7-methyl-3-methyleneocta-1,6-diene

Endpoint summary

Description of key information

Less than 50% viable cells (25.9 ± 2.6 %) were obtained after exposure to myrcene in the in vitro human epidermis model EPISKIN.

In an in vivo eye irritation GLP study conducted according to the OECD Guideline 405, mean individual scores at 24, 48 and 72 h of 3 animals after myrcene exposure were 0, 0, 0 for cornea score; 0, 0, 0 for iris score; 2, 1.7, 2 for conjunctivae score and 2, 1, 1 for chemosis score.

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:

from June 22 to July 27, 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study well conducted and in compliance with GLP.

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: ECVAM protocol version 1.8 of february 2009

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Species:

human

Details on test animals or test system and environmental conditions:

Three-dimensional human epidermis model, supplied by SkinEthic Laboratories, Nice, France constituted by:
- a collagen type I matrix, coated with type IV collagen
- a differentiated and stratified epidermis model from human keratinocytes, obtained after 13-day culture period.
All biological components of the epidermis and the kit culture medium have been tested for the absence of viruses, bacteria and mycoplasma

Type of coverage:

open

Vehicle:

unchanged (no vehicle)

Controls:

other: not applicable

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 10 µL

Duration of treatment / exposure:

15 ± 0.5 min

Number of animals:

3 epidermis/product

Details on study design:

Before the beginning of the study, the non-specific MTT reduction by the test item was checked by incubating MTT solution and 10 µL test item for 3 hours ± 30 minutes and checking the colour of the solution.

Application of the test item and control:
- negative control: 10 µL of PBS+ to each epidermis surface
- positive control: 10 µL of SDS solution at 5% (w/v) in distilled water; after 7 minutes contact time, an intermediate re-spreading is done
- test substance: 10 µL of the test item, as supplied
- contact timepoint for all products: 15 ± 0.5 minutes at room temperature

Rinsing:
- epidermis rinsed thoroughly with 25 mL of PBS+
- remaining product removed with a cotton-bud

Incubation: plates incubated during 42 ± 1 hours in CO2 incubator with maintenance medium

MTT assay:
- epidermis incubated for 3 hours ± 5 minutes in MTT solution in the CO2 incubator
- formazan extraction in 500 µL of acidic isopropanol stored at 4 hours ± 30 minutes at room temperature, protected from light or during 70 ± 5 hours at 4 °C
- measurement of OD at 570 nm

Irritation / corrosion parameter:

other: other: viability % (MTT assay)

Value:

25.9

Remarks on result:

other:

Remarks:

Basis: mean. Time point: 15 min. Reversibility: no data. Remarks: ± 2.6. (migrated information)

Irritant / corrosive response data:

No data

Other effects:

No data

Negative control (PBS+): mean OD = 0.821

Positive control (5% SDS): % viability (MTT assay) = 18.7 ± 3.0

Test item: % viability (MTT assay) = 25.9 ± 2.6

Table 1: MTT conversion assay in living epidermis

		O.D. 1	O.D. 2	Mean	Standard deviation	Viability %	Mean Viability %	Standard deviation
Negative control	Epidermis 1	0.768	0.808	0.788	0.028	96.0	100	0.044
	Epidermis 2	0.855	0.864	0.860	0.006	104.7
	Epidermis 3	0.819	0.811	0.815	0.006	99.3
Positive control	Epidermis 1	0.124	0.145	0.135	0.015	16.4	18.7	0.030
	Epidermis 2	0.158	0.131	0.145	0.019	17.6
	Epidermis 3	0.191	0.172	0.182	0.013	22.1
Test item	Epidermis 1	0.195	0.206	0.201	0.008	24.4	25.9	0.026
	Epidermis 2	0.207	0.268	0.238	0.043	28.9
	Epidermis 3	0.198	0.202	0.200	0.003	24.4

Interpretation of results:

irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The percentage of viability is below 50%. Therefore, myrcene is classified as skin irritant, R38, according to Directive 67/548/EEC and in category 2 according to CLP Regulation (EC) N° 1272/2008.

Executive summary:

A GLP study conducted in vitro with human epidermis model EPISKIN to assess the irritancy potential of myrcene (similarly to ECVAM protocol version 1.8 of February 2009).

10 µL of the test item was applied directly on epidermis for 15 min on 3 epidermis. Positive control was 10 µL of 5% (w/v) SDS solution and negative control was 10 µL of PBS (each tested on 3 epidermis). MTT conversion assay was peformed to evaluate the percentage of cellular viability of the epidermis.

Positive control had a percentage of viability of 18.7± 3.0 and test item 25.9 ± 2.6.

As the percentage of vaibility is below 50%, the test item is considered as irritating.

Therefore, myrcene is classified as skin irritant, R38, according to Directive 67/548/EEC and in category 2 according to CLP Regulation (EC) N° 1272 /2008.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (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:

From May 10 to 25, 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted in compliance with OECD guideline 405 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:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Elevage de Gerome, Quartier Labaste, France
- Age at study initiation: 11, 13 or 14 weeks
- Weight at study initiation: 2.29-3.56 kg
- Housing: Each animal housed in an individual box
- Diet (e.g. ad libitum): Foodstuff (SDS - C15); ad libitum
- Water (e.g. ad libitum): Tap water (public distribution system); ad libitum
- Acclimation period: Five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17-23 °C
- Humidity (%): 30-70%
- Air changes (per hr): 15/hour
- Photoperiod (hrs dark / hrs light): 12 hours dark / 12 hours light

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

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

Duration of treatment / exposure:

No washing was done

Observation period (in vivo):

Eight days

Number of animals or in vitro replicates:

Three males

Details on study design:

SCORING SYSTEM:

Chemosis
No swelling …0
Slight swelling, including the nictitating membrane…1
Swelling with eversion of the eyelid...2
Swelling with eyelid half-closed…3
Swelling with eyelid more than half-closed…4

Discharge
No discharge…0
Slight discharge (normal slight secretion in the inner corner not to be taken into account)…1
Discharge with moistening of the eyelid and neighboring hairs…2
Discharge with moistening of the eyelids and large areas around the eye…3

Redness
Blood vessel normal…0
Vessels significantly more prominent than normal…1
Vessels individually distinguishably with difficulty
Generally red coloration….2
Generally deep red coloration….3

Iris
Normal…0
Iris significantly more wrinkled than normal, congestion, swelling of the iris which continues to react to light, even slowly…1
No reaction to light, haemorrhage, significant damage (any or all of these characteristics)…2

Cornea: degree of opacity
No modification visible either directly or after instillation of fluorescein (no loss of glint or polish)…0
Translucent areas (diffuse or disseminated), iris details clearly visible…1
Early identifiable translucent area, iris details slightly obscured…2
Opalescent area, no iris details visible, pupil outline scarcely distinguishable…3
Total corneal opacity, completely obscuring the iris and pupil…4

Cornea: extent of opacity
Opaque area present but covering one quarter or less…1
Between one quarter and half…2
Between half and three quarters…3
Between three quarters and the entire surface…4

Irritation parameter:

chemosis score

Basis:

animal #1

Remarks:

mean individual score

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 7 days

Irritation parameter:

conjunctivae score

Basis:

animal #1

Remarks:

mean individual score

Time point:

24/48/72 h

Score:

2

Max. score:

3

Reversibility:

fully reversible within: 8 days

Irritation parameter:

iris score

Basis:

animal #1

Remarks:

mean individual score

Time point:

24/48/72 h

Score:

0

Max. score:

2

Reversibility:

other: not applicable

Irritation parameter:

cornea opacity score

Basis:

animal #1

Remarks:

mean individual score

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: not applicable

Irritation parameter:

chemosis score

Basis:

animal #2

Remarks:

mean individual score

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 4 days

Irritation parameter:

conjunctivae score

Basis:

animal #2

Remarks:

mean individual score

Time point:

24/48/72 h

Score:

1.66

Max. score:

3

Reversibility:

fully reversible within: 8 days

Irritation parameter:

iris score

Basis:

animal #2

Remarks:

mean individual score

Time point:

24/48/72 h

Score:

0

Max. score:

2

Reversibility:

other: not applicable

Irritation parameter:

cornea opacity score

Basis:

animal #2

Remarks:

mean individual score

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: not applicable

Irritation parameter:

chemosis score

Basis:

animal #3

Remarks:

mean individual score

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 4 days

Irritation parameter:

conjunctivae score

Basis:

animal #3

Remarks:

mean individual score

Time point:

24/48/72 h

Score:

2

Max. score:

3

Reversibility:

fully reversible within: 8 days

Irritation parameter:

iris score

Basis:

animal #3

Remarks:

mean individual score

Time point:

24/48/72 h

Score:

0

Max. score:

2

Reversibility:

other: not applicable

Irritation parameter:

cornea opacity score

Basis:

animal #3

Remarks:

mean individual score

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: not applicable

Irritant / corrosive response data:

- Moderate redness of the conjunctivae associated with slight to severe chemosis was noted after 1 hour of instillation; irritation completely resolved within 8 days.
- See table 1 for more details

Other effects:

No data

Table 1: Individual and mean scores of conjunctivae, iris and cornea

 

Animal	Time after treatment	Conjunctivae		Iris	Cornea
		Chemosis	Redness	Lesion	Opacity
1	1 hour	2	2	0	0
	24 hours	2	2	0	0
	48 hours	2	2	0	0
	72 hours	2	2	0	0
	Day 4	2	2	0	0
	Day 7	0	1	0	0
	Day 8	0	0	0	0
	Mean (24, 48 and 72 hours)	2	2	0	0
2	1 hour	1	2	0	0
	24 hours	1	2	0	0
	48 hours	1	2	0	0
	72 hours	1	1	0	0
	Day 4	0	1	0	0
	Day 7	0	1	0	0
	Day 8	0	0	0	0
	Mean (24, 48 and 72 hours)	1	1.66	0	0
3	1 hour	3	2	0	0
	24 hours	1	2	0	0
	48 hours	1	2	0	0
	72 hours	1	2	0	0
	Day 4	0	1	0	0
	Day 7	0	1	0	0
	Day 8	0	0	0	0
	Mean (24, 48 and 72 hours)	1	2	0	0

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

Instillation of myrcene resulted in moderate redness of the conjunctivae associated with slight to severe chemosis in all treated animals after 1 hour of instillation. The irritation completely resolved within 8 days.
Myrcene is classified as ‘Irritating to eyes (Category 2)’ according to CLP Regulation (EC) N° 1272/2008 but not classified according to Directive 67/548/EEC.

Executive summary:

In an eye irritation study conducted according to Guideline OECD 405 and in compliance with GLP, three male rabbits of the New Zealand White strain were exposed to 0.1 mL of undiluted myrcene in one of the eyes while the other eye corresponded to the control. The eyes were examined for irritation scores at 1 hour and 1, 2, 3, 4, 7 and 8 days after dosing.

 

Instillation of myrcene resulted in moderate redness of the conjunctivae associated with slight to severe chemosis in all treated animals after 1 hour of instillation. The irritation completely resolved within 8 days. Mean individual scores at 24, 48 and 72 hours after exposure for the 3 animals were 0, 0, 0 for cornea score; 0, 0, 0 for iris score; 2, 1.7, 2 for conjunctivae score and 2, 1, 1 for chemosis score.

 

Therefore, myrcene is classified as ‘Irritating to eyes (Category 2)’ according to CLP Regulation (EC) N° 1272/2008 but not classified according to Directive 67/548/EEC.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Additional information

In a GLP study conducted similarly to ECVAM protocol version 1.8 of February 2009 with human epidermis model EPISKIN in vitro, MTT conversion showed less than 50% viable cells (25.9 ± 2.6 %); therefore myrcene was considered to be skin irritant. Myrcene is classified as irritating to skin, R38, according to Directive 67/548/EEC and in category 2 according to CLP Regulation (EC) N° 1272/2008/).

In an in vivo eye irritation study conducted according to the OECD Guideline 405 and in compliance with GLP, mean individual scores at 24, 48 and 72 hours after exposure for the 3 animals were 0, 0, 0 for cornea score; 0, 0, 0 for iris score; 2, 1.7, 2 for conjunctivae score and 2, 1, 1 for chemosis score. Myrcene was therefore found to be eye irritant (Category 2) according to CLP Regulation (EC) N°1272 /2008 but not classified according to Directive 67/548/EEC.

Justification for classification or non-classification

As MTT conversion showed less than 50% viable cells in in vitro human epidermis model EPISKIN, myrcene was considered as skin irritant.

In an in vivo eye irritation study conducted according to the OECD Guideline 405 and in compliance with GLP, a mean individual (24, 48 and 72 h) conjunctivae score of 2 was found in 2 animals, leading to category 2 classification according to CLP Regulation (EC) N° (1272-2008) but not classified according to the criteria of Annex VI to the Directive 67/548/EEC. However, as the CLP Regulation is the most recent classification, myrcene is considered as eye irritating and classified category 2 according to the criteria of this classification.