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Description of key information

Skin sensitising potential of the test substance was assessed on the source substance DL-Citronellol by an activation of keratinocytes study and an LLNA study.

The activation of keratinocytes study concluded that the substance shows no indication of being a skin sensitiser.

Skin sensitisation was assessed in an LLNA (OECD 429) where approximately 25µl of a 2.5, 5, 10, 25 or 50% w/v preparation of the test substance in 1:3 Et0H:DEP was applied to the dorsal surface of each ear for 3 consecutive days. The EC3 value was calculated to be 43.5% w/v (1 0875µg/cm²). Therefore, the substance is classified as Skin sensitiser 1B.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
9 December 2013 - 10 August 2015
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Related information:
Composition 1
Reference:
Composition 0
Qualifier:
no guideline available
Principles of method if other than guideline:
Not specified
GLP compliance:
not specified
Remarks:
not known
Type of study:
activation of keratinocytes
Test material information:
Composition 1
Key result
Parameter:
other: EC 1.5
Run / experiment:
1
Value:
> 2 000
Remarks on result:
no indication of skin sensitisation
Key result
Parameter:
other: EC 3.0
Run / experiment:
1
Value:
> 2 000
Remarks on result:
no indication of skin sensitisation
Key result
Parameter:
other: mean IC50
Run / experiment:
1
Value:
453.14
Remarks on result:
no indication of skin sensitisation
Other effects / acceptance of results:
The KeratinoSens assay was accepted when the positive control (cinnamic aldehyde) caused an in vitro score that fell within two standard deviations of the historical mean. Additionally, the results of the three definitive trials for each plate were assessed, but not necessarily deemed valid, using similar criteria outlined in the validation ring trial. Those acceptance criteria included: 1) variability in DMSO solvent control wells for each definitive assay was <20%; and 2) the positive control produced a statistically significant induction above 1.5 fold below 64 μM in each definitive assay.
Interpretation of results:
GHS criteria not met
Conclusions:
According to the test results, there is no indication of skin sensitisation in contact with the test substance.
Endpoint:
skin sensitisation: in vitro
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
9 December 2013 - 10 August 2015
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the hypothesis that source substances and target substance have similar physical-chemical properties and (eco)toxicological properties because they are either stereoisomers of the target substance, are hydrolysed to the same substance or their chemical structure differs only by an additional double bond. This prediction is supported by data on the substances themselves.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The target substance, L-Citronellol, is a mono-constituent substance (EC No. 231-415-7, CAS no. 7540-51-4 consisting of a C8 carbon backbone, methyl substituents at C3 and C7, one double bond and a hydroxyl group. The substance is optically active, comprising a single, pure enantiomeric laevo form.

The source substance, DL-Citronellol, is a mono-constituent substance (EC No. 203-375-0, CAS no. 106-22-9, consisting of a C8 carbon backbone, methyl substituents at C3 and C7, one double bond and a hydroxyl group. The substance is an equimolar mixture of two optical isomers (enantiomers).

The source substance, citronellyl acetate, is a mono-constituent substance (EC No. 205-775-0, CAS no. 150-84-5) consisting of a C8 carbon backbone, methyl substituents at C3 and C7, one double bond and an acetate group.

The source substance, geraniol and it’s isomer, consist of a C8 carbon backbone, methyl substituents at C3 and C7, two double bonds and a hydroxyl group. The only difference between the isomers is the position of the first double bond.

The source substance, geraniol and nerol, is a multi-constituent substance of E/Z isomers (EC No. 906-125-5). The constituents consist of a C8 carbon backbone, methyl substituents at C3 and C7, two double bonds and a hydroxyl group.

The source substance, geraniol, is a mono-constituent substance (EC No. 203-377-1, CAS no. 106-24-1), consisting of a C8 carbon backbone, methyl substituents at C3 and C7, two double bonds and a hydroxyl group. Geraniol is a pure form of the E-isomer.

The source substance, nerol, is a mono-constituent substance (EC No. 203-378-7, CAS no. 106-25-2), consisting of a C8 carbon backbone, methyl substituents at C3 and C7, two double bonds and a hydroxyl group. Nerol is a pure form of the Z-isomer.
The source and target substances are both of high purity with a low concentration of impurities.

3. ANALOGUE APPROACH JUSTIFICATION
The read across hypothesis is based on structural similarity where the source substances only differ in the enantiomeric ratio or an additional double bond. Another source substance is expected to be hydrolysed to the same structure as the target substance.
In a non-chiral environment the target and source chemical DL-Citronellol will have identical properties, but in the chiral environment of living organisms the enantiomers may possess different carcinogenicity and teratogenicity (in a chiral environment, stereoisomers might experience selective absorption, protein binding, transport, enzyme interactions and metabolism, receptor interactions, and DNA binding). All endpoints read-across from DL-Citronellol are considered to be acceptable for this substance assuming that 50% of the target compound is available in the test material.
The source substance citronellyl acetate is read-across from as part of a weight of evidence approach in the repeated dose toxicity endpoint. As this substance is hydrolysed to Citronellol within 2 hours, this read-across endpoint is acceptable in the weight of evidence approach used.
The source substances geraniol, nerol and the reaction mass of geraniol/nerol differ from the target substance only by an additional double bond at C2. These structures are considered to represent a worst case scenario due to the additional potential reactive feature of the second double bond. The genotoxicity, repeated dose and reproductive toxicity endpoints read-across from these substances are therefore acceptable as a worst case assumption.

4. DATA MATRIX
Please refer to the data matrix included in the read-across justification document attached in Section 13.2.
Reason / purpose:
read-across source
Related information:
Composition 1
Reference:
Composition 0
Qualifier:
no guideline available
Principles of method if other than guideline:
Not specified
GLP compliance:
not specified
Type of study:
activation of keratinocytes
Test material information:
Composition 1
Key result
Parameter:
other: EC 1.5
Run / experiment:
1
Value:
> 2 000
Remarks on result:
no indication of skin sensitisation
Key result
Parameter:
other: EC 3.0
Run / experiment:
1
Value:
> 2 000
Remarks on result:
no indication of skin sensitisation
Key result
Parameter:
other: mean IC50
Run / experiment:
1
Value:
453.14
Remarks on result:
no indication of skin sensitisation
Other effects / acceptance of results:
The KeratinoSens assay was accepted when the positive control (cinnamic aldehyde) caused an in vitro score that fell within two standard deviations of the historical mean. Additionally, the results of the three definitive trials for each plate were assessed, but not necessarily deemed valid, using similar criteria outlined in the validation ring trial. Those acceptance criteria included: 1) variability in DMSO solvent control wells for each definitive assay was <20%; and 2) the positive control produced a statistically significant induction above 1.5 fold below 64 μM in each definitive assay.
Interpretation of results:
GHS criteria not met
Conclusions:
According to the test results, there is no indication of skin sensitisation in contact with the test substance.
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 July 2004 - 3 August 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Related information:
Composition 1
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
GLP compliance:
yes
Type of study:
mouse local lymphnode assay (LLNA)
Test material information:
Composition 1
Specific details on test material used for the study:
Name: dI-Citronellol
Colour: Colourless
Physical state: Liquid
Storage conditions: Ambient temperature in the dark



Species:
mouse
Strain:
CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Females nulliparous and non-pregnant: not specified]
- Age at study initiation:
8-12 weeks of age
- Diet: ad libitum
- Water: ad libitum
- Acclimation period:
5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C):
22±3°C
- Humidity (%):
30-70
- Air changes (per hr):
A minimum of 15 changes per hour
- Photoperiod (hrs dark / hrs light):
Artificial, giving 12 hours light, 12 hours dark
Vehicle:
other: 1:3 ethanol:diethylphthalate
Concentration:
25µl of a 2.5, 5, 10, 25 or 50% w/v preparation of the test substance in 1:3 Et0H:DEP
No. of animals per dose:
4
Details on study design:
The vehicle for the positive control substance was acetone:olive oil (4:1).
Groups of four female mice were used for this study. Approximately 25µl of a 2.5, 5, 10, 25 or 50% w/v preparation of the test substance in 1:3 Et0H:DEP was applied, using a variable volume micro-pipette, to the dorsal surface of each ear. A vehicle control group was similarly treated using 1 :3 Et0H:DEP alone. The procedure was repeated daily for 3
consecutive days.
Three days afier the third application, all the animals were injected, via the tail vein, with 250µl of phosphate buffered saline (PBS) containing 20µCi of a 2.0Ci/mmol specific activity
3H-methyl thymidine.
A single cell suspension was prepared by mechanical disaggregation of lymph node. The lymph node suspensions were transferred to scintillation vials and 10ml of scintillant
(Optiphase) was added prior to β-scintillation counting.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Positive control results:
The application of hexylcinnamaldehyde at concentrations of 5%, 10% and 25% w/v in acetone:olive oil (4: 1) resulted in a greater than 3-fold increase in isotope incorporation at both 10% and 25% concentrations. Therefore, hexylcinnamaldehyde was shown to be a skin sensitiser, confirming the validity of the protocol used for the study.
Key result
Parameter:
EC3
Remarks:
% w/v
Value:
43.5
Key result
Parameter:
EC3
Remarks:
µg/cm²
Value:
10 875
Parameter:
SI
Remarks:
Test:control ratio
Value:
1
Test group / Remarks:
10% test concentration
Parameter:
SI
Remarks:
Test:control ratio
Value:
1.3
Test group / Remarks:
25% test concentration
Parameter:
SI
Remarks:
Test:control ratio
Value:
3.6
Test group / Remarks:
50% test concentration
Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
In conclusion, the test substance diluted in vehicle 1 :3 Et0H:DEP is likely to be a skin sensitiser under the conditions of the test. The EC3 value was calculated to be 43.5% w/v (1 0875µg/cm²).
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
28 July 2004 - 3 August 2004
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the hypothesis that source substances and target substance have similar physical-chemical properties and (eco)toxicological properties because they are either stereoisomers of the target substance, are hydrolysed to the same substance or their chemical structure differs only by an additional double bond. This prediction is supported by data on the substances themselves.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The target substance, L-Citronellol, is a mono-constituent substance (EC No. 231-415-7, CAS no. 7540-51-4 consisting of a C8 carbon backbone, methyl substituents at C3 and C7, one double bond and a hydroxyl group. The substance is optically active, comprising a single, pure enantiomeric laevo form.

The source substance, DL-Citronellol, is a mono-constituent substance (EC No. 203-375-0, CAS no. 106-22-9, consisting of a C8 carbon backbone, methyl substituents at C3 and C7, one double bond and a hydroxyl group. The substance is an equimolar mixture of two optical isomers (enantiomers).

The source substance, citronellyl acetate, is a mono-constituent substance (EC No. 205-775-0, CAS no. 150-84-5) consisting of a C8 carbon backbone, methyl substituents at C3 and C7, one double bond and an acetate group.

The source substance, geraniol and it’s isomer, consist of a C8 carbon backbone, methyl substituents at C3 and C7, two double bonds and a hydroxyl group. The only difference between the isomers is the position of the first double bond.

The source substance, geraniol and nerol, is a multi-constituent substance of E/Z isomers (EC No. 906-125-5). The constituents consist of a C8 carbon backbone, methyl substituents at C3 and C7, two double bonds and a hydroxyl group.

The source substance, geraniol, is a mono-constituent substance (EC No. 203-377-1, CAS no. 106-24-1), consisting of a C8 carbon backbone, methyl substituents at C3 and C7, two double bonds and a hydroxyl group. Geraniol is a pure form of the E-isomer.

The source substance, nerol, is a mono-constituent substance (EC No. 203-378-7, CAS no. 106-25-2), consisting of a C8 carbon backbone, methyl substituents at C3 and C7, two double bonds and a hydroxyl group. Nerol is a pure form of the Z-isomer.
The source and target substances are both of high purity with a low concentration of impurities.

3. ANALOGUE APPROACH JUSTIFICATION
The read across hypothesis is based on structural similarity where the source substances only differ in the enantiomeric ratio or an additional double bond. Another source substance is expected to be hydrolysed to the same structure as the target substance.
In a non-chiral environment the target and source chemical DL-Citronellol will have identical properties, but in the chiral environment of living organisms the enantiomers may possess different carcinogenicity and teratogenicity (in a chiral environment, stereoisomers might experience selective absorption, protein binding, transport, enzyme interactions and metabolism, receptor interactions, and DNA binding). All endpoints read-across from DL-Citronellol are considered to be acceptable for this substance assuming that 50% of the target compound is available in the test material.
The source substance citronellyl acetate is read-across from as part of a weight of evidence approach in the repeated dose toxicity endpoint. As this substance is hydrolysed to Citronellol within 2 hours, this read-across endpoint is acceptable in the weight of evidence approach used.
The source substances geraniol, nerol and the reaction mass of geraniol/nerol differ from the target substance only by an additional double bond at C2. These structures are considered to represent a worst case scenario due to the additional potential reactive feature of the second double bond. The genotoxicity, repeated dose and reproductive toxicity endpoints read-across from these substances are therefore acceptable as a worst case assumption.

4. DATA MATRIX
Please refer to the data matrix included in the read-across justification document attached in Section 13.2.
Reason / purpose:
read-across source
Related information:
Composition 1
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
GLP compliance:
yes
Type of study:
mouse local lymphnode assay (LLNA)
Test material information:
Composition 1
Species:
mouse
Strain:
CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Females nulliparous and non-pregnant: not specified]
- Age at study initiation:
8-12 weeks of age
- Diet: ad libitum
- Water: ad libitum
- Acclimation period:
5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C):
22±3°C
- Humidity (%):
30-70
- Air changes (per hr):
A minimum of 15 changes per hour
- Photoperiod (hrs dark / hrs light):
Artificial, giving 12 hours light, 12 hours dark
Vehicle:
other: 1:3 ethanol:diethylphthalate
Concentration:
25µl of a 2.5, 5, 10, 25 or 50% w/v preparation of the test substance in 1:3 Et0H:DEP
No. of animals per dose:
4
Details on study design:
The vehicle for the positive control substance was acetone:olive oil (4:1).
Groups of four female mice were used for this study. Approximately 25µl of a 2.5, 5, 10, 25 or 50% w/v preparation of the test substance in 1:3 Et0H:DEP was applied, using a variable volume micro-pipette, to the dorsal surface of each ear. A vehicle control group was similarly treated using 1 :3 Et0H:DEP alone. The procedure was repeated daily for 3
consecutive days.
Three days afier the third application, all the animals were injected, via the tail vein, with 250µl of phosphate buffered saline (PBS) containing 20µCi of a 2.0Ci/mmol specific activity
3H-methyl thymidine.
A single cell suspension was prepared by mechanical disaggregation of lymph node. The lymph node suspensions were transferred to scintillation vials and 10ml of scintillant
(Optiphase) was added prior to β-scintillation counting.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Positive control results:
The application of hexylcinnamaldehyde at concentrations of 5%, 10% and 25% w/v in acetone:olive oil (4: 1) resulted in a greater than 3-fold increase in isotope incorporation at both 10% and 25% concentrations. Therefore, hexylcinnamaldehyde was shown to be a skin sensitiser, confirming the validity of the protocol used for the study.
Key result
Parameter:
EC3
Remarks:
% w/v
Value:
43.5
Key result
Parameter:
EC3
Remarks:
µg/cm²
Value:
10 875
Parameter:
SI
Remarks:
Test:control ratio
Value:
1
Test group / Remarks:
10% test concentration
Parameter:
SI
Remarks:
Test:control ratio
Value:
1.3
Test group / Remarks:
25% test concentration
Parameter:
SI
Remarks:
Test:control ratio
Value:
3.6
Test group / Remarks:
50% test concentration
Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
In conclusion, the test substance diluted in vehicle 1 :3 Et0H:DEP is likely to be a skin sensitiser under the conditions of the test. The EC3 value was calculated to be 43.5% w/v (1 0875µg/cm²).
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

A skin sensitiser is defined as a substance that will lead to an allergic response following skin contact.

Substances are classified as skin sensitisers (Category 1) if there is evidence in humans that the substance can lead to sensitisation by skin contact in a substantial number of persons, or if there are positive results from an appropriate animal test.

Substances may also be classified into:

sub-category 1A: substances showing a high frequency of occurrence in humans and/or a high potency in animals can be presumed to have the potential to produce significant sensitisation in humans) or sub-category 1B: substances showing a low to moderate frequency of occurrence in humans and/or a low to moderate potency in animals can be presumed to have the potential to produce sensitisation in humans.

A substance is classified as skin sensitiser category 1A if the EC3 value is ≤ 2 % and as skin sensitiser category 1B if the EC3 value is > 2%. In the LLNA study the EC3 value was calculated to be 43.5% w/v (1 0875µg/cm²). Therefore, the substance is classified as Skin sensitiser 1B.