2-methylcyclohexyl acetate

Endpoint summary

Description of key information

Skin irritation/corrosion: Key study: Test method OECD 439. GLP study. The relative mean tissue viability obtained after 15 minutes treatment with 2-methylcyclohexylacetate compared to the negative control tissues was 88% and was considered to be non-irritant to the skin.

Eye irritation/corrosion: Key study: Test method OECD 437. GLP study. The mean in vitro irritancy score was 11 after 10 minutes of treatment with 2-methylcyclohexyl acetate and was considered to be not severe irritant or corrosive to the eye.

Key study: Test method OECD 405. GLP study. Instillation of 0.1 mL of 2-methylcyclohexylacetate into eyes of rabbits resulted in effects on the iris and conjunctivae. 2-methylcyclohexyl acetate was determined to be classified as irritating to eyes (category 2) according to CLP Regulation (EC) No 1272/2008.

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:

April 2, 2012 - April 29, 2012.

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Cell type:

non-transformed keratinocytes

Details on animal used as source of test system:

EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2, Batch no: 12-EKIN-016). This model is a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes. The keratinocytes were cultured for 13 days, which results in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

Justification for test system used:

In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).

Vehicle:

unchanged (no vehicle)

Details on test system:

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37.0 ºC
- Temperature of post-treatment incubation (if applicable): 37.0 ºC
REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: After the exposure period of 15 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test substance. 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.
- Observable damage in the tissue due to washing: none
DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader.
- Wavelength: 570 nm
NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION: 3

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 μl

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 25 μl

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 25 μl
- Concentration (if solution): 5% (aq) Sodium dodecyl sulphate

Duration of treatment / exposure:

15 min

Duration of post-treatment incubation (if applicable):

44 h

Number of replicates:

3

Type of coverage:

open

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean

Value:

>= 87.4 - <= 88.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100 ± 18

Positive controls validity:

valid

Remarks:

4 ± 4

Other effects / acceptance of results:

The relative mean tissue viability obtained after 15 minutes treatment with 2-methylcyclohexylacetate compared to the negative control tissues was 88%. Since the mean relative tissue viability for 2-methylcyclohexylacetate was above 50% 2-methylcyclohexyl acetate is considered to be non-irritant.

Mean tissue viability (percentage of control):

	A	B	C	Mean ± SD
Negative control	79	111	110	100 ± 18
2-methylcyclohexyl acetate	88	93	82	88 ± 6
Positive control	5	3	4	4 ± 4

Mean absorption:

	A	B	C	Mean ± SD
Negative control	0.614	0.864	0.861	0.780 ± 0.143
2-methylcyclohexyl acetate	0.686	0.728	0.638	0.684 ± 0.045
Positive control	0.040	0.026	0.032	0.033 ± 0.007

 

OD = optical density

SD = Standard deviation

Triplicate exposures are indicated by A, B and C.

Acceptability of the assay:

The absolute mean OD570 (optical density at 570 nm) of the three tissues of the negative control were within the laboratory historical control data range and the Standard Deviation value (SD) of the % viability was <18.

The mean relative tissue viability of the positive control was <40% relative to the negative control and the Standard Deviation value (SD) of the % viability should be was <18.

The SD calculated from individual % tissue viabilities of the three identically treated replicates was <18.

According to the technical data, safety sheet and certificate of analysis of the reconstructed human epidermis (data available from Skinethic), used skin met all acceptability criteria.

Interpretation of results:

not irritating

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

2-methylcyclohexyl acetate was determined to be non-irritant in the in vitro skin irritation test on a human three dimensional epidermal model (EPISKIN Small Model).

Executive summary:

An in-vitro skin irritation test was performed on 2 -methylcyclohexyl acetate in accordance with OECD Guideline 439. 2-methylcyclohexylacetate was applied undiluted (25 μl) directly on top of the skin tissue (human three dimensional epidermal model (EPISKIN Small Model)) for 15 minutes. After a post-incubation period of approximately 44 hours, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment. Skin irritation is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 15 minutes treatment with 2-methylcyclohexylacetate compared to the negative control tissues was 88%. Since the mean relative tissue viability was above 50% after 15 minutes treatment, 2-methylcyclohexylacetate was considered to be non-irritant.

According to the technical data, safety sheet and certificate of analysis of the reconstructed human epidermis (data available from Skinethic), used skin met all acceptability criteria.

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:

June 8, 2012 - June 28, 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Kisslegg, Germany
- Age at study initiation: at least 6 weeks old
- Weight at study initiation: at least 1.0 kg
- Housing: Individually, in cages with perforated floors (dimensions 67 x 62 x 55 cm) and shelters (dimensions 40 x 32 x 23 cm).
- Diet (e.g. ad libitum): Ad libitum.
- Water (e.g. ad libitum): Ad libitum.
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 24°C
- Humidity (%): 40 - 70%
- Air changes (per hr): 15 room air changes/hour
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 mL

Duration of treatment / exposure:

24h

Observation period (in vivo):

7 days

Number of animals or in vitro replicates:

3

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): The eyes were not rinsed with tap water since no residual test substance was present.

SCORING SYSTEM: Grading of ocular lesions according to OECD 405 and EU method B.5.

TOOL USED TO ASSESS SCORE: fluorescein

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

other: mean 24-72 h

Score:

0

Max. score:

4

Reversibility:

other: not applicable

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

other: mean 24-72 h

Score:

0

Max. score:

4

Reversibility:

other: not applicable

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

other: mean 24-72 h

Score:

0

Max. score:

4

Reversibility:

other: not applicable

Irritation parameter:

iris score

Basis:

animal #1

Time point:

other: mean 24-72 h

Score:

0

Max. score:

2

Reversibility:

other: not applicable

Irritation parameter:

iris score

Basis:

animal #2

Time point:

other: mean 24-72 h

Score:

0

Max. score:

2

Reversibility:

other: not applicable

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

other: mean 24-72 h

Score:

3

Max. score:

3

Reversibility:

fully reversible within: 7 days

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

other: mean 24-72 h

Score:

2

Max. score:

3

Reversibility:

fully reversible within: 7 days

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

other: mean 24-72 h

Score:

1.7

Max. score:

3

Reversibility:

fully reversible within: 7 days

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

other: mean 24-72 h

Score:

1.3

Max. score:

4

Reversibility:

fully reversible within: 7 days

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

other: mean 24-72 h

Score:

0.3

Max. score:

4

Reversibility:

fully reversible within: 72 hours

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

other: mean 24-72 h

Score:

0.3

Max. score:

4

Reversibility:

fully reversible within: 72 hours

Irritant / corrosive response data:

Instillation of 0.1 mL of 2-methylcyclohexylacetate into one eye of each of three rabbits resulted in effects on the iris and conjunctivae. Iridial irritation grade 1 was observed in two animals, one hour after instillation only. The irritation of the conjunctivae consisted of redness, chemosis and discharge and completely resolved within 7 Days in all animals.

Other effects:

- Ophthalmoscopic findings: No staining of (peri) ocular tissues by the test substance was observed and no test substance remnants were seen. There was no evidence of ocular corrosion.
- Other observations: No symptoms of systemic toxicity were observed in the animals during the test period and no mortality occurred.

Mean value eye irritation scores:

Animal	Mean 24, 48 and 72 hours
	Corneal opacity	Iris	Conjunctivae
			Redness	Chemosis
294	0.0	0.0	3.0	1.3
293	0.0	0.0	2.0	0.3
296	0.0	0.0	1.7	0.3

Interpretation of results:

irritating

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

Instillation of 0.1 mL of 2-methylcyclohexylacetate into one eye of each of three rabbits resulted in effects on the iris and conjunctivae. The test substance was determined to be classified as irritating to eyes (category 2) according to CLP Regulation (EC) No 1272. 2008.

Executive summary:

An acute eye irritation/corrosion study was performed on 2 -methylcyclohexyl acetate in accordance with OECD Guideline 405. Single samples of 0.1 mL of 2-methylcyclohexylacetate were instilled into one eye of each of three rabbits. Observations were made 1, 24, 48 and 72 hours and 7 days after instillation. Iridial irritation grade 1 was observed in two animals, only one hour after instillation (fully reversible within 24 hours). The irritation of the conjunctivae consisted of redness, chemosis and discharge and completely resolved within 7 Days in all animals. No corneal opacity was observed and there was no evidence of ocular corrosion. No symptoms of systemic toxicity were observed in the animals during the test period and no mortality occurred. Based on these results, 2 -methylcyclohexyl acetate was determined to be classified as irritating to eyes (category 2) according to CLP Regulation (EC) No 1272/2008.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Additional information

Skin irritation/corrosion:

Key study: An in-vitro skin irritation test was performed on 2 -methylcyclohexyl acetate in accordance with OECD Guideline 439. 2-methylcyclohexylacetate was applied undiluted (25 μl) directly on top of the three skin tissues (human three dimensional epidermal model (EPISKIN Small Model)) for 15 minutes. Negative control (phosphate buffered saline) and positive control (5% (aq) sodium dodecyl sulphate) were used as reference. The relative mean tissue viability obtained after 15 minutes treatment compared to the negative control tissues was 88% and therefore, 2-methylcyclohexylacetate was considered to be non-irritant to the skin.

Eye irritation/corrosion:

Key study: The screening for the eye irritancy potential of 2-methylcyclohexyl acetate using the Bovine Corneal Opacity and Permeability test (BCOP test) was performed in accordance with OECD Guideline 437. The test substance was applied undiluted (750 μl) directly on top of three corneas for 10 minutes. Negative controls (physiological saline) and positive controls (10% w/v Benzalkonium Chloride) were used as reference. The mean in vitro irritancy score was 11 after 10 minutes of treatment with 2-methylcyclohexyl acetate, and therefore the test item was considered to be not severe irritant or corrosive to the eye.

Key study: An acute eye irritation/corrosion study was performed on 2 -methylcyclohexyl acetate in accordance with OECD Guideline 405. Single samples of 0.1 mL of 2-methylcyclohexylacetate were instilled into one eye of each of three rabbits. Observations were made 1, 24, 48 and 72 hours and 7 days after instillation. Iridial irritation grade 1 was observed in two animals, only one hour after instillation (fully reversible within 24 hours). The irritation of the conjunctivae consisted of redness, chemosis and discharge and completely resolved within 7 Days in all animals. 2 -methylcyclohexyl acetate was determined to be classified as irritating to eyes (category 2) according to CLP Regulation (EC) No 1272/2008.

Justification for selection of skin irritation / corrosion endpoint:

Only one study available.

Justification for selection of eye irritation endpoint:

Only one in-vivo study is available.

Effects on eye irritation: irritating

Justification for classification or non-classification

Skin irritation/corrosion: Based on the available data (OECD 439), 2 -methylcyclohexyl acetate is not classified for skin irritation in accordance with CLP Regulation (EC) No 1272/2008.

Eye irritation/corrosion. Based on the available data (OECD 437 and OECD 405), 2 -methylcyclohexyl acetate is classified as irritating to eyes (category 2) according to CLP regulation (EC) No 1272/2008, since the test item produced at least in 2 of 3 tested animals a positive response of conjunctival redness ≥ 2 (calculated as the mean scores following grading at 24, 48 and 72 hours after installation of the test material and fully reversible within an observation period of 21 days).