4-(1-methoxy-1-methylethyl)-1-methylcyclohexene

Endpoint summary

Description of key information

Skin corrosion: Not corrosive based on absence of eye irritation in rabbit eye and absence of irritant effects at 100% application in guinea pig (skin sensitisation).

Skin irritation: Skin irritant (OECD TG 439): Irritant
Eye irritation (OECD TG 405): Not irritating
Respiratory irritation (based on absence of human data indicating such effects, absence of effects in long-term inhalation study and absence of skin corrosion and severy eye irritation): Not a respiratory irritant.

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:

20 April, 2015 - 22 May, 2015

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:

(2013)

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:

non-transformed keratinocytes

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Small ModelTM
- Supplier : SkinEthic Laboratories, Lyon, France
- EpiSkinTM Tissues (0.38cm2) lot number: 15-EKIN-020

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

Test material
- Applied volume: 25 μL into 12-well plates on top of the skin tissues

Duration of treatment / exposure:

15 min

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean compared to the negative control

Value:

7.2

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with Orange Flower Ether compared to the negative control tissues was 7.2%. Since the mean relative tissue viability for Orange Flower Ether was below 50% it is considered to be irritant.

Direct MTT Reduction

Orange Flower Ether was checked for colour interference in aqueous conditions and possible direct MTT reduction by adding the test substance to MTT medium. Because no colour changes were observed it was concluded that Orange Flower Ether did not interact with the MTT endpoint.

 

Test Item, Positive Control Item and Negative Control Item

The relative mean viability of the test item treated tissues was 7.2 % after a 15 -minute exposure period and 42 hours post-exposure incubation period.

 

Quality Criteria

The relative mean tissue viability for the positive control treated tissues was 24% relative to the negative control treated tissues.The positive control acceptance criterion was therefore satisfied.

The mean OD570 for the negative control treated tissues was 0.957 and the standard deviation was 0.023. The negative control acceptance criterion was therefore satisfied. The standard deviation value of the percentage viability of three tissues treated identically was less than 5%, indicating that the test system functioned properly.

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	0.983	0.957	0.023		100
	0.946
	0.940
Positive Control Item	0.271	0.226	0.057	28	24
	0.245			26
	0.162			17
Test Item	0.060	0.069	0.010	6.1	7.2
	0.066			7.0
	0.080			8.5

SD = Standard deviation

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

Interpretation of results:

other: Criteria for Cat 2 are met

Remarks:

in accordance with EU CLP (1272/2008 and its updates)

Conclusions:

The relative mean tissue viability after 15 minutes treatment with Orange Flower Ether compared to the negative control tissue was 7.2%. Since the mean relative tissue viability for the substance was below 50% after 15 minutes treatment, the substance is considered to be irritant. The substance needs to be classified as Cat 2 and Cat 2A for EU CLP and GHS, respectively.

Executive summary:

The skin irritation potential of the test substance was tested 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. The positive control had a mean cell viability of 24% 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 7.2%. Since the mean relative tissue viability for the test substance was below 50% after 15 minutes treatment the substance is considered to be irritant. Based on these results the substance needs to be classified as Cat 2 and Cat 2A for EU CLP and GHS, respectively.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vivo

Remarks:

See justification of information

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 February 1980 - 27 February 1980

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Justification for type of information:

An in vitro eye irritation was not conducted because information from an OECD TG 405 was availabel carried out before 2016.

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
- Weight at study initiation: 2.0 – 3.5 kg
- Housing: singly under standard laboratory conditions (as specified in “guide for the care and use of laboratory animals” DHEW Publication No. 78-23, Revised 1978). The suspended pans beneath the cage were littered with prepared bedding (Easi Litter)
- Diet: 100 g Purina rabbit Chow supplemented by whole oats daily
- Water: ad libitum

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

Amount applied (volume or weight with unit): 0.1 mL

Duration of treatment / exposure:

Single instillation to the right eye

Observation period (in vivo):

7 days

Number of animals or in vitro replicates:

6

Details on study design:

REMOVAL OF TEST SUBSTANCE: substance was not removed

SCORING SYSTEM: according to the Method of Draize at 24, 48 and 72 hours and at 4 and 7 days after dosings.

TOOL USED TO ASSESS SCORE: fluorescein

Irritation parameter:

cornea opacity score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

iris score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

conjunctivae score

Basis:

animal: #1, #2, #3

Time point:

24/48/72 h

Score:

0.66

Max. score:

3

Reversibility:

fully reversible within: 72 hours

Irritation parameter:

conjunctivae score

Basis:

animal: #4, #6

Time point:

24/48/72 h

Score:

0.33

Max. score:

3

Reversibility:

fully reversible within: 48 hours

Irritation parameter:

conjunctivae score

Basis:

animal #5

Time point:

24/48/72 h

Score:

0

Max. score:

3

Irritation parameter:

chemosis score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

4

Interpretation of results:

other: criteria are not met

Remarks:

according to EU CLP (1272/2008 and its updates)

Conclusions:

Under the test conditions the test substance is not considered to be an eye irritant in accordance with EU CLP and GHS criteria.

Executive summary:

To determine the eye irritation potential 0.1 mL of the test substance was instilled once into the right eye of each of six New Zealand White rabbits. Both eyes were examined before testing, at 24, 48 and 72 hours and at 4 and 7 days. The irritation was scored by the method of Draize. The corneal opacity, iritis and chemosis scores were determined to be 0 for all animals. The conjunctival redness scores were determined to be 0.66, 0.66, 0.66, 0.33, 0, and 0.33 for animal 1, 2, 3, 4, 5, and 6, respectively. Based on these scores the test substance was considered not to be irritating to the eyes in accordance with EU CLP and GHS criteria..

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Additional information

Skin corrosion: Skin corrosion is not anticipated because the substance is not an eye irritant and did not show irritation at 100% in a guinea pig Buehler test.

Skin irritation: The possible skin irritation potential of the test substance was tested 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. The positive control had a mean cell viability of 24% 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 7.2%. Since the mean relative tissue viability for the test substance was below 50% after 15 minutes treatment the substance is considered to be irritant. Based on these results the substance needs to be classified as Cat 2 and 2A in accordance with EU CLP and GHS, respectively.

Eye irritation OECD TG 405

To determine the eye irritation potential 0.1 mL of the test substance was instilled once into the right eye of each of six New Zealand White rabbits. Both eyes were examined before testing, at 24, 48 and 72 hours and at 4 and 7 days. The irritation was scored by the method of Draize. The corneal opacity, iritis and chemosis scores were determined to be 0 for all animals. The conjunctival redness scores were determined to be 0.66, 0.66, 0.66, 0.33, 0, and 0.33 for animal 1, 2, 3, 4, 5, and 6, respectively. Based on these scores the test substance was considered not to be irritating to the eyes.Based on these results the substance does not need to be classified according the EU CLP and GHS.

Respiratory irritation

For assessing respiratory irritation human data are key for assessing this endpoint. Animal data may be used to indicate effects (ECHA guidance, R7.2.12.1, 2015). No human data are available that indicate respiratory effects. In a 90-day inhalation study with an analogue Terpineol-multi no respiratory irritation was seen. In addition, the substance is not corrosive or a severe eye irritant and therefore respiratory irritation is not anticipated either (ECHA guidance, R7.2.13.1, 2015).

Justification for classification or non-classification

Skin corrosion: The substance does not need to be classified for corrosion because no corrosion is anticipated based on e.g. absence of eye irritation and absenc of skin irritation in a guinea pig Buehler test in accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation No. 1272/2008 and its updates.

Skin irritation: Based on the positive results in the skin irritation test the substance needs to be classified as a skin irritant. According to EU Classification, Labeling and Packaging of Substances and Mixtures (CLP) Regulation No. 1272/2008 and its updates: this results in skin irritation, Category 2, H315: Causes skin irritation.

Eye irritation: Based on the negative results in the eye irritation tests classification for eye irritation is not warranted in accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation No. 1272/2008 and its updates.

Respiratory irritation: The substance does not need to be classified for respiratory irritation in absence of human data and other data in accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation No. 1272/2008 and its updates.