Bicyclo[3.1.1]hept-2-ene-2-ethanol, 6,6-dimethyl-, 2-acetate, (1R,5S)-

Endpoint summary

Description of key information

In an in vivo skin irritation study (similar to OECD Guideline No 404), the overall mean scores obtained with nopl acetate at 24, 48 and 72 h were 1.2 and 0.9 for erythema and oedema, respectively.
In a BCOP test (Bovine Corneal Opacity and Permeability) performed according to OECD Guideline No 437, negative results were obtained with nopyl acetate. In an in vitro eye irritation study on human reconstructed corneal epithelium model, the exposure time to nopyl acetate that caused 50% of cell mortality was 141.01 min.

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:

experimental study

Adequacy of study:

key study

Study period:

01-30 March 1977

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Study similar to OECD Guideline No 404 with deviations: no data about purity and no certificate of analysis of the test substance; no details on test animals and environmental conditions; animals were not observed for 14 days to study the reversibility; individual animal weights not reported; different irritation scoring system

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

yes

Remarks:

no data about purity and no certificate of analysis; no details on test animals and environmental conditions; reversibility of irritation and systemic effects not followed; individual animal weights not reported; different irritation scoring system

Principles of method if other than guideline:

not applicable

GLP compliance:

no

Remarks:

pre-dating GLP regulation

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
Age at study initiation: 9-12 weeks

Type of coverage:

semiocclusive

Preparation of test site:

other: clipped

Vehicle:

unchanged (no vehicle)

Controls:

other: cyclamen aldehyde and diethyl phthalate were used as standards for comparison

Amount / concentration applied:

TEST MATERIAL
Amount(s) applied (weight with unit): 0.5 mL
Concentration (if solution): 100 %

Duration of treatment / exposure:

4 h

Observation period:

72 h

Number of animals:

8

Details on study design:

TEST SITE
Area of exposure: dorsum of rabbits.
Type of wrap if used: occlusive patches were prepared by heat-sealing 1" x 1" 24 ply gauze pads on to 1.25" x 1.25" squares of polythene sheeting, which was then attached to 3.5" x 1" strips of adhesive tape.

REMOVAL OF TEST SUBSTANCE
After removal of the patches, the treated site was wiped to remove the residual material.

SCORING SYSTEM:
Treated sites were scored for erythema, oedema, cracking and scaling and any other feature using an 8-point anchored ordinate scale ranging from "a" (very slight) to "h" (severe). For better interpretation, an equivalence with OECD guideline 404 scoring system was made (see table 7.3.1/3).

Irritation parameter:

erythema score

Basis:

mean

Remarks:

(8 animals)

Time point:

24/48/72 h

Score:

1.2

Max. score:

4

Reversibility:

no data

Irritation parameter:

edema score

Basis:

mean

Remarks:

(8 animals)

Time point:

24/48/72 h

Score:

0.9

Max. score:

4

Reversibility:

no data

Irritant / corrosive response data:

Nopyl acetate produced marginal to slight oedema and erythema, and slight to fairly distinct cracking and scaling.

Overall level of irritation was less than that of the control, cyclamen aldehyde, which produced fairly distinct to fairly well-developed erythema, fairly distinct to distinct oedema and slight cracking and scaling.

Response to nopyl acetate was greater than that produced by the control, diethyl phthalate, which produced only a marginal response.

Other effects:

none

Table 7.3.1/1: equivalence in scoring systems

Study scaling	OECD 404 scaling
a: marginal/very slight b: slight	1
c: fairly distinct d: quite distinct	2
e: becoming well developed f: well developed	3
g: becoming severe h: severe	4

Table 7.3.1/2: irritation scores

Animal number	4 h		24 h		48 h		72 h
	E	O	E	O	E	O	E	O
118	a	a	a	b	b	a	a	a
138	b	a	b	a	b	b	a	a
141	a	a	a	-	a	-	a	a
140	a	a	a	a	b	b	a	b
142	a	-	a	-	b	-	b	-
143	a	a	a	a	a	a	a	a
144	a	a	c	b	c	c	b	b
132	b	b	c	c	c	b	c	b

Table 7.3.1/3: equivalence in OECD 404 scaling

Animal number	4 h		24 h		48 h		72 h		Individual means at 24-48-72 h
	E	O	E	O	E	O	E	O	E	O
118	1	1	1	1	1	1	1	1	1	1
138	1	1	1	1	1	1	1	1	1	1
141	1	1	1	0	1	0	1	1	1	0.3
140	1	1	1	1	1	1	1	1	1	1
142	1	0	1	0	1	0	1	0	1	0
143	1	1	1	1	1	1	1	1	1	1
144	1	1	2	1	2	2	1	1	1.7	1.3
132	1	1	2	2	2	1	2	1	2	1.3
					Overall mean				1.2	0.9

E: erythema

O: oedema

Interpretation of results:

not classified

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Nopyl acetate induced only slight skin irritation in rabbits. As overall mean scores for erythema and oedema were lower than 2, nopyl acetate is not classified for skin irritation/corrosion according to Directive 67/548/EEC and CLP Regulation (EC) No 1272/2008.

Executive summary:

In a primary dermal irritation study (similar to OECD Guideline No 404), eight New Zealand White rabbits were dermally exposed to 0.5 mL of nopyl acetate (100 %) under a semi-occlusive dressing for 4 h. After removal of the residual test item, treated sites were scored for erythema, oedema, cracking and scaling and any other feature using an 8-point anchored ordinate scale ranging from very slight to severe at 0, 24, 48 and 72 h after exposure. Cyclamen aldehyde and diethyl phthalate were used as standards for comparison.

 

Nopyl acetate produced marginal to slight oedema and erythema, and slight to fairly distinct cracking and scaling. Overall level of irritation was less than that of the control, cyclamen aldehyde, which produced fairly distinct to fairly well-developed erythema, fairly distinct to distinct oedema and slight cracking and scaling. Response to nopyl acetate was greater than that produced by the control, diethyl phthalate, which produced only a marginal response. When using an equivalence between this scoring scale and the OECD 404 scoring system, the overall mean scores at 24, 48 and 72 h were 1.2 and 0.9 for erythema and oedema, respectively.

 

Nopyl acetate induced only slight skin irritation in rabbits. As overall mean scores for erythema and oedema were lower than 2, nopyl acetate is not classified for skin irritation/corrosion according to Directive 67/548/EEC and CLP Regulation (EC) No 1272/2008.

Reference 2

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1972

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Study similar to OECD Guideline No 402 with deviations: no data about purity and no test substance certificate of analysis ; no data on age, gender and source of animals; no data on housing and environmental conditions; observation period: 7 days instead of 14; performed on abraded skin.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

equivalent or similar to guideline

Guideline:

other: 402 (Acute dermal toxicity)

Deviations:

yes

Remarks:

no data about purity and no certificate of analysis of test substance; no data on age, gender and source of animals; no data on housing and environmental conditions; observation period: 7 days; performed on abraded skin

Principles of method if other than guideline:

not applicable

GLP compliance:

no

Remarks:

pre-dating GLP regulation

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
Weight at study initiation: 1.9-2.4 kg

Controls:

not specified

Amount / concentration applied:

2000 mg/kg bw
- Area of exposure: clipped abraded abdominal skin
- Type of wrap if used: wrapped with binders of rubber dam, gauze and adhesive tape

Duration of treatment / exposure:

24 h

Observation period:

7 days

Number of animals:

10

Details on study design:

Mortality and toxic effects were observed for a period of 7 days.
Necropsy of survivors performed: yes
Other examinations performed:dermal reactions: erythema, edema and atonia

Irritation parameter:

other: see irritant/corrosive response data

Basis:

other: see irritant/corrosive response data

Time point:

24/48/72 h

Remarks on result:

no indication of irritation

Remarks:

see irritant/corrosive response data

Irritant / corrosive response data:

Dermal reactions:
- slight erythema in 8, 4 and 1 animal on Days 1, 2 and 3, respectively; moderate erythema in 1 animal on Day 1
- slight edema in 8 and 4 animals on Days 1 and 2, respectively; moderate edema in 1 animal on Day 1
- complete recovery within 4 days
- no signs of atonia were observed during the study

Other effects:

no data

no data

Conclusions:

In an acute dermal toxicity study (limit test), which is a worse case of exposure to skin irritation, only few rabbits showed slight signs of erythema and edema. These effects were totally reversed within 4 days.
Therefore, nopyl acetate induced only slight and reversible skin irritation and is not classified for skin corrosion/irritation according to Directive 67/548/EEC and CLP Regulation (EC) No 1272/2008.

Executive summary:

In an acute dermal toxicity study (limit test), a group of 10 New Zealand White rabbits were administered a single dermal dose of nopyl acetate at 2000 mg/kg bw on clipped abraded abdominal skin using an occlusive patch for 24 h. Animals were then observed for mortality, clinical signs, bodyweights and dermal reactions for 7 days and were all macroscopically necropsied after sacrifice.

 

No deaths occurred throughout the study. Normal body weight gain was observed in all animals. At necropsy, macroscopic examination of main organs showed no abnormalities. Dermal reactions noted were slight to moderate erythema and edema, which completely recovered to normal within four days.

Only few rabbits showed slight signs of erythema and edema which were totally reversed within 4 days. Therefore, nopyl acetate induced only slight and reversible skin irritation and is not classified for skin corrosion/irritation according to Directive 67/548/EEC and CLP Regulation (EC) No 1272/2008.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 May 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted in compliance with OECD Guideline 437 without any deviation.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 437

Deviations:

no

Principles of method if other than guideline:

not applicable

GLP compliance:

yes (incl. QA statement)

Species:

other: bovine eye

Strain:

not specified

Details on test animals or tissues and environmental conditions:

TEST SYSTEM
Bovine eyes (from cattle less than 12 months old) collected at the slaughterhouses of La Talaudière - France (42350) and carried in a stable medium (Hanks medium buffered with sodium bicarbonate), at room temperature, and used within 4 h after killing the animals.

Vehicle:

unchanged (no vehicle)

Controls:

other: yes, 2 groups (positive and negative controls) containing 3 corneas each.

Amount / concentration applied:

TEST MATERIAL
Amount(s) applied (volume or weight with unit): 750 ± 8 µL

Concentrations:
- test material: undiluted
- negative control: 0.9 % (w/v) sodium chloride
- positive control: 10 % (w/v) sodium hydroxide

Duration of treatment / exposure:

10 ± 1 min

Observation period (in vivo):

Corneal opacity was measured after 2 h ± 10 min of incubation.
Corneal permeability was measured after 90 ± 5 min of incubation.

Number of animals or in vitro replicates:

Total: 9 corneas (3 corneas/group)

Details on study design:

Details of test procedure: bovine eyes recovered from a slaughterhouse were inspected and undamaged corneas were dissected and mounted in specially constructed BCOP corneal holders. After 1 h incubation in media, the basal opacity of each cornea was recorded using an opacitometer. 750 ± 8 µL of the test item and of each control were applied directly on the cornea using a positive-displacement micropipette and incubated in a water-bath at 32 ± 1 °C, in a vertical position (screw upward) for 10 ± 1 min. Before reading the final opacity, the corneas were rinsed and incubated for 2 h ± 10 min in fresh media to equilibrate. Then the permeability of each cornea was determined with a fluorescein solution after an incubation of 90 ± 5 min. An aliquot (360 µL) of the media from the posterior compartment was read in a plate reader at 490 nm to determine the permeability of the cornea to the fluorescein solution. The opacity and permeability values were combined to obtain an in vitro irritancy score (IVIS).

Incubation temperature: 32 ± 1 °C

Calibration information for devices used for measuring opacity and permeability (e.g., opacitometer and spectrophotometer): opacitometer (OPKIT) was calibrated with specific calibrators before each measurement of the corneal opacity. Values of opacity after three calibrations were 73-77, 147-153 and 220-240, respectively.

Controls: 0.9 % (w/v) sodium chloride and 10 % (w/v) sodium hydroxide were used as negative and positive controls, respectively.

Irritation parameter:

in vitro irritation score

Run / experiment:

1

Value:

ca. -1.6

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Run / experiment:

2

Value:

ca. -3.5

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Run / experiment:

3

Value:

ca. -4.3

Negative controls validity:

valid

Positive controls validity:

valid

Table 7.3.2/1: Opacity and optical density

Treatment	Opacity				Optical density		IVIS
	Before treatment (OPT0)	After 2 h (OPT2)	Difference (OPT2-OPT0)	Corrected	OD	Corrected	Score
Negative control	1	8	7		0.037
	0	3	3		0.018
	1	5	4		0.034
Mean				4.7	0.03
Standard deviation				2.1	0.01
10 % (w/v) NaOH	3	126	123	118.3	7.848	7.818	235.6
	4	130	126	121.3	7.888	7.858	239.2
	1	96	95	90.3	7.864	7.834	207.8
Mean				110.0	7.837		227.6
Standard deviation				17.1	0.020		17.2
Test item	0	3	3	-1.7	0.034	0.004	-1.6
	1	2	1	-3.7	0.038	0.008	-3.5
	3	3	0	-4.7	0.051	0.021	-4.3
Mean				-3.3	0.011		-3.2
Standard deviation				1.5	0.009		1.4

In vitro irritancy score (IVIS) = (OPT2-OPT0) + (15 x OD)

O.D. of the fluorescein solution (8 µg/mL): 1.490

Interpretation of results:

not classified

Remarks:

Migrated information (IVIS<55.1) Criteria used for interpretation of results: EU

Conclusions:

IVIS of nopyl acetate was 0.0. Therefore it is not classified as corrosive or severe irritant for the isolated bovine cornea, after a 10 min contact period.

Executive summary:

In an in vitro eye irritation study performed according to OECD Guideline No 437 and in compliance with GLP, 750 ± 8 µL of nopyl acetate were applied directly to isolated bovine corneas (3 corneas/group), mounted in specially constructed BCOP corneal holders, and incubated in a water-bath at 32 ± 1°C for 10 ± 1 min. Before reading the final opacity, the corneas were rinsed and incubated for 2 h in fresh media to equilibrate. Then the permeability of each cornea was determined with a fluorescein solution after an incubation of 90 min. An aliquot (360 µL) of the media from posterior compartment was read in a plate reader at 490 nm to determine the permeability of the cornea to the fluorescein solution. The opacity and permeability values were combined to obtain an in vitro irritancy score (IVIS). 0.9% (w/v) sodium chloride and 10% (w/v) sodium hydroxide were used as negative and positive controls, respectively.

The IVIS of the test item and positive control were 0.0 and 227.6 ± 17.2 respectively. Optical density values for the negative control corneas (nutritive medium) and fluorescein solution (8 µg/mL) were within the desired range and thus confirmed the validity of the test.

IVIS of nopyl acetate is 0.0. Therefore it is not classified as corrosive or severe irritant for the isolated bovine cornea, after 10 min of contact.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15-16 May 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted in accordance with generally accepted scientific standards and described in sufficient details.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

no guideline available

Principles of method if other than guideline:

Evaluation of the primary ocular irritation after application of test item on a reconstructed human corneal epithelium model by quantification of cellular viability by MTT reduction test (Mosmann, T. 1983) and determination of exposure time that causes 50% of cell mortality (T50).

GLP compliance:

yes (incl. QA statement)

Species:

human

Strain:

not specified

Details on test animals or tissues and environmental conditions:

not applicable

Vehicle:

unchanged (no vehicle)

Controls:

other: negative control: sodium chloride (0.9 % w/v) in sterile water; positive control: 1.5 % (w/w) SDS in sterile water

Amount / concentration applied:

TEST MATERIAL
Amount(s) applied (volume or weight with unit): 30 µL

Concentrations:
- test material undiluted
- negative control: sodium chloride (0.9 % w/v)
- positive control: 1.5 % (w/w) SDS solution

Duration of treatment / exposure:

Test item: 10 ± 2 min, 1 h ± 10 min and 3 h ± 30 min
Positive control: 10 ± 2 min and 1 h ± 10 min
Negative control: 3 h ± 30 min

Observation period (in vivo):

Post-exposure period: approximately 2 h

Number of animals or in vitro replicates:

Test item, negative control and positive control were tested in duplicate.

Details on study design:

TEST SYSTEM:
Cell system used: Human reconstructed corneal epithelium model of 0.5 cm² (5 days of culture). When cultivated at the air-liquid interface in a chemically defined medium, the immortalized human corneal epithelial cells from the cell line HCE (Human Corneal Epithelial cells) reconstruct a corneal epithelial tissue (mucosa), devoided of stratum corneum, ultra-structurally (tissue morphology and thickness) similar to the corneal mucosa of the human eye.
Source: SkinEthic Laboratories (Lyon, France)

CONTROLS AND REPLICATES:
Negative control: sodium chloride at 0.9% w/v in sterile water.
Positive control: sodium dodecyl sulfate at 1.5% w/w in sterile water.
The test item and the negative and positive controls were tested in duplicate.

METHODOLOGY:
- Treatment
Upon arrival the epithelia were transferred to new maintenance medium, 500 µL per well in 24-well plates, and incubated at 37°C ± 1°C in a CO2 incubator (5 ± 1% CO2, 95 ± 5% humidity). The medium was renewed 24 hours later (500 µL per well) and 30 µL of the test material or 30 µL of each control were topically applied on the tissue surface.
The tissues treated with the test item were incubated in the CO2 incubator for 10 ± 2 min, 1 hour ± 10 min or 3 hours ± 30 min. The tissues treated with the positive contol were incubated for 10 ± 2 min or 1 hour ± 10 min. The tissues exposed to the negative control were incubated for 3 hours ± 30 min. Two epithelia were used for each treatment and exposure period.

- Viability measurement using the MTT assay
(MTT reduction into formazan by cellular enzymes)
At the end of the treatment period, the epithelia were rinsed with 10 mL of PBS+ (Phosphate Buffered Saline) and transferred into wells containing 500 µL of a MTT solution at 0.5 mg/mL [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]. The tissues were incubated for 1 hour ± 10 min in the CO2 incubator.
Each epithelium was then removed from the MTT solution, blotted on absorbent paper and transferred to isopropanol (1 mL per well - 24-well plates). After 1 hour ± 10 min of gentle stirring protected from light, 200 µL aliquots per well were transferred to 96-well plates before measuring optical density (OD) at 570 nm with isopropanol as the blank, using a microtiter plate reader.

- Expression of viability
Viability was expressed as % relative to the negative control: [test item mean OD/negative control mean OD] x 100. The exposure time that reduced cell viability to 50% of negative control level (T50) was determined by linear regression analysis.

- Non-specific MTT reduction
Before being assayed, the test item was tested for its potential interaction with the MTT reagent. 10 µL of the test item was added to 2 mL of a MTT solution at 0.3 mg/mL in each well of a 12-well plate. After mixing and incubation for 3 hours ± 5 min at 37°C ± 1°C (no coloration means no interaction between the test item and the MTT reagent).

Irritation parameter:

other: % viability

Run / experiment:

10 min

Value:

105

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

other: % vaibility

Run / experiment:

60 min

Value:

118

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

other: % viability

Run / experiment:

180 min

Value:

19

Negative controls validity:

valid

Positive controls validity:

valid

Table 7.3.2/2: Optical density readings and % viability

	Contact Timepoint (min.)	O.D. 1	O.D. 2	Mean O.D.	% viability
Negative control	180	1.179	1.125	1.152	100%
Positive control	10	1.093	1	1.047	91%
	60	0.054	0.03	0.042	4%
Test item	10	1.213	1.197	1.205	105%
	60	1.366	1.342	1.354	118%
	180	0.218	0.209	0.214	19%

O.D.: Optical density

Interpretation of results:

slightly irritating

Remarks:

Migrated information Criteria used for interpretation of results: other: See table 7.3.2/1

Conclusions:

The T50 value of nopyl acetate was found between 60 and 180 min and it was considered as slightly irritant when applied on a human reconstructed corneal epithelium. Therefore, without clear correspondance between the results of this test and common classification criteria, a conservative approach is adopted and nopyl acetate is classified as irritant "R36: irritating to eyes" and irritating to eyes Category 2 according to Directive 67/548/EEC and CLP Regulation (EC) No 1272/2008, respectively.

Executive summary:

In an in vitro eye irritation study performed in compliance with GLP, 30 µL of nopyl acetate were applied to human reconstructed corneal epithelium model (in duplicate) for 10 min ± 2 min, 1 h ± 10 min and 3 h ± 30 min at room temperature. Exposure time for negative control (sodium chloride at 0.9 % w/v) was 3 h ± 30 min at room temperature. Exposure times for positive control (SDS 1.5% w/w) were 10 min ± 2 min and 1 h ± 10 min. At the end of the treatment period, the epithelia were rinsed, transferred to well plates containing a MTT solution for 1 h ± 10 min. Each epithelium was then removed from the MTT solution and transferred to isopropanol (1 mL per well). After stirring for 1 h ± 10 min, aliquots (200 μL/well) were transferred to 96-well plates and optical density was recorded at 570 nm with a plate reader. For each treated tissue the viability was expressed as mean percentage of cellular viability relative to the negative control and the exposure time that reduced cell viability to 50% of negative control level (T₅₀) was determined by linear regression analysis.

T₅₀ value for the test item was 141.01 min. Optical density value for the negative control was 1.152 and T₅₀ value for positive control was 33.49 min and thus confirmed the validity of the test.

The T₅₀ value of nopyl acetate was between 60 and 180 min, thus it is slightly irritant when applied on a human reconstructed corneal epithelium. Therefore, without clear correspondance between the results of this test and common classification criteria, a conservative approach is adopted and nopyl acetate is classified "R36: irritating to eyes" according to Directive 67/548/EEC and irritating to eyes Category 2 according to CLP regulation (EC) No 1272/2008.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In a skin irritation study performed similarly to OECD 404 Guideline on eight New Zealand White rabbits, nopyl acetate produced marginal to fairly distinct erythema, marginal to slight oedema, and slight to fairly distinct cracking and scaling. When using an equivalence between this scoring scale and OECD 404 scoring system, the overall mean scores at 24, 48 and 72 h were 1.2 and 0.9 for erythema and oedema, respectively. The result was negative. There is no requirement to conduct additional skin irritation tests with nopyl acetate, as negative results were obtained in an appropriate test system.

In an in vitro eye irritation study performed according to OECD 437 Guideline and in compliance with GLP, 750 µL of nopyl acetate were applied directly to isolated bovine corneas (32°C for 10 min). The opacity and permeability values were combined to obtain an in vitro irritancy score (IVIS).

IVIS nopyl acetate = 0.0.

In an in vitro eye irritation study performed in compliance with GLP, 30 µL of nopyl acetate were applied to a human reconstructed corneal epithelium model (in duplicate) for 10 min, 1 h and 3 h at room temperature. For each treated tissue the viability was expressed as the mean percentage of cellular viability relative to the negative control and the exposure time that causes 50% of cell mortality (T50) was determined by linear regression analysis.

T50 nopyl acetate = 141.01 min. The substance was considered as slightly irritating to eyes.

Justification for classification or non-classification

In an in vivo skin irritation study (similar to OECD Guideline 404), nopyl acetate induced only slight skin irritation in rabbits. As the overall mean scores for erythema and oedema were lower than 2, nopyl acetate is not classified for skin irritation/corrosion according to Directive 67/548/EEC and CLP Regulation (EC) No. (1272-2008).

In an in vitro eye irritation study on a human reconstructed corneal epithelium model, the exposure time that caused 50% of cell mortality (T50) was 141.01 min. The T50 value of nopyl acetate was between 60 and 180 min therefore it was considered as slightly irritant. Without clear correspondance between results of this test and common classification criteria, a conservative approach is adopted and nopyl acetate is classified as irritant "R36: irritating to eyes" according to Directive 67/548/EEC and irritating to eyes Category 2 according to CLP Regulation (EC) No 1272/2008.