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Toxicological information

Skin sensitisation

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Administrative data

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
Cross-reference
Reason / purpose:
reference to same study
Reference
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).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2005

Materials and methods

Test guidelineopen allclose all
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

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
Name: dI-Citronellol
Colour: Colourless
Physical state: Liquid
Storage conditions: Ambient temperature in the dark



In vivo test system

Test animals

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

Study design: in vivo (LLNA)

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)

Results and discussion

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.

In vivo (LLNA)

Resultsopen allclose all
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

Applicant's summary and conclusion

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).