Registration Dossier

Administrative data

Description of key information

Skin sensitisation: Skin sensitiser 1B based on read-across from Limonene, which was tested in an OECD TG 429

Respiratory sensitisation (in absence of human data and absence of respiratory sensitisation alerts): not respiratory sensitising

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Read-across information
Justification for type of information:
The read-across justification is presented in the Endpoint summary Skin sensitisation. The accompanying files are also attached there.
Reason / purpose:
read-across source
Key result
Parameter:
EC3
Value:
22
Remarks on result:
other: read-across from Limonene
Parameter:
SI
Value:
1.3
Test group / Remarks:
10% v/v
Remarks on result:
other: 10%
Parameter:
SI
Value:
3.4
Test group / Remarks:
25% v/v
Remarks on result:
other: 25%
Parameter:
SI
Value:
4
Test group / Remarks:
50% v/v
Remarks on result:
other: 50%
Parameter:
SI
Value:
8.8
Test group / Remarks:
75% v/v
Remarks on result:
other: 75%
Parameter:
SI
Value:
6.5
Test group / Remarks:
100% v/v
Remarks on result:
other: 100%
Parameter:
other: disintegrations per minute (DPM)
Value:
2 511
Test group / Remarks:
vehicle
Remarks on result:
other: read-across from Limonene
Parameter:
other: disintegrations per minute (DPM)
Value:
3 319
Test group / Remarks:
10% v/v
Remarks on result:
other: read-across from Limonene
Parameter:
other: disintegrations per minute (DPM)
Value:
8 554
Test group / Remarks:
25% v/v
Remarks on result:
other: read-across from Limonene
Parameter:
other: disintegrations per minute (DPM)
Value:
9 916
Test group / Remarks:
50% v/v
Remarks on result:
other: read-across from Limonene
Parameter:
other: disintegrations per minute (DPM)
Value:
22 063
Test group / Remarks:
75% v/v
Remarks on result:
other: read-across from Limonene
Parameter:
other: disintegrations per minute (DPM)
Value:
16 259
Test group / Remarks:
100% v/v
Remarks on result:
other: read-across from Limonene
Interpretation of results:
other: Skin sensitiser Cat. 1B
Remarks:
according to EU CLP (EC No. 1272/2008 and its amendments).
Conclusions:
The substance is a skin sensitiser 1B, with an EC3 value of 22%, based on read across from Limonene.
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From May 07 to 18, 2004
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
no certificate of analysis of the test substance
Justification for type of information:
This information is used for read across to Terpene hydrocarbon alcohols.
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
no certificate of analysis of the test substance
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
other: CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River UK Ltd, UK
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 17.7-20 g
- Housing: Housed in a group of 4 mice (maximum) per cage
- Diet (e.g. ad libitum): Diet (RM1) (Special Diet Services Limited, Witham, UK), ad libitum
- Water (e.g. ad libitum): Mains water, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30-70%
- Air changes (per hour): 15/hour (minimum)
- Photoperiod (hours dark / hours light): 12 hours dark / 12 hours light
Vehicle:
other: ethanol/diethyl phthalate (3: 1 v/v)
Concentration:
0, 10, 25, 50, 75 or 100% v/v in ethanol/diethyl phthalate (3: 1 v/v)
No. of animals per dose:
Four females
Details on study design:
MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: One or more concentrations of the test substance should elicit a 3-fold or greater increase in isotope incorporation relative to the vehicle control group.

TREATMENT PREPARATION AND ADMINISTRATION: 25 µL of test material at concentrations of 0 (vehicle control), 10, 25, 50, 75 or 100% v/v in ethanol/diethyl phthalate (3: 1 v/v) were applied to the dorsal surface of each ear on Days 1, 2 and 3. On Day 6, 250 μL phosphate buffered saline (PBS) containing 20 μCi of 3H-methyl thymidine (specific gravity: 2.0 Ci/mmol) was injected into the tail vein of each experimental mouse. Five hours later, all mice were killed by inhalation of halothane vapour followed by cervical dislocation and the draining auricular lymph node of each ear was excised into PBS. A single cell suspension of lymph node cells (LNC) was prepared by gentle separation through 200-mesh stainless steel gauze. LNC were washed thrice with 10 mL of PBS and precipitated with 5% trichloroacetic acid (TCA) at 4 °C. Pellets were re-suspended in 1 mL TCA and transferred to scintillation vials containing 10 mL of scintillation fluid (Optiphase) for 3H-counting. The number of radioactive disintegrations per minute (DPM) was measured using a Packard Tri-Carb Liquid Scintillation Counter.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
No data
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:
EC3
Value:
22
Parameter:
SI
Value:
1.3
Test group / Remarks:
10% v/v
Remarks on result:
other: 10%
Parameter:
SI
Value:
3.4
Test group / Remarks:
25% v/v
Remarks on result:
other: 25%
Parameter:
SI
Value:
4
Test group / Remarks:
50% v/v
Remarks on result:
other: 50%
Parameter:
SI
Value:
8.8
Test group / Remarks:
75% v/v
Remarks on result:
other: 75%
Parameter:
SI
Value:
6.5
Test group / Remarks:
100% v/v
Remarks on result:
other: 100%
Parameter:
other: disintegrations per minute (DPM)
Value:
2 511
Test group / Remarks:
vehicle
Remarks on result:
other: 0%
Parameter:
other: disintegrations per minute (DPM)
Value:
3 319
Test group / Remarks:
10% v/v
Parameter:
other: disintegrations per minute (DPM)
Value:
8 554
Test group / Remarks:
25% v/v
Parameter:
other: disintegrations per minute (DPM)
Value:
9 916
Test group / Remarks:
50% v/v
Parameter:
other: disintegrations per minute (DPM)
Value:
22 063
Test group / Remarks:
75% v/v
Parameter:
other: disintegrations per minute (DPM)
Value:
16 259
Test group / Remarks:
100% v/v

- 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:
other: Skin sensitiser Cat. 1B
Remarks:
according to EU CLP (EC No. 1272/2008 and its amendments).
Conclusions:
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% 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). The substance is classified as a Skin sensitiser Category 1B according to EU CLP.
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 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% 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.

Endpoint:
skin sensitisation: in vitro
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
an in vitro skin sensitisation study does not need to be conducted because adequate data from an in vivo skin sensitisation study are available
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

The Terpene hydrocarbon alcohols skin sensitisation potential is based on read-across from Limonene. The executive summary of the source information is presented below followed by the read-across rationale.

Limonene and its skin sensitisation

For Limonene, a local lymph node assay is available performed in CBA/Ca strain mice according to OECD Guideline 429 and in compliance with GLP criteria. In this study, doses of 25 µL of d-limonene were applied to groups of mice (4 females/dose) 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 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% 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. Based upon these results d-limonene was concluded to be skin sensitising.

The skin sensitizing properties Terpene hydrocarbon alcohols using read across from d-Limonene (CAS# 5989-27-5)

Introduction and hypothesis for the analogue approach

Terpene hydrocarbon alcohols have the followingconstituent types of substances: Solely hydrocarbons-terpene type, Alcohol-type, Ketone-type and Ether-type all having a saturated or unsaturated cyclic hydrocarbon backbone.For this substance no skin sensitisation information is available. In accordance with Article 13 of REACH, lacking information can be generated by other means than experimental testing, i.e. applying alternative methods such as QSARs, grouping and read-across. For assessing the skin sensitisation potential the information from the constituent Limonene is used, which can represent the Terpene hydrocarbon alcohols.

Hypothesis: Terpene hydrocarbon alcohols have the same skin sensitisation potential as Limonene.

Available information: Limonene is tested in an LLNA (OECD TG 429, Rel. 1), in which the following concentrations are used: 0 (vehicle control), 10, 25, 50, 75 or 100%. Stimulation indexes were 1, 1.3, 3.4, 4.0, 8.8 or 6.5, respectively. The EC3 was calculated as 22% v/v (5500 µg/cm2).

Target chemical and source chemical(s)

Constituent types of the target substance and chemical structures of the source substances are shown in the data matrix, including physico-chemical properties and toxicological information, thought relevant for skin sensitisation.

Purity / Impurities

Constituent types of the target substanceare covered by the presented constituent types, there are no other constituent that impacts the skin sensitisation potential.

Analogue approach justification

According to Annex XI 1.5 read across can be used to replace testing when the similarity can be based on a common backbone and a common functional group. When using read across the result derived should be applicable for C&L and/or risk assessment and it should be presented with adequate and reliable documentation, which is presented below.

Analogue selection: For Terpene hydrocarbon alcohols Limonene is a representative for the Solely hydrocarbons-terpene type and the Ether type that have structural features for skin sensitisation and therefore the approach is conservative for those constituents without these features. For Limonene reliable LLNA information is available.

Structural similarities and differences: The Terpene hydrocarbon alcohol constituent: Solely hydrocarbon-terpene types have a terpene type of hydrocarbon backbone: a double bond with a methyl group. Limonene represents this group. It is a skin sensitiser because after air oxidation this terpene bond can form hydro-peroxides. Not all Terpene hydrocarbons are sensitisers e.g. the Alcohol-type and Ketone-type are not necessarily skin sensitiser.

Dermal absorption: Skin absorption will be very similar for all Terpene hydrocarbon alcohol constituent types and will not affect the read across. Metabolism: The air oxidation of the terpene double result in hydro-peroxides, which are the sensitising reactants.

Skin sensitisation reactivity: The major constituent types Alcohols (40-90%) and Ketone-type (0-17%) have no skin sensitisation potential. The Solely hydrocarbon-terpene type has skin sensitisation potential. The formed hydro-peroxides after air oxidation are very likely cause this skin sensitisation reaction for Limonene (EC3 22%) but also for Terpinolene (EC3 8%, ECHA website, EC. No. 209-578-0 and Cas no. 586-62-9). For the aromatic ether impurity, cis Anethole present at maximum 4% ( Cas no. 25679-28-1) a Michael addition reaction is causing a more moderate skin sensitisation reaction (EC3 calculated as 2.3% by Roberts et al, 2007, ICCVAM, 2009).

Conversion: In the Terpene hydrocarbon alcohols the presence of the Solely hydrocarbons is maximally 15%. Limonene is representing this category and has an EC3 of 148.7% (EC3 of 22.3%/0.15 fraction present in the Terpene hydrocarbon alcohol substance). When using the lower EC3 of 8% of Terpinolene the EC3 for the whole substance would be 53% (8%/0.15). Even when using the cis-Anethole with the calculated EC3 of 2.3% (2.3%/0.04 fraction of cis-Anethole present), the EC3 would result in 57.5% for the whole substance. The EC3 of Limonene of 22% for the Terpene hydrocarbon alcohols is therefore sufficiently conservative.

Uncertainty of the prediction: There are no uncertainties other than those already addressed above.

Data matrix

The relevant information on physico-chemical properties and toxicological characteristics are presented in the Data Matrix.

Conclusions on skin sensitisation for hazard and risk assessment

For Terpene hydrocarbon alcohols no skin sensitisation information is available but for some constituents such information is present of which Limonene is selected. When using read across the result derived should be applicable for C&L and/or risk assessment and be presented with adequate and reliable documentation.This documentation is presented in the current text. Limonene has an EC3 of 22.3% and is considered a skin sensitiser 1B. This information can be used for read across to Terpene hydrocarbon alcohols which mainly consists of constituent types that are not sensitising but contains circa 15% Solely hydrocarbon-terpene type that will have a similar sensitising potency. In view of this the EC3 of Limonene can be used for read across to Terpene hydrocarbon alcohols.

Final conclusion:Terpene hydrocarbon alcohols is considered to have skin sensitizing properties, based on read-across, the conservative EC3 is 22%, which result in a skin sensitiser 1B.

Data matrix supporting the Terpene hydrocarbon alcohol skin sensitisation potential by usingread acrossfrom Limonene

Terpene hydrocarbon alcohols

Terpineol hydrocarbon alcohols

Limonene

 

Target

Source

Structure

Not applicable

CAS

Not applicable

5989-27-5

EC No.

945-149-0

227-813-5

Reach registration

2018

Registered

Molecular weight

136-154

152

Phys-chem properties

 

 

Appearance

Liquid

Liquid

Log Kow

4.9 (3.3-5.5; IFF)

4.4 (ECHA site)

Identity, Constituent type (%)

100%

 

Solely hydrocarbons

 

 

Limonene-type

0-15

>80

Alcohol type

 

 

Tertiary alcohols (e.g Terpineol)

40-90

 

Secondary alcohols (e.g. Borneol)

7-40

 

Ketone type

-

 

Camphor-Type

0-17%

 

Ether type

 

 

Aromatic ether type

<4%

 

Human health

 

 

Skin sensitisation: EC3 in %

22

(Read across)

22

(LLNA, OECD TG 429)

References:

Roberts DW, Patlewicz G, Kern PS, Gerberick F, Kimber I, Dearman RJ, Ryan CA, Basketter DA, Aptula AO. Mechanistic applicability domain classification of a local lymph node assay dataset for skin sensitization.Chemical Research in Toxicology. 2007 Jul;20(7):1019-30.

 

(ICCVAM), 2009. ICCVAM Test Method Evaluation Report. The Reduced Murine Local Lymph Node Assay: An Alternative Test Method Using Fewer Animals to Assess the Allergic Contact Dermatitis Potential of Chemicals and Products. NIH Publication Number 09-6439. Research Triangle Park, NC: National Institute of Environmental Health Sciences. Available at:http://iccvam.niehs.nih.gov/methods/immunotox/LLNA-LD/TMER.htm

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

The substance is not a respiratory sensitiser in absence of human data indicating such effects. In addition, the respiratory sensitisation is assessed using the integrated evaluation strategy for respiratory sensitisation data in the ECHA guidance (R7A, Fig. 7.3-2, 2014).

1) The substance is a skin sensitiser;

2) The substance does not belong to the di-isocyanates;

3) The substance has no structural alerts or is structurally related to chemicals causing respiratory sensitisation as presented in Table R.7.3-1 in the ECHA guidance of 2008 or those provided in the following document: http://ec.europa.eu/health/scientific_committees/docs/annex6_respiratory.pdf

Therefore the substance is considered to be not a respiratory sensitiser.

Justification for classification or non-classification

The substance is a skin sensitiser based on read across from Limonene. It is a skin sensitiser Category 1B and shall be labelled with 'H317: May cause an allergic skin reaction', according to EU CLP (EC No. 1272/2008 and its amendments).

In absence of human data indicating respiratory sensitisation and using the ITS in the ECHA guidance (R.7a, 2014) the substance is not considered to be a respiratory sensitiser in accordance with the criteria outlined in the EU CLP (EC 1272/2008 and its amendments).