Reaction mass of (4R)-isopropenyl-1-methylcyclohexene and (4S)-isopropenyl-1-methylcyclohexene and 1-isopropyl-4-methylcyclohexa-1,3-diene and 1-isopropyl-4-methylcyclohexa-1,4-diene and 4-isopropylidene-1-methylcyclohexene

Endpoint summary

Description of key information

In in vivo studies conducted according to or similarly to OECD guideline 404 on d-limonene in rabbits, reversible signs of skin irritation were observed.

In an in vitro study conducted in accordance with OECD Guideline 492, REACTION MASS OF DL-LIMONENE, ALPHA- GAMMA- TERPINENES, TERPINOLENE was found not irritating to eyes.

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:

supporting study

Justification for type of information:

D-Limonene is one of the main constituents of multiconstituent substance REACTION MASS OF DL-LIMONENE, ALPHA- GAMMA- TERPINENES, TERPINOLENE. Therefore, data on d-limonene can be used for extrapolation to REACTION MASS OF DL-LIMONENE, ALPHA- GAMMA- TERPINENES, TERPINOLENE. See read-across justification document in section 13.

Reason / purpose for cross-reference:

read-across source

Irritation parameter:

erythema score

Basis:

mean

Remarks:

all animals

Time point:

other: 24, 48 and 72 h

Score:

1.92

Max. score:

4

Reversibility:

not fully reversible within: 7 days

Irritation parameter:

edema score

Basis:

mean

Remarks:

all animals

Time point:

other: 24, 48 and 72 h

Score:

1.33

Max. score:

4

Reversibility:

not fully reversible within: 7 days

Irritant / corrosive response data:

- Slight desquamation from skin surface was observed in 2/4 animals on Day 7
- See table 1 for individual animal scores

Other effects:

No data

Table 1: Skin irritation data for individual rabbits

 

Rabbits No.		#1*	#2	#3	#4*
Erythema	1 hour	1	1	0	2
	24 hours	2	2	1	2
	48 hours	2	2	2	2
	72 hours	2	2	2	2
	Day 7	2	1	2	2
Mean (24 hours + 48 hours + 72 hours)		2	2	1.67	2
Oedema	1 hour	4	3	3	3
	24 hours	2	1	2	2
	48 hours	1	1	1	2
	72 hours	1	0	1	2
	Day 7	1	0	2	2
Mean (24 hours + 48 hours + 72 hours)		1.33	0.67	1.33	2

* slight desquamation from skin surface after 7 days

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

d-limonene is classified as ‘R38 Irritating to skin’ according to Directive 67/548/EEC but it is not classified according to CLP Regulation (EC) No. 1272/2008.

Executive summary:

In a primary dermal irritation study performed according to OECD guideline 404 and in compliance with GLP, four albino rabbits were dermally exposed to 0.5 mL of undiluted d-limonene, under a semi-occlusive patch for 4 hours to intact skin of the flank. Animals were then observed for 7 days. Irritation was scored by the method of Draize at 1, 24, 48 and 72 hours and 7 days after exposure.

 

Mean individual scores at 24, 48 and 72 h after exposure for the 4 animals were 2, 2, 1.67, 2 for erythema score and 1.33, 0.67, 1.33, 2 for oedema score. Slight desquamation was observed in 2/4 animals on day 7. In this study, d-limonene was found to be skin irritant when applied topically to the rabbit.

 

Therefore, d-limonene is classified as ‘R38 Irritating to skin’ according to Directive 67/548/EEC but it is not classified according to CLP Regulation (EC) No. 1272 /2008.

Reference 2

Endpoint:

skin irritation: in vivo

Type of information:

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

Adequacy of study:

supporting study

Justification for type of information:

D-Limonene is one of the main constituents of multiconstituent substance REACTION MASS OF DL-LIMONENE, ALPHA- GAMMA- TERPINENES, TERPINOLENE. Therefore, data on d-limonene can be used for extrapolation to REACTION MASS OF DL-LIMONENE, ALPHA- GAMMA- TERPINENES, TERPINOLENE. See read-across justification document in section 13.

Reason / purpose for cross-reference:

read-across source

Irritation parameter:

erythema score

Basis:

animal #1

Remarks:

mean individual score

Time point:

other: 24, 48 and 72 h

Score:

2

Max. score:

4

Reversibility:

not fully reversible within: 7 days

Irritation parameter:

edema score

Basis:

animal #1

Remarks:

mean individual score

Time point:

other: 24, 48 and 72 h

Score:

1.33

Max. score:

4

Reversibility:

not fully reversible within: 7 days

Irritation parameter:

erythema score

Basis:

animal #2

Remarks:

mean individual score

Time point:

other: 24, 48 and 72 h

Score:

2

Max. score:

4

Reversibility:

not fully reversible within: 7 days

Irritation parameter:

edema score

Basis:

animal #2

Remarks:

mean individual score

Time point:

other: 24, 48 and 72 h

Score:

2

Max. score:

4

Reversibility:

not fully reversible within: 7 days

Irritation parameter:

erythema score

Basis:

animal #3

Remarks:

mean individual score

Time point:

other: 24, 48 and 72 h

Score:

2

Max. score:

4

Reversibility:

not fully reversible within: 7 days

Irritation parameter:

edema score

Basis:

animal #3

Remarks:

mean individual score

Time point:

other: 24, 48 and 72 h

Score:

1.33

Max. score:

4

Reversibility:

not fully reversible within: 7 days

Irritant / corrosive response data:

- Desquamation from skin surface was observed in all animals on Day 7
- See table 1 for individual animal scores

Other effects:

No data

Table 1: Skin irritation data for individual rabbits

 

Rabbits No.		1*	2**	3*
Erythema	1 hour	2	2	1
	24 hours	2	2	2
	48 hours	2	2	2
	72 hours	2	2	2
	Day 7	1	2	1
Mean (24 hours + 48 hours + 72 hours)		2	2	2
Oedema	1 hour	3	4	3
	24 hours	2	2	2
	48 hours	1	2	1
	72 hours	1	2	1
	Day 7	1	3	1
Mean (24 hours + 48 hours + 72 hours)		1.33	2	1.33

* marked desquamation from skin surface after 7 days

** desquamation from skin surface after 7 days

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

d-limonene is classified as ‘R38 Irritating to skin’ according to Directive 67/548/EEC but it is not classified according to CLP Regulation (EC) No. 1272/2008.

Executive summary:

In a primary dermal irritation study performed according to OECD guideline 404 and in compliance with GLP, three albino rabbits were dermally exposed to 0.5 mL of undiluted d-limonene, under a semi-occlusive patch for 4 hours to intact skin of the flank. Animals were then observed for 7 days. Irritation was scored by the method of Draize at 1, 24, 48 and 72 hours and 7 days after exposure.

 

Mean individual scores at 24, 48 and 72 h after exposure for the 3 animals were 2, 2, 2 for erythema score and 1.33, 2, 1.33 for oedema score. Signs of erythema and oedema, as well as desquamation from skin surface, were observed in all animals on Day 7. In this study, d-limonene was found to be skin irritant when applied topically to the rabbit.

 

Therefore, d-limonene is classified as ‘R38 Irritating to skin’ according to Directive 67/548/EEC but it is not classified according to CLP Regulation (EC) No. 1272/2008.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (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:

19 May 2016 - 02 June 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

other: reconstructed human Cornea-like Epithelium

Strain:

other: reconstructed human Cornea-like Epithelium

Details on test animals or tissues and environmental conditions:

EpiOcularTM OCL-212-ver2.0, supplied by MatTek Corporation

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

The test item was applied, as supplied, at the dose of 50 µL

Duration of treatment / exposure:

30 minutes

Duration of post- treatment incubation (in vitro):

A 12-minute post exposure immersion period at room temperature and 1 hour and 58 minures post exposure incubation at standard culture conditions.

Number of animals or in vitro replicates:

2 replicates

Irritation parameter:

other: mean percent tissue viability

Value:

76.69

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

percent tissue viability =34.89%

Irritant / corrosive response data:

REACTION MASS OF DL-LIMONENE, ALPHA-GAMMA-TERPINENES, TERPINOLENE IS CONSIDERED AS NON IRRITANT AND NON CORROSIVE.

Interpretation of results:

GHS criteria not met

Conclusions:

In accordance with Regulation EC No. 1272/2008, test item REACTION MASS OF DL-LIMONENE, ALPHA- GAMMA-TERPINENES, TERPINOLENE does not require classification for eye irritation or serious eye damage. No hazard statement and signal word are required.

Executive summary:

The aim of the study was to evaluate the eye hazard potential of test item REACTION MASS OF DL-LIMONENE, ALPHA- GAMMA-TERPINENES, TERPINOLENE after topical administration on in vitro reconstructed human cornea-like epithelium tissues (EpiOcular^TMtissue model).

 

Test item REACTION MASS OF DL-LIMONENE, ALPHA- GAMMA-TERPINENES, TERPINOLENE was applied, as supplied, at the dose of 50 µL, to 2 PBS pre-treated RhCE (EpiOcular^TMtissue model) during 30 minutes at 37°C, 5% CO₂, 95% humidity (standard culture conditions). The exposure period was followed by extensive rinsing with PBS at room temperature, a 12 minutes post-exposure immersion period at room temperature and a 1 -hour and 56 -minute post-exposure incubation at standard culture conditions. The tissue viability was measured by performing a MTT assay. The experimental protocol was established in accordance with OECD Guideline No. 492 adopted 28 July 2015.

 

The mean percent tissue viability of the RhCE replicates treated with test item REACTION MASS OF DL-LIMONENE, ALPHA- GAMMA-TERPINENES, TERPINOLENE was 76.69%, versus 34.89% in the positive control (Methyl acetate).

 

Therefore, in accordance with Regulation EC No. 1272/2008, test item REACTION MASS OF DL-LIMONENE, ALPHA- GAMMA-TERPINENES, TERPINOLENE does not require classification for eye irritation or serious eye damage. No hazard statement and signal word are required.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

29-30 January 2013

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: the result does not allow to classify the substance according to CLP/GHS criteria

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
- Concentration: 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), from immortalized human corneal epithelial cells from the cell line HCE (Human Corneal Epithelial cells), cultivated in chemically defined medium
- Source: SKINETHIC Laboratories (Lyon, France)

METHODOLOGY:
- Non-specific MTT reduction: 10 µL of the test item was added to each well of a 12-wells plate containing 2 mL of MTT solution (0.3 mg/mL) and colour of the solution was checked after incubation for 3 h ± 5 min at 37 ± 1 °C, 5 ± 1 % CO2, 95 ± 5 % humidity (CO2 incubator).
- MTT conversion assay: The epithelial units were transferred from the kit into maintenance medium filled wells and pre-incubated at 37 ± 1 °C until the following day. After pre-incubation, 30 µL of test material, in duplicate, were applied to the epithelium model for 10 ± 2 min, 1 h ± 10 min and 3 h ± 30 min at room temperature. Contact timepoint for negative control was 3 h ± 30 min and for positive controls were 10 ± 2 min and 1 h ± 10 min. At the end of each incubation period, each epithelium was rinsed under a jet of PBS+ (10 mL), transferred into a new well containing MTT solution and incubated for 1 h ± 10 min in CO2 incubator. Each epithelium was then transferred into a new 24 wells plate containing isopropanol (1 mL). After agitating for 1 h ± 10 min, each extract (200 μL/well) was transferred into a 96 wells microplate and optical density was recorded at 570 nm with a plates reader (blank = isopropanol). For each treated tissue the viability was expressed as 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.

Irritation parameter:

other: T50

Value:

ca. 85.6

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: another study is necessary to conclude on classification.

Irritant / corrosive response data:

Negative control:
- Mean OD: 1.092

T50 values:
- Positive control: 33.49 min
- Test material: 42.88 min

- See table 7.3.2/2 for more data

Other effects:

- Non-specific MTT reduction: REACTION MASS OF DL-LIMONENE, ALPHA- GAMMA- TERPINENES, TERPINOLENE did not interact with MTT

None

Interpretation of results:

study cannot be used for classification

Conclusions:

The T50 value of the test item, REACTION MASS OF DL-LIMONENE, ALPHA- GAMMA- TERPINENES, TERPINOLENE, was between 60 and 180 min therefore it was considered as slightly irritant when applied on a human reconstructed corneal epithelium. However, this result does not allow to classify the substance according to CLP/GHS criteria. Another study has to be conducted.

Executive summary:

In an in vitro eye irritation study performed in compliance with GLP, 30 µL of the test item, REACTION MASS OF DL-LIMONENE, ALPHA- GAMMA- TERPINENES, TERPINOLENE, was applied to the human reconstructed corneal epithelium model (in duplicate) for 10 ± 2 min, 1 h ± 10 min and 3 h ± 30 min at room temperature. Contact timepoint for negative control (sodium chloride 0.9 % w/v) was 3 h ± 30 min; contact timepoints for positive control (SDS 1.5% w/w) were 10 ± 2 min and 1 h ± 10 min. At the end of each incubation period, each epithelium was rinsed, transferred to new well containing MTT solution and incubated for 1 h ± 10 min in CO2 incubator. Each epithelium was then transferred into a new well plate containing isopropanol (1 mL). After agitating for 1 h ± 10 min, the extract (200 μL/well) was transferred into a 96 wells microplate and optical density was recorded at 570 nm with a plates 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 causes 50% of cell mortality (T50) was determined by linear regression analysis.

T50 value for the test item was 85.6 min. Optical density value for the negative control was 1.092 and T50 value for positive control was 19.5 min and thus confirmed the validity of the test.

Therefore, the T50 value of test item REACTION MASS OF DL-LIMONENE, ALPHA- GAMMA- TERPINENES, TERPINOLENE was between 60 and 180 min therefore it was considered as slightly irritant when applied on a human reconstructed corneal epithelium. However, this result does not allow to classify the substance according to CLP/GHS criteria. Another study has to be conducted.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In a primary dermal irritation study performed according to OECD guideline 404 and in compliance with GLP, three albino rabbits were dermally exposed to d-limonene, under a semi-occlusive patch for 4 hours. Mean individual scores at 24, 48 and 72 h after exposure for the 3 animals were 2, 2, 2 for erythema score and 1.33, 2, 1.33 for oedema score. Signs of erythema and oedema, as well as desquamation from skin surface, were observed in all animals on Day 7. This test was repeated under the same conditions. In this second experiment: mean individual scores at 24, 48 and 72 h after exposure for 4 animals were 2, 2, 1.67, 2 for erythema score and 1.33, 0.67, 1.33, 2 for oedema score. Slight desquamation was observed in 2/4 animals on day 7.

Test item REACTION MASS OF DL-LIMONENE, ALPHA- GAMMA-TERPINENES, TERPINOLENE was applied, as supplied, at the dose of 50 µL, to 2 PBS pre-treated RhCE (EpiOcular^TMtissue model) during 30 minutes at 37°C, 5% CO₂, 95% humidity (standard culture conditions). The exposure period was followed by extensive rinsing with PBS at room temperature, a 12 minutes post-exposure immersion period at room temperature and a 1 -hour and 56 -minute post-exposure incubation at standard culture conditions. The tissue viability was measured by performing a MTT assay. The experimental protocol was established in accordance with OECD Guideline No. 492 adopted 28 July 2015.

The mean percent tissue viability of the RhCE replicates treated with test item REACTION MASS OF DL-LIMONENE, ALPHA- GAMMA-TERPINENES, TERPINOLENE was 76.69%, versus 34.89% in the positive control (Methyl acetate).

Justification for classification or non-classification

An harmonised classification is available for d-limonene and it is classified as skin irritant. Therefore, as the registered substance contains more than 10% of dl-limonene, it should be classified as skin irritant according to the rules of classification for mixtures of CLP Regulation (EC) No. 1272/2008.

In an in vitro study conducted in accordance with OECD Guideline 492, REACTION MASS OF DL-LIMONENE, ALPHA- GAMMA- TERPINENES, TERPINOLENE was found not irritating to eyes.