Reaction mass of 1-methyl-4-(1-methylethyl)-7-oxabicyclo[2.2.1]heptane and cineole

Endpoint summary

Description of key information

d-Limonene, one of the main impurities of the registered substance, is classifed as skin senitiser 1B. Therefore, according to the rules of classification criteria of Regulation (EC) No 1272/2008 (CLP), Reaction mass of 1,4 -cineole and 1,8 -cineole is classified as skin sensitiser 1B.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vivo (LLNA)

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 impurities of substance REACTION MASS OF 1,4-CINEOLE AND 1,8-CINEOLE.

Reason / purpose for cross-reference:

read-across source

Positive control results:

Mean DPM
- Vehicle (acetone/olive oil 4:1 v/v) = 2263
- Test material (5 % w/v) = 5496
- Test material (10 % w/v) = 7571
- Test material (25 % w/v) = 24752

Stimulation Index
- Test material (5 % w/v) = 2.4
- Test material (10 % w/v) = 3.3
- Test material (25 % w/v) = 10.9

Key result

Parameter:

SI

Remarks on result:

other: - Test material (10% v/v) = 1.3 - Test material (25% v/v) = 3.4 - Test material (50% v/v) = 4.0 - Test material (75% v/v) = 8.8 - Test material (100% v/v) = 6.5

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: - Vehicle = 2511 - Test material (10% v/v) = 3319 - Test material (25% v/v) = 8554 - Test material (50% v/v) = 9916 - Test material (75% v/v) = 22063 - Test material (100% v/v) = 16259

Dermal reactions: Application of the test substance did not result in an increase in visual levels of irritancy to the skin on or around the ear area for the duration of the study.

Table 1 - Skin sensitisation potential of d-limonene in ethanol/diethyl phthalate (3:1 v/v)

 

Concentration of test substance (% v/v)	Number of lymph nodes assayed	Disintegrations per min (dpm)	dpm per lymph node	Test:control ratio
0 (vehicle only)	8	2511	314	Not applicable
10	8	3319	415	1.3
25	8	8554	1069	3.4
50	8	9916	1240	4.0
75	8	22063	2758	8.8
100	8	16259	2032	6.5
EC3	Estimated to be 22% (5500 µg/cm2)

 

Table 2 - Skin sensitisation potential of the positive control substance (hexylcinnamaldehyde)

 

Concentration of hexylcinnamaldehyde (% w/v)	Number of lymph nodes assayed	Disintegrations per min (dpm)	dpm per lymph node	Test:control ratio
0 (vehicle only)	8	2263	283	Not applicable
5	8	5496	687	2.4
10	8	7571	946	3.3
25	8	24752	3094	10.9

Interpretation of results:

sensitising

Remarks:

Migrated information

Conclusions:

d-Limonene is classified as ‘R43 May cause sensitisation by skin contact’, according toDirective 67/548/EEC and in ‘Category 1’ according to CLP Regulation (EC) No 1272/2008.

Executive summary:

In a local lymph node assay performed in CBA/Ca strain mice according to OECD guideline 429 and in compliance with GLP, groups of mice (4 females/dose) were applied with 25 µL of d-limonene at concentrations of 0 (vehicle control), 10, 25, 50, 75 or 100% v/v in ethanol/diethyl phthalate (3: 1 v/v) to the dorsal surface of each ear for three consecutive days. On Day 6, all animals were injected with 3H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised and measured for radioactivity expressed as number of disintegrations per minute (DPM). Historic data of hexylcinnamaldehyde (5, 10 and 25 % w/v) in acetone/olive oil (4:1 v/v) was used as the data for the positive control group.

 

Mean DPM for 0, 10, 25, 50, 75 or 100% d-limonene were observed to be 2511, 3319, 8554, 9916, 22063 or 16259 dpm, respectively. Stimulation index for 10, 25, 50, 75 or 100% of d-limonene were calculated to be 1.3, 3.4, 4.0, 8.8 or 6.5, respectively. The estimated concentration giving rise to a 3 fold increase in lymphocyte proliferation (EC3) was 22% v/v (5500 µg/cm2). No increase in visual levels of irritancy to the ear skin was observed during the study.

 

Therefore, d-limonene is classified as ‘R43 May cause sensitisation by skin contact’, according to Directive 67/548/EEC and in ‘Category 1’ according to CLP Regulation (EC) No 1272/2008.

Reference 2

Endpoint:

skin sensitisation: in vivo (LLNA)

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 impurities of substance REACTION MASS OF 1,4-CINEOLE AND 1,8-CINEOLE.

Reason / purpose for cross-reference:

read-across source

Positive control results:

Not applicable

Key result

Parameter:

SI

Remarks on result:

other: 1, 1.84, 2.44 and 3.95 at 0, 25, 50 and 100% v/v, respectively

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: 476, 877, 1164 and 1882 at 0, 25, 50 and 100% v/v, respectively

Table 1: Local lymph node assay responses to limonene

Exposure Concentration (% v/v)	dpm/node	Stimulation index (SI)	EC3 (%)
0	476	1	68.5
25	877	1.84
50	1164	2.44
100	1882	3.95

Interpretation of results:

sensitising

Remarks:

Migrated information

Conclusions:

The calculated EC3-value for d-limonene was found to be 68.5%. Therefore, d-limonene was found to be sensitising and should be clasified as skin sensitiser according to Directive 67/548/EEC and CLP Regulation (EC) No 1272/2008.

Executive summary:

In a skin sensitization study conducted following a method similar to OECD guidelines 429, four groups of CBA/Ca strain female mice (4/concentration) were exposed topically on the dorsum of both ears to 25 µL of vehicle (acetone/olive oil, 4:1 v/v), 25, 50 and 100% v/v of d-limonene daily for 3 consecutive days. Five days following initiation of exposure all mice were injected 250 µL of phosphate buffered saline (PBS) containing 20 µCi of [3H] methyl thymidine (3H-TdR). The animals were then sacrificed after 5 hours and suspension of lymph node cells was prepared from auricular lymph nodes. The incorporation of 3H-TdR was measured by β-scintillation counting as disintegrations per minute (dpm) per node for each experimental group. A Stimulation Index of 3 or greater was considered to be indicative of a potential to cause contact sensitization.

d-Limonene at exposure concentration of 0 (vehicle), 25, 50 and 100% v/v resulted in 476, 877, 1164 and 1882 dpm/node and stimulation index of 1, 1.84, 2.44 and 3.95, respectively.

The calculated EC3-value for d-limonene was found to be 68.5%. Therefore, d-limonene was found to be sensitising and should be clasified as skin sensitiser according to Directive 67/548/EEC and CLP Regulation (EC) No 1272/2008.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The registered substance contains d- and l-limonene at concentrations potentially higher than 1% (w/w), which is the generic concentration limit that triggers classification for skin sensitisation of mixtures. As shown by the data on d- and l-limonene, and according to the current proposed harmonised classification of d-limonene, these impurities are classified as Skin Sens. 1B, H317 which leads to the classification of the registered substance as Skin Sens. 1B, H317 according to the rules of classification criteria of Regulation (EC) No. 1272/2008 (CLP).