Reaction mass of (3R,3aS,6R,7R,8aS)-3,6,8,8-tetramethyloctahydro-1H-3a,7-methanoazulen-6-yl rel-acetate and rel-(1aR,4aR,8aR)-2,4a,8,8-tetramethyl-1,1a,4,4a,5,6,7,8-octahydrocyclopropa[d]naphthalene and rel-(3R,3aS,7S,8aS)-3,6,8,8-tetramethyl-2,3,4,7,8,8a-hexahydro-1H-3a,7-methanoazulene

Endpoint summary

Description of key information

Skin corrosion: The substance is not corrosive based on read across from the analogue Longifolene Coeur tested in an OECDTG 404 study.

Skin irritation: The substance is a skin irritant based on read across from Longifolene Coeur tested in an OECD TG 404 study.

Eye irritation: The substance is not eye irritating based on read across from Cedryl Acetate 'mono' (tested in an OECD TG 405) and Longifolene (tested in an OECD TG 438)

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vivo

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2018

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Read-across information.

Justification for type of information:

The read across justification is presented in the Endpoint summary Irritation. The accompanying files are also attached there.

Reason / purpose for cross-reference:

read-across source

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 14 days

Remarks on result:

other: read-across from Longifolene Coeur

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

1.67

Max. score:

4

Reversibility:

fully reversible within: 14 days

Remarks on result:

other: read-across from Longifolene Coeur

Irritation parameter:

erythema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

2.33

Max. score:

4

Reversibility:

fully reversible within: 14 days

Remarks on result:

other: read-across from Longifolene Coeur

Irritation parameter:

edema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

2.67

Max. score:

4

Reversibility:

fully reversible within: 7 days

Remarks on result:

other: read-across from Longifolene Coeur

Irritation parameter:

edema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 7 days

Remarks on result:

other: read-across from Longifolene Coeur

Irritation parameter:

edema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

1.67

Max. score:

4

Reversibility:

fully reversible within: 7 days

Remarks on result:

other: read-across from Longifolene Coeur

Interpretation of results:

other: Skin irritant Category 2

Remarks:

according to Regulation (EC) No. 1272/2008 and its amendments.

Conclusions:

Moderate erythema (score 2), and moderate to severe edema (score 1-3) is determined. The irritant effects were reversible within 14 days. The substance is not a skin irritant, based on the results of the source substance.

Reference 2

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

Justification for type of information:

This information is used for read across to Cedryl Acetate EOA

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 a skin irritant

Remarks:

according to EU CLP (EC No. 1272/2008 and its amendments).

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).

Reference 3

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 April 2001 - 16 May 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The in vivo test was performed before the REACH regulation came into force, requesting in vitro studies. This information is used for read across to Cedryl Acetate EOA

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Version / remarks:

17 July 1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)

Version / remarks:

31 July 1992

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Elevage Scientifique des Dombes, F-01400 Chatillon sur Chalaronne, France
- Age at study initiation: 11 weeks
- Weight at study initiation: 2.1 - 2.2 kg
- Housing: Individually in stainless steel cages.
- Diet: Free access to pelleted standard Provimi Kliba 3418 rabbit maintenance diet (Provimi Kliba AG, CH-4303 Kaiseraugst).
- Water: Free access to community tap water from Füllinsdorf
- Acclimation period: no data.

ENVIRONMENTAL CONDITIONS (set to maintain)
- Temperature (°C): 20 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): 10 - 14
- Photoperiod (hrs dark / hrs light): 12/12

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

unchanged (no vehicle)

Controls:

other: Adjacent areas of the untreated skin of each animal served as controls.

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 0.5 mL

Duration of treatment / exposure:

4 hours

Observation period:

14 days

Number of animals:

1 male and 2 females

Details on study design:

TEST SITE:
Four days before treatment, the left flank was was clipped with an electric clipper, exposing an area of approximately 100 square centimeters (10x10 cm). The skin of the animals was examined one day before treatment, and regrown fur of all animals was again clipped.

TREATMENT
On the day of treatment, 0.5 mL of the test substance was places on a surgical gauze patch (ca. 2.5 cm x 2.5 cm). This gauze patch was applied to approx. 6 square centimeters of the intact skin of the clipped area. The patch was covered with a semi-occlusive dressing. The dressing was wrapped around the abdomen and enchored with tape.

REMOVAL OF TEST SUBSTANCE
After 4 hours the dressing was removed and the skin was flushed with lukewarm tap water to clean the application site.

OBSERVATIONS
- Mortality/Viability and Clinical signs: Daily from delivery of the animals to the termination of the test.
- Body Weight: At the start of acclimatization, on the day of application and at termination of observation.
- Irritation: The skin reactions were assessed at approximately 1, 24, 48 and 72 hours and at 7, 10 and 14 days after the removal of the dressings and test substance. The irritation scores and a description of all other (local) effects were recorded.

SCORING SYSTEM:
The irritation was assessed according to the numerical scoring system listed in the EEC Commission Directive 92/69/EEC, July 31, 1992.
If evident, corrosive or staining properties of the test substance were described and recorded.

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 14 days

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

1.67

Max. score:

4

Reversibility:

fully reversible within: 14 days

Irritation parameter:

erythema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

2.33

Max. score:

4

Reversibility:

fully reversible within: 14 days

Irritation parameter:

edema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

2.67

Max. score:

4

Reversibility:

fully reversible within: 7 days

Irritation parameter:

edema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 7 days

Irritation parameter:

edema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

1.67

Max. score:

4

Reversibility:

fully reversible within: 7 days

Irritant / corrosive response data:

At 1 hour, in one animal moderate erythema was seen; at the same time in 2/3 severe swelling was seen. At 24/48/72 hours well-defined erythema were seen in all animals with maximum score 2; at this time point 2/3 animals showed moderate to severe swelling with average score of 2.33 and one animal scored 1.33. All skin reactions were clear 14 days after treatment.

Other effects:

No clinical signs of systemic toxicity were observed in the animals during the study and no mortality occurred.
Scaling was evident at the test site of all animals from 7 to 10 days after treatment. In addition, the skin of one female was noted to be dry and inelastic at these readings.
No staining by the test substance of the treated skin was observed. No irreversible alterations of the treated skin were observed nor were corrosive effects evident on the skin.
The body weights of all animals were considered to be within the normal range of variability.

Interpretation of results:

other: Skin irritant Category 2

Remarks:

according to EU CLP (EC No. 1272/2008 and its amendments).

Conclusions:

In this skin irritation study with rabbits, performed according to OECD 404 guideline and GLP principles, moderate erythema (score 2) were seen and moderate to severe edema (score 1-3) were seen at 24/48 and 72hours. Two out of three animals have an average edema score of = 2.33, while the other animal scored an average of 1.33. The irritant effects were reversible within 14 days. This means that the substance is a skin irritant.

Executive summary:

The substance was tested for 4 hours in a skin irritation test in 3 rabbits according to OECD TG 404 test guideline and GLP principles.

No clinical signs of systemic toxicity were observed in the animals during the study and no mortality occurred. In this study moderate erythema (score 2) were seen and moderate to severe edema (score 1 -3) were seen at 24/48 and 72hours. Two out of three animals have an average edema score of = 2.33, while the other animal scored average of 1.33 for edema. The irritant effects were reversible within 14 days. This means that the substance is a skin irritant.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vivo

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2018

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Read-across information.

Justification for type of information:

The read across justification is presented in the Endpoint summary Irritation. The accompanying files are also attached there.

Reason / purpose for cross-reference:

read-across source

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

fully reversible

Remarks on result:

other: read-across from Cedryl Acetate mono'

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

fully reversible

Remarks on result:

other: read-across from Cedryl Acetate 'mono'

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

fully reversible

Remarks on result:

other: read-across from Cedryl Acetate 'mono'

Irritation parameter:

cornea opacity score

Basis:

animal #4

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

fully reversible

Remarks on result:

other: read-across from Cedryl Acetate 'mono'

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

fully reversible

Remarks on result:

other: read-across from Cedryl Acetate 'mono'

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

fully reversible

Remarks on result:

other: read-across from Cedryl Acetate 'mono'

Irritation parameter:

iris score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

fully reversible

Remarks on result:

other: read-across from Cedryl Acetate 'mono'

Irritation parameter:

iris score

Basis:

animal #4

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

fully reversible

Remarks on result:

other: read-across from Cedryl Acetate 'mono'

Irritation parameter:

conjunctivae score

Remarks:

(redness)

Basis:

animal #1

Time point:

24/48/72 h

Score:

1.33

Max. score:

3

Reversibility:

fully reversible

Remarks on result:

other: read-across from Cedryl Acetate 'mono'

Irritation parameter:

conjunctivae score

Remarks:

(redness)

Basis:

animal #2

Time point:

24/48/72 h

Score:

1.67

Max. score:

3

Reversibility:

fully reversible

Remarks on result:

other: read-across from Cedryl Acetate 'mono'

Irritation parameter:

conjunctivae score

Remarks:

(redness)

Basis:

animal #3

Time point:

24/48/72 h

Score:

1.33

Max. score:

3

Reversibility:

fully reversible

Remarks on result:

other: read-across from Cedryl Acetate 'mono'

Irritation parameter:

conjunctivae score

Remarks:

(redness)

Basis:

animal #4

Time point:

24/48/72 h

Score:

1

Max. score:

3

Reversibility:

fully reversible

Remarks on result:

other: read-across from Cedryl Acetate 'mono'

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible

Remarks on result:

other: read-across from Cedryl Acetate 'mono'

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible

Remarks on result:

other: read-across from Cedryl Acetate 'mono'

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible

Remarks on result:

other: read-across from Cedryl Acetate 'mono'

Irritation parameter:

chemosis score

Basis:

animal #4

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible

Remarks on result:

other: read-across from Cedryl Acetate 'mono'

Interpretation of results:

other: Not an eye irritant

Remarks:

according to Regulation (EC) No. 1272/2008 and its amendments.

Conclusions:

No effects were observed on the cornea and iris, whereas some minor effects were noted on the conjunctiva in the first days. All effects were reversible. The substance is not an eye irritant, based on the results of the source substance.

Reference 2

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

Justification for type of information:

This information is used for read across to Cedryl Acetate EOA

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 #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

fully reversible

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

fully reversible

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

fully reversible

Irritation parameter:

cornea opacity score

Basis:

animal #4

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

fully reversible

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

2

Reversibility:

fully reversible

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

fully reversible

Irritation parameter:

iris score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

fully reversible

Irritation parameter:

iris score

Basis:

animal #4

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

fully reversible

Irritation parameter:

conjunctivae score

Remarks:

(redness)

Basis:

animal #1

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 #3

Time point:

24/48/72 h

Score:

1.33

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

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible

Irritation parameter:

chemosis score

Basis:

animal #2

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

Irritation parameter:

chemosis score

Basis:

animal #4

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 an eye irritant

Remarks:

according to Regulation (EC) No. 1272/2008 and its amendments.

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.

Reference 3

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23-12-2016 to 08-02-2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

This information is used for read across to Cedryl Acetate EOA

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

GLP compliance:

yes (incl. QA statement)

Remarks:

Triskelion B.V., Utrechtseweg 48, 3700 AV, Zeist

Species:

other: Eyes of male or female chickens (ROSS, spring chickens)

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Slaughterhouse v.d. Bor, Nijkerkerveen, The Netherlands
- Characteristics of donor animals: Approximately 7 weeks old, male or female chickens, body weight range approximately 1.5-2.5 kg, were used as eye donors.
- Storage, temperature and transport conditions of ocular tissue: Heads of the animals were cut off immediately after sedation of the animals by electric shock and incision of the neck for bleeding, and before they reached the next station on the process line. The heads were placed in small plastic boxes on a bedding of paper tissues moistened with isotonic saline. Next, they were transported to the testing facility. During transportation, the heads were kept at ambient temperature.
- Time interval prior to initiating testing: Within 2 hours after kill, eyes were carefully dissected and placed in a superfusion apparatus.
- Indication of any existing defects or lesions in ocular tissue samples: No
- Indication of any antibiotics used: No

Vehicle:

unchanged (no vehicle)

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES
Within 2 hours after kill, eyes were carefully dissected and placed in a superfusion apparatus using the following procedure: First the eye-lids were carefully removed without damaging the cornea and a small drop of Fluorescein sodium 2.0% w/v was applied to the corneal surface for a few seconds and subsequently rinsed off with isotonic saline at ambient temperature. Next, the head with the fluorescein-treated cornea was examined with a slit-lamp microscope (Slit-lamp 900 BP, Haag-Streit AG, Liebefeld-Bern, Switzerland) to ensure that the cornea was not damaged. If undamaged (e.g., fluorescein retention and corneal opacity scores of ≤ 0.5), the eye was further dissected from the head without damaging the eye or cornea. Care was taken to remove the eye-ball from the orbit without cutting off the optical nerve too short. The enucleated eye was placed in a stainless steel clamp with the cornea positioned vertically and transferred to a chamber of the superfusion apparatus. The clamp holding the eye was positioned in such a way that the entire cornea was supplied with isotonic saline from a bent, stainless steel tube, at a target rate of 0.10-0.15 mL/min. The chambers of the superfusion apparatus as well as the saline were temperature controlled at approximately 32 °C (water pump set at 36.4 °C). After placing in the superfusion apparatus, the eyes were examined again with the slit-lamp microscope to ensure that they were not damaged. An accurate measurement was taken at the corneal apex of each eye. Eyes with a corneal thickness deviating more than 10% of the average corneal thickness of the eyes, eyes showing opacity (score higher than 0.5), or were unacceptably stained with fluorescein (score higher than 0.5) indicating the cornea to be permeable, or eyes that showed any other signs of damage, were rejected as test eyes and replaced.

EQUILIBRATION AND BASELINE RECORDINGS
Each eye provided its own baseline values for corneal swelling, corneal opacity and fluorescein retention. For that purpose, after an equilibration period of 45-60 minutes, the corneal thickness of the eyes was measured again to determine the zero reference value for corneal swelling calculations.

NUMBER OF REPLICATES
Negative control: 1
Positive control: 3
Test group: 3

NEGATIVE CONTROL USED
Physiological saline

POSITIVE CONTROL USED
Benzalkonium Chloride 5%

APPLICATION DOSE AND EXPOSURE TIME
30 μL for 10 seconds

OBSERVATION PERIOD
240 minutes

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: 20 mL saline. After rinsing, each eye in the holder was returned to its chamber.
- Indicate any deviation from test procedure in the Guideline: none

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Slit-lamp microscope examination
- Damage to epithelium based on fluorescein retention: Slit-lamp microscope examination
- Swelling: measured with optical pachymeter on a slit-lamp microscope; slit-width setting: set at 0.095 mm
- Others: After the final examination, the test substance treated eyes, the negative and positive control eyes were preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde. The corneas were embedded in paraffin wax, sectioned at ca 4 μm and stained with PAS (Periodic Acid-Schiff). The microscopic slides were subjected to histopathological examination.

SCORING SYSTEM:
Defined scoring scales were used for each parameter to define the severity of effects into four categories (I-IV).
- Mean corneal swelling (%): According to OECD 438 guideline. Examination of the eyes after 0, 30, 75, 120, 180, and 240 minutes
- Mean maximum opacity score: According to OECD 438 guideline. Examination of the eyes after 0, 30, 75, 120, 180, and 240 minutes
- Mean fluorescein retention score at 30 minutes post-treatment: According to OECD 438 guideline.

DECISION CRITERIA: According to OECD 438 guideline

Irritation parameter:

percent corneal swelling

Run / experiment:

slit-lamp examination

Value:

3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: maximum mean values

Irritation parameter:

cornea opacity score

Run / experiment:

slit-lamp examination

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: maximum mean values

Irritation parameter:

fluorescein retention score

Run / experiment:

slit-lamp examination

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Slit-lamp examination: The test substance caused no or very slight corneal swelling (mean of 3%). The negative control eye did not show any corneal effect and demonstrated that the general conditions during the tests were adequate. The positive control BAC 5% caused severe corneal effects and demonstrated the ICE test valid to detect severe eye irritants.

Microscopic examination of the corneas treated with the test substance did not reveal any abnormalities. Microscopic examination of the cornea treated with the negative control (saline) did not reveal any abnormalities. Microscopic examination of the corneas treated with the positive control BAC 5% revealed slight, moderate or severe erosion and very slight or slight vacuolation of the epithelium and the epithelium partly detached from the basement membrane.

Interpretation of results:

other: Not an eye irritant

Remarks:

according to Regulation (EC) No. 1272/2008 and its amendments.

Conclusions:

The test substance caused no or very slight corneal swelling. Microscopic examination of the corneas did not reveal any abnormalities.

Executive summary:

In accordance to OECD guideline 438 and GLP, the test substance was examined for its in vitro eye irritating potential using the Isolated Chicken Eye (ICE) Test. In the ICE test, 3 eyes were exposed to 30 µL test substance for 10 seconds. In addition, one negative control eye (30 µL saline) and three positive control eyes (30 µL Benzalkonium Chloride (BAC)) were tested. After the exposure the eyes were rinsed with 20 mL saline and were examined at approximately 0, 30, 75, 120, 180, and 240 minutes after treatment. The test substance caused no or very slight corneal swelling (mean of 3%). The negative control eye did not show any corneal effect and demonstrated that the general conditions during the tests were adequate. The positive control BAC 5% caused severe corneal effects and demonstrated the ICE test valid to detect severe eye irritants. Microscopic examination of the corneas treated with the test substance did not reveal any abnormalities. Microscopic examination of the cornea treated with the negative control (saline) did not reveal any abnormalities. Microscopic examination of the corneas treated with the positive control BAC 5% revealed slight, moderate or severe erosion and very slight or slight vacuolation of the epithelium and the epithelium partly detached from the basement membrane. Based on these results, the test substance is considered to be not eye irritating.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Additional information

Corrosion and Irritation to skin and eye is assessed based on read-across from Cedryl Acetate 'mono' and Longifolene Coeur. The executive summaries of the source information is presented below followed by the read-across rationale.

Skin irritation of Cedryl Acetate 'mono'

Cedryl Acetate 'mono' 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.

Skin irritation of Longifolene Coeur

Longifolene Coeur was tested for 4 hours in a skin irritation test in 3 rabbits according to OECD TG 404 test guideline and GLP principles. No clinical signs of systemic toxicity were observed in the animals during the study and no mortality occurred. In this study moderate erythema (score 2) were seen and moderate to severe edema (score 1 -3) were seen at 24/48 and 72hours. Two out of three animals have an average edema score of = 2.33, while the other animal scored average of 1.33 for edema. The irritant effects were reversible within 14 days. This means that the substance is a skin irritant.

Eye irritation of Cedryl Acetate 'mono'

Cedryl Acetate 'mono' undiluted, was tested in an eye irritation test in four female rabbits, in accordance with OECD TG 405 (1981) and under GLP conditions. No effects were observed on the cornea and iris. Minimal effects were seen in the conjunctivae and slight chemosis was seen all scores were below 2 during 24 to 72 hours. All effects were reversible. Based on these scores the substance is not an eye irritant.

Eye irritation Longifolene Coeur

Longifolene Coeur was examined for its in vitro eye irritating potential using the Isolated Chicken Eye (ICE) Test in accordance to OECD guideline 438 and GLP. In the ICE test, 3 eyes were exposed to 30 µL test substance for 10 seconds. In addition, one negative control eye (30 µL saline) and three positive control eyes (30 µL Benzalkonium Chloride (BAC)) were tested. After the exposure the eyes were rinsed with 20 mL saline and were examined at approximately 0, 30, 75, 120, 180, and 240 minutes after treatment. The test substance caused no or very slight corneal swelling (mean of 3%). The negative control eye did not show any corneal effect and demonstrated that the general conditions during the tests were adequate. The positive control BAC 5% caused severe corneal effects and demonstrated the ICE test valid to detect severe eye irritants. Microscopic examination of the corneas treated with the test substance did not reveal any abnormalities. Microscopic examination of the cornea treated with the negative control (saline) did not reveal any abnormalities. Microscopic examination of the corneas treated with the positive control BAC 5% revealed slight, moderate or severe erosion and very slight or slight vacuolation of the epithelium and the epithelium partly detached from the basement membrane. Based on these results, the test substance is considered to be not eye irritating.

Skin and eye irritation of Cedryl Acetate EOA based on read across from data available for Cedryl Acetate ‘mono’ (CAS #77-54-3) and Longifolene Coeur (CAS #475-20-7)

 

Introduction and hypothesis for the analogue approach         

Cedryl acetate EOA consists of one major, two minor constituents and a number of impurities, all containing a hydrocarbon fused-ring system.Half of the constituents have an acetate attached to this ring the other half does not have this ester group.

For Cedryl Acetate EOA there are no data on skin and eye irritation available.In accordance with Article 13 of REACH, lacking information can be generated by i.e. applying alternative methods such QSARs, grouping and read-across.For assessing the skin and eye irritation of Cedryl Acetate EOA, the analogue approach is selected because for one of its constituents, Cedryl Acetate ‘mono’, and for one close structural analogue, Longifolene Coeur, skin and eye irritation data is available which can be used for read across.

Hypothesis:Cedryl acetate EOA has the same skin irritation properties as Longifolene Coeur and has the same and eye irritating properties as Cedryl acetate ‘mono’ and Longifolene Coeur.

Available information:

Skin irritation/corrosion: For Cedryl Acetate ‘mono’ in vitro data is available from a study according to OECD TG 439 (Rel. 1). In this study, the substance was concluded to be not a skin irritant. For Longifolene, in vivo data is available from a study according to OECD TG 404 (Rel. 1). In this study, the substance was concluded to be a skin irritant Cat. 2.

Eye irritation: For Cedryl Acetate ‘mono’ in vivo data is available from a study according to OECD TG 405 (Rel. 1). In this study, the substance was concluded to be not an eye irritant. For Longifolene in vitro data is available from a study according to OECD TG 438 (Rel. 1). In this study, the substance was concluded not to be an eye irritant.

Target chemical and source chemical(s)

Chemical structures of the target chemical and the source chemical(s) are shown in the data matrix, including physico-chemical properties and available toxicologicalinformation.

Purity / Impurities

The unidentified impurities of Cedryl Acetate EOA are not considered to have a significant influence on the mutagenic potential.

Analogue approach justification

According to Annex XI 1.5 read across can be used to replace testing when the similarity can be based on a common backbone and a common functional group. When using read across the result derived should be applicable for C&L and/or risk assessment and it should be presented with adequate and reliable documentation.

Analogue selection: For Cedryl Acetate EOA,Cedryl Acetate ‘mono’ was selected as an analogue because it is its key constituent of Cedryl Acetate EOA: it has the same backbone and an acetate moiety similar to half its constituents. Longifolene Coeur was selected as an analogue because it has a similar backbone but no acetate functionality and covers the other half of the Cedryl Acetate EOA constituents.

Structural similarities and differences: The Cedryl Acetate EOA components all have a hydrocarbon fused-ring system. The source Cedryl Acetate ‘mono’ represents the subgroup of Cedryl Acetate EOA with the acetate group present, while Longifolene Coeur represents the one with a hydrocarbon backbone with only a double bond as functional group. The different positions of the acetate and double bond are considered to have minimal influence on the reactivity of these groups.

Bioavailability: The source chemicals and the target chemical subgroups are expected to have similar skin and eye tissue absorption based on the similarity in chemical structure and physico-chemical properties. All constituents have low (estimated) water solubility and high (estimated) log Kow values >5 and therefore bioavailability is expected to be limited.

Reactivity: The reactivity of Cedryl Acetate EOA and its analogues are considered similar in view of the similar functional groups. The acetate bond is not showing additional reactivity because Longifolene Coeur was a skin irritant in an OECD TG 404 while Cedryl Acetate is not. This may be due to different type of testing: in vivo versus in vitro, respectively.

Uncertainty of the prediction: The skin irritation results of both analogues are not alike. For the risk assessment the conservative result will be selected: a skin irritant Cat 2. This means that this uncertainty is covered. There are no other remaining uncertainties other than those discussed above. 

Data matrix

The relevant information on physico-chemical properties and toxicological characteristics are presented in the data matrix below.

Conclusions on irritation for hazard and risk assessment

For Cedryl Acetate EOA no skin and eye irritation information is available. Read across is used to fill this data gap. When using read across the result derived should be applicable for C&L and/or risk assessment and be presented with adequate and reliable documentation. In the present document the read across is justified. For skin irritation Longifolene Coeur is used for read across, resulting in skin irritation for Cedryl Acetate EOA. For eye irritation both Cedryl Acetate ‘mono’ and Longifolene Coeur are not eye irritants and therefore Cedryl Acetate EOA is not considered an eye irritant either.

Final conclusion: Cedryl Acetate EOA is a skin irritant Cat 2 and not an eye irritant.

 

Data matrix to support the read across to Cedryl acetate EOA from Cedryl acetate and Longifolene Coeur for skin and eye irritation

Common names	Cedryl Acetate EOA							Cedryl acetate	Longifolene Coeur
	Target	Target	Target	Target	Target	Target	Target	Source	Supporting source
	Cedryl Acetate type			Longifolene type
Chemical structures
Typical concentration (%)	30-45 (major)	<10	<10	15-30 (minor)	8-16 (minor)	<10	<10
CAS no				32435-95-3	22567-43-7			77-54-3	475-20-7
Einecs	944-520-4							201-036-1	207-491-2
REACH		2018						2018	2018
Molecular weight	264	264	264	204	204	204	202	264	204
Phys-Chem data*
Log Kow	5.33	5.33	5.94	6.12	5.74	5.82	6.19	5.33 (6 exp.)	5.48 (5 exp.)
Human health
Skin irritation	Skin irritation Cat 2 (Read across)	Skin irritation Cat 2 (Read across)	Skin irritation Cat 2 (Read across)	Skin irritation Cat 2 (Read across)	Skin irritation Cat 2 (Read across)	Skin irritation Cat 2 (RA)	Skin irritation Cat 2 (RA)	No skin irritation (OECD TG 439)	Skin irritation Cat 2 (OECD TG 404)
Eye irritation	No eye irritation (Read across)	No eye irritation (Read across)	No eye irritation (Read across)	No eye irritation (Read across)	No eye irritation (Read across)	No eye irritation (RA)	No eye irritation (RA)	No eye irritation (OECD TG 405 )	No eye irritation (OECD TG 438)

*Physico-chemical data are based on EpiSuite; RA=Read across

 

Justification for classification or non-classification

Based on the results, the substance needs to be classified as Skin irritant Category 2, and shall be labelled with 'H315: Causes skin irritation', according to EU CLP (EC No. 1272/2008 and its amendments).

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