Rosetal A

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 January 2003 - 15 April 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Information is used for read across to Rosetal A (target)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

2002

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of (2E)-3,7-dimethylocta-2,6-dien-1-ol and (2Z)-3,7-dimethylocta-2,6-dien-1-ol

EC Number:

906-125-5

Molecular formula:

Unspecified

IUPAC Name:

Reaction mass of (2E)-3,7-dimethylocta-2,6-dien-1-ol and (2Z)-3,7-dimethylocta-2,6-dien-1-ol

Test material form:

liquid

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Remarks:

Ola/Hsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Interfauna UK Limited, Blackthorne, Bicester, Oxon, UK
- Females nulliparous and non-pregnant: not specified
- Males for the positive control study
- Age at study initiation: young adults
- Weight at study initiation: 19.0 - 23.7 g
- Housing: a maximum of 4 mice was housed per cage, in cages suitable for animals of this strain and weight range
- Diet (ad libitum): RM1, supplied by Special Diets Services Limited, Witham, Essex, UK. Each batch routinely analysed for composition and for contaminants.
- Water (ad libitum): mains water, supplied by an automatic system. Periodically analysed for contaminants
- Acclimation period: The animals were housed under the experimental conditions for at least 5 days, prior to the start of dosing.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 30-70%
Temperature and humidity where recorded daily and where within the specified ranges.
- 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:diethyl phtalate

Concentration:

2.5%, 5%, 10%, 25%, 50% w/v preparation of the test substance in the vehicle

No. of animals per dose:

4

Details on study design:

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- 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 the preparation of the test substance in the vehicle was applied, using a variable volume micro-pipette, to the dorsal surface of each ear. The vehicle control group was similarly treated using the vehicle alone. The test method was repeated daily for 3 consecutive days.
Three days after the third application, all the animals were injected, via the tail vein, with approximately 250 µl of phosphate buffered saline (PBS) containing approximately 20 µCi of a 2.0Ci/mmol specific activity 3H-methyl thymidine. Approximately 5 hours later, the animals were humanely killed by inhalation of halothane vapour followed by cervical dislocation. The draining auricular lymph nodes were removed from each animal and together with the nodes from the other animals in the group, were placed in a container of PBS.
A single cell suspension was prepared by mechanical disaggregation of lymph nodes through a 200-mesh stainless steel gauze. The cell suspensions were then washed three times by centrifugation with approximately 10 ml of PBS. Approximately 3 ml of 5% w/v trichloroacetic acid (TCA) was added and after overnight precipitation at 4°C, the samples were pelleted by centrifugation and the supernatant was discarded. The cells were then resuspended in approximately 1 ml of TCA.
The lymph node suspensions were transferred to scintillation vials and 10 ml of scintillant (Optiphase) was added prior to β-scintillation counting using a Packard Tri-Carb 2500TR Liquid Scintillation Counter.

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 1%, 3% and 10% w/v in acetone resulted in a greater than 3-fold increase in isotope incorporation at the 3% and 10% w/v concentrations. Therefore, hexylcinnamaldehyde was shown to be a skin sensitiser confirming the validity of the protocol used for the study.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

EC3

Value:

11.4

Results 2

Parameter:

SI

Value:

1.74

Test group / Remarks:

2.5% test substance

Remarks on result:

other: 2.5%

Results 3

Parameter:

SI

Value:

2.42

Test group / Remarks:

5% test substance

Remarks on result:

other: 5%

Results 4

Parameter:

SI

Value:

2.81

Test group / Remarks:

10% test substance

Remarks on result:

other: 10%

Results 5

Parameter:

SI

Value:

4.79

Test group / Remarks:

25% test substance

Remarks on result:

other: 25%

Results 6

Parameter:

SI

Value:

6.06

Test group / Remarks:

50% test substance

Remarks on result:

other: 50%

Any other information on results incl. tables

The application of geraniol at concentrations of 2.5%, 5%, 10%, 25% and 50% in 1:3 ethanol:diethyl phthalate resulted in an increase in isotope incorporation which was greater than 3-fold at the 25% an 50% w/v concentrations,  SI > 3. Consequently, the test substance was shown to be a potential skin sensitiser. The estimated concentration giving rise to a 3-fold increase in lymphocyte proliferation (EC3) was 11.4% w/v.  

Effects of bodyweights or dermal irritation are not mentioned.

Applicant's summary and conclusion

Interpretation of results:

other: Substance is a skin sensitiser (1B) in accordance with EU CLP (1272/2008 and its amendments)

Conclusions:

The substance is a skin sensitiser (1B) in the Local Lymph Node Assay (OEDC guideline TG 429)

Executive summary:

This Local Lymph Node Assay (OECD TG 429) was performed to determine the sensitising potential of the substance in mice. Groups of 4 mice were treated with test concentrations of 2.5, 5, 10, 25 and 50% w/v ethanol:diethyl phthalate 1:3, or a vehicle treated control. Hexylcinnamaldehyde (HCA), freshly prepared as 1, 3 and 10% w/v dilution in acetone, was used as positive control. Clinical observations and bodyweights were recorded, and lymph node proliferation was determined using 3HTdR incorporation.

The number of radioactive disintegrations per minute (dpm) reflect the proliferation reponse of lymph node cells, and were 330, 574, 800, 926, 1582 and 2000 dpm/lymph node for the vehicle control, 2.5%, 5%, 10%, 25%, and 50% concentration groups, respectively. This corresponds with a lymph node proliferation of n.a., 1.74, 2.42, 2.81, 4.79 and 6.06, respectively, for the substance-treated groups, calculated as the Stimulation Index (SI). This SI was greater than 3-fold at the 25% an 50% w/v concentrations. 

An EC3 value of 11.4% was calculated. 

Based in the EC3 > 2% as stated in Annex I of 1272/2008/EC, the substance will be classified as Category 1B.