Eye irritation (OECD TG 405): 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:
14 March, 2016 - 21 March, 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:
(2015)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
(2012)
Deviations:
no
GLP compliance:
yes
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-011
- 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: 36.3 - 37.3°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 thecellular 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:
Cedryl Acetate 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 Cedryl Acetate 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, 25 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 for approximately 22 hours 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. Twenty five μL of the undiluted test item was added into 12-well plates on top of the skin tissues. Three tissues were treated with 25 μL PBS (negative control) and 3 tissues with 25 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 isopropanol (Merck, Darmstadt, Germany). Tubes were stored refrigerated and protected from light for 70 hours. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate 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: 25 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: other: relative mean viability
Value:
73
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
The relative mean tissue viability compared to the negative control tissues (100%).
Other effects / acceptance of results:
Direct MTT Reduction
Cedryl Acetate was checked for colour interference in aqueous conditions and possible direct MTT reduction by adding the test item to MTT medium. Because no colour changes were observed it was concluded that Cedryl Acetate did not interact with the MTT endpoint.

Test Item, Positive Control Item and Negative Control Item
The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with Cedryl Acetate compared to the negative control tissues was 73%.

Quality Criteria
The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 44%. The positive control meets the validity criterion meets the validity criterion even though it is just outside the historical control range, which has not affected the result of the results for the test substance.The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range. The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 44%. The positive control meets the validity criterion meets the validity criterion even though it is just outside the historical control range, which has not affected the result of the results for the test substance.The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range.

Mean OD570 Values and Percentage Viabilities for the Negative Control Item, Positive Control Item and Test Item:

Item

OD570 of

tissues

Mean OD562

of triplicate

tissues

± SD of

OD570

Relative

individual

tissue

viability (%)

Relative

mean

viability (%)

Negative

Control Item

1.066

1.047

0.022

100

1.052

1.024

Positive Control Item

0.398

0.462

0.078

37

44

0.549

52

0.437

43

Test Item

0.715

0.759

0.100

67

73

0.689

65

0.874

85

SD = Standard deviation

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

Interpretation of results:
other: not irritating
Remarks:
based on CLP criteria
Conclusions:
The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the substance compared to the negative control tissues was 73%. Since the mean relative tissue viability for the substance was above 50% the substance is considered to be non-irritant and therefore does not need to be classified in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).
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 25 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 44% after 15 minutes exposure. The standard deviation value of the percentage viability of three tissues treated identically was less than 5%, 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 73%. Since the mean relative tissue viability for the substance was above 50% after 15 minutes treatment the substance is considered to be not irritant and therefore does not need to be classified in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).

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
Study period:
4 April 2000 - 17 April 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
In accordance with GLP conditions
Qualifier:
according to guideline
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Version / remarks:
(1981)
Deviations:
no
GLP compliance:
yes
Species:
rabbit
Strain:
other: Albino Chbb:HM(SPF) - Littlerussian
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: BI Pharma KG, 88397 Biberach
- Age at study initiation: no information available
- Weight at study initiation: Body weights were 1.9 - 2.0 kg
- Housing: Individually
- Diet: ad libitum pelleted complete rabbit diet (Altromin 2123)
- Water: free access to domestic water (acidified with hydrochloric acid)
- Acclimation period: no information available

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20
- Humidity (%): 55+/-15
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12
Vehicle:
unchanged (no vehicle)
Controls:
other: Untreated eye served as negative control
Amount / concentration applied:
0.1 mL
Duration of treatment / exposure:
Single instillation on Day 1
Observation period (in vivo):
7 days
Number of animals or in vitro replicates:
4 (female)
Details on study design:
REMOVAL OF TEST SUBSTANCE
-Washing: No

SCORING SYSTEM: Irritation was assessed in accordance with the scoring system as included in OECD TG 405 (1981).

TOOL USED TO ASSESS SCORE: fluorescein
Irritation parameter:
cornea opacity score
Basis:
animal: all
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
fully reversible
Irritation parameter:
iris score
Basis:
animal: all
Time point:
24/48/72 h
Score:
0
Max. score:
2
Reversibility:
fully reversible
Irritation parameter:
conjunctivae score
Remarks:
(redness)
Basis:
animal: #1 and #3
Time point:
24/48/72 h
Score:
1.33
Max. score:
3
Reversibility:
fully reversible
Irritation parameter:
conjunctivae score
Remarks:
(redness)
Basis:
animal #2
Time point:
24/48/72 h
Score:
1.67
Max. score:
3
Reversibility:
fully reversible
Irritation parameter:
conjunctivae score
Remarks:
(redness)
Basis:
animal #4
Time point:
24/48/72 h
Score:
1
Max. score:
3
Reversibility:
fully reversible
Irritation parameter:
chemosis score
Basis:
animal: #1, #2 and #4
Time point:
24/48/72 h
Score:
1
Max. score:
4
Reversibility:
fully reversible
Irritation parameter:
chemosis score
Basis:
animal #3
Time point:
24/48/72 h
Score:
1
Max. score:
4
Reversibility:
fully reversible
Irritant / corrosive response data:
No or only very mild (at 1 hour after instillation) cornea and iris effects were noted in the study. Redness and chemosis of the conjunctiva was more apparent but disappeared in the first few days. All irritation effects were reversible within the observation period. For animal 3 this was seen after 72 hours, for the other 3 animals this was seen on day 7.
Other effects:
- Lesions and clinical observations: No lesions of the cornea were observed in any of the animals after 7 days.

Individual scores for the treated animals:

 

Time after administration

 

1 hour

24 hours

48 hours

72 hours

Animal:

1

2

3

4

1

2

3

4

1

2

3

4

1

2

3

4

Cornea score (opacity)

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Iris score

0

1

1

1

0

0

0

0

0

0

0

0

0

0

0

0

Conjunctivae score (redness)

2

2

2

3

1

2

2

2

2

2

1

1

1

1

1

0

Chemosis score

2

2

2

3

2

2

1

2

1

1

1

1

0

0

1

0

Interpretation of results:
other: not classified
Remarks:
based on CLP criteria
Conclusions:
In this eye irritation study, no effects were observed on the cornea and iris. Some effects were noted on the conjunctiva in the first days. All effects were reversible. Based on the individual mean scores for the four animals at 24, 48 and 72 hours, it was established that none meet the classification criteria as outlined in Annex I of the CLP Regulation (1272/2008/EC). Therefore, the substance is considered not to be an eye irritant.
Executive summary:

Undiluted Cedryl acetate was tested in an eye irritation test in four female rabbits, in accordance with OECD TG 405 (1981) and under GLP conditions. A 21 day observation period was used to score ocular lesions to the cornea, iris and conjunctiva in accordance with the scoring system in the OECD guideline. No effects were observed on the cornea and iris. Some effects were noted on the conjunctiva in the first days. All effects were reversible. Based on the individual mean scores for the four animals at 24, 48 and 72 hours, it was established that none meet the classification criteria as outlined in Annex I of CLP (1272/2008/EC). Therefore, Cedryl acetate is considered not to be an eye irritant.

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

Additional information

In vitro 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 25 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 44% after 15 minutes exposure. The standard deviation value of the percentage viability of three tissues treated identically was less than 5%, 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 73%. Since the mean relative tissue viability for the substance was above 50% after 15 minutes treatment the substance is considered to be not irritant.

In vivo eye irritation

Undiluted Cedryl acetate was tested in an eye irritation test in four female rabbits, in accordance with OECD TG 405 (1981) and under GLP conditions. A 21 day observation period was used to score ocular lesions to the cornea, iris and conjunctiva in accordance with the scoring system in the OECD guideline. No effects were observed on the cornea and iris. Some effects were noted on the conjunctiva in the first days. All effects were reversible. Based on the individual mean scores for the four animals at 24, 48 and 72 hours, it was established that none meet the classification criteria as outlined in Annex I of CLP (1272/2008/EC). Therefore, Cedryl acetate is considered not to be an eye irritant.

Justification for classification or non-classification

Based on the negative results found in the in vitro skin irritation test, the substance does not need to be classified as a skin irritant in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).

Based on the negative results found in the in vivo eye irritation test, the substance does not need to be classified as an eye irritant in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).