Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted between 22 January 2015 and 24 February 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was considered to be a reliability 1 as it has been conducted according to OECD Test guideline 429 using a local lymph node method and in compliance with GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: Liquid
Details on test material:
Identification: FRET 08-0338
Chemical name: 4,7-Methano-1H-inden-5-ol, octahydro-3,4,5-trimethyl and 4,7-Methano-1H-inden-5-ol,octahydro-2,4,5-trimethyl and isomers
CAS number: 1340502-93-3 & 1340502-69-3
Intended use: Industrial fragrance
Appearance: Clear liquid
Storage conditions: Room temperature in the dark
Supplier: Sponsor
Sample receipt: 10 November 2014

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
Healthy female CBA/Ca mice (a total of 29 females) were obtained from Charles River UK Ltd.
The mice were in the weight range 15.9 to 21.7 g and approximately eight to twelve weeks of age prior to dosing on Day 1. They were acclimatised to the experimental environment for at least 6 days prior to the start of the study.
Each animal was assigned an alpha-numeric code and identified uniquely within the study by tail marking. Each cage label was colour-coded and was identified uniquely with the study number, test substance concentration and animal mark(s).

Animal housing, diet and water supply
Animals were housed inside a barriered rodent facility (Building F21, Room 058/059 and 061/062). The facility was designed and operated to minimise the entry of external biological and chemical agents and to minimise the transference of such agents between rooms.
The animals were allocated without conscious bias to cages within the treatment groups. They were housed two or three animals per cage, in solid bottomed polycarbonate cages with a stainless steel mesh lid. Each cage contained a quantity of autoclaved wood flake bedding, additionally Nestlets and a plastic shelter were included for environmental enrichment.
Certificates of analysis for woodflake bedding and Nestlets were lodged in Huntingdon Life Sciences Archives.
The animal room was kept at positive pressure with respect to the outside by its own supply of filtered fresh air, which was passed to atmosphere and not re-circulated. The temperature and relative humidity controls were set to maintain the range of 19 to 23°C and 40 to 70% respectively. Any minor deviations from these ranges would not have had an adverse effect on the animals and would not affect the integrity or validity of the study. Artificial lighting was controlled to give a cycle of 12 hours continuous light and 12 hours continuous dark per 24 hours.
Periodic checks were made on the number of air changes in the animal rooms. Temperature and humidity were monitored daily and records are archived with the departmental raw data.
Alarms were activated if there was any failure of the ventilation system, or temperature limits were exceeded. A stand-by electricity supply was available to be automatically brought into operation should the public supply fail.
The animals were allowed free access to a standard rodent diet (Harlan Teklad 2014C Diet). This diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
Potable water taken from the public supply was freely available via polycarbonate bottles fitted with sipper tubes.
Each batch of diet was analysed routinely by the supplier for various nutritional components and chemical and microbiological contaminants. Supplier’s analytical certificates were scrutinised and approved before any batch of diet was released for use. The quality of the water supply is governed by regulations published by the Department for Environment, Food and Rural Affairs. Certificates of analysis were received routinely from the water supplier. Since the results of these various analyses did not provide evidence of contamination that might have prejudiced the study, they are not presented.
No other specific contaminants that were likely to have been present in the diet or water were analysed, as none that may have interfered with or prejudiced the outcome of the study was known.

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
10, 25 and 50% v/v
No. of animals per dose:
Groups of five mice were treated at one of three concentrations of the test substance.
Details on study design:
Concentration selection
Preliminary investigations
Preliminary investigations were performed to ensure the highest concentration to be used on the main study did not result in systemic toxicity or excessive local irritation. Although the material could be dosed as supplied as a precautionary measure the preliminary investigation started at 50% (v/v) in AOO (4:1 v/v).
Results of the preliminary investigation dosed at 50% showed minimal signs of ear swelling (below 25%) in one mouse and only slight erythema on Days 2 and 3 which had resolved by Day 4. Therefore as there was no swelling a further preliminary phase was dosed with the test material as supplied.
Results of the preliminary investigation dosed with the test material as supplied showed minimal signs of swelling of the ears (below 25%) in both mice, however well-defined to moderate or severe erythema was recorded on Days 2 and 3 for both mice therefore as severe erythema was noted and following the OECD guidelines dosing the test material as supplied was not undertaken.

Main study
The results of the preliminary investigations indicated that 50% v/v was a suitable high concentration for administration in the main phase of the study. The low and intermediate concentrations were selected from the concentration series given in regulatory guidelines and the concentrations administered on the main study were:
10, 25 and 50% v/v in AOO
The positive control group received 25% v/v hexylcinnamic aldehyde in AOO based on previous experience at this laboratory.
Prior to dose administration the Study Director authorised the choice of vehicle and dose levels in the raw data.

Administration of test substance
Preliminary investigation
Two female mice (per concentration) received a daily application of 25 µL of appropriate concentration of the test substance to the dorsal surface of each ear for three consecutive days (Days 1-3). The test substance was applied to the dorsal surface of each ear using an automatic micropipette and was spread over the entire dorsal surface of the ear using the tip of the pipette.

Main phase
Groups of five mice were treated at one of three concentrations of the test substance. The mice were treated by daily application of 25 µL of the appropriate concentration of the test substance to the dorsal surface of each ear for three consecutive days (Days 1-3). The test substance was applied to the dorsal surface of each ear using an automatic micropipette and was spread over the entire dorsal surface of the ear using the tip of the pipette. Further groups of five mice received the vehicle alone or the positive control substance in the same manner.

Administration of 3H-methyl Thymidine
In the main phase of the study, five days following the first topical application of test substance (Day 6) all mice were injected via the tail vein with 250 µL of phosphate buffered saline containing 3H-methyl Thymidinea (3HTdR: 80 µCi/mL) giving a nominal 20 µCi to each mouse. The injection into the tail vein was carried out using a plastic syringe and needle after the mouse had been heated in a warming chamber.

Observations
Clinical signs
All animals were observed daily for signs of ill health or toxicity. The ears were examined for signs of irritation.

Measurement of ear swelling
On the preliminary phase of the study, ear thickness was measured for both ears of each mouse on Days 1 (pre-dose), 3 (pre-dose) and 6 (pre-terminal). Measurements were taken in triplicate by placing a thickness gauge in the centre of each ear. The gauge was completely removed from the ear and replaced for each measurement. The reported ear thickness on each occasion was the mean of the three measurements.

Body weight
The weight of each mouse was recorded on arrival (data not reported), Day 1 (before dosing) and prior to termination (Day 6).

Terminal studies
Termination
The mice in the preliminary investigations were humanely killed by carbon dioxide asphyxiation on Day 6 of the study. The carcasses were discarded and no further investigations were carried out with these animals.
In the main phase of the study, five hours following the administration of 3HTdR on Day 6 all mice were humanely killed by carbon dioxide asphyxiation. The draining auricular lymph nodes were excised for each experimental animal and placed in 1.0 mL of PBS. The carcasses were then discarded and no further investigations were carried out. The following procedures were carried out with the lymph nodes from these animals only.

Preparation of single cell suspensions
A single cell suspension of lymph node cells (LNC) was prepared by gentle mechanical disaggregation through a stainless steel gauze (200 mesh size). The LNC were then washed by adding 10 mL PBS, pelleted at 190 x g for 10 minutes and re-suspended. The cells were washed twice again and re-suspended in 3 mL trichloroacetic acid (TCA: 5%) following the final wash.

Determination of incorporated 3H-methyl Thymidine
After overnight incubation (minimum of 18 hours) with 5% TCA at 4°C, the precipitate was recovered by centrifugation and re-suspended in 1 mL 5% TCA and transferred to 10 mL Ultima gold scintillation fluid on Day 7. The 3HTdR incorporation was measured by β-scintillation counting. The proliferative response of LNC was expressed as radioactive disintegrations per minute per lymph node (dpm/node).

Data treatment
Stimulation Index
Results for each treatment group were expressed as the Stimulation Index (SI). This was derived by dividing the mean dpm/mouse for each treated group and the positive control group by the mean dpm/mouse in the vehicle control group.
The individual dpm data was analysed statistically and the results of the statistical analysis are appended to the report.
If the SI is 3 or more, the test substance is regarded as a skin sensitizer with a consideration given to dose response and statistical significance.
The positive control group is expected to give an SI of 3 or more to demonstrate the validity of the study.

Estimated Concentration of Three
The Estimated Concentration of Three (EC3) is the concentration of test substance which would result in a SI of 3 and, where data permits, this value will be calculated (Ryan 2007).
The results for this study included one concentration which had an SI greater than 3 and one concentration which had an SI of less than 3, therefore the EC3 value was calculated according to the following formula:

EC3 = c + [(3 – d) / b / d)] x (a – c)
Where:
a = concentration giving SI greater than 3
b = SI at concentration a
c = concentration giving SI less than 3
d = SI at concentration c.
Positive control substance(s):
other: 25% v/v hexylcinnamic aldehyde

Results and discussion

In vivo (LLNA)

Results
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
The SI (test/control ratios) obtained for 10, 25 and 50% v/v FRET 08-0338 were 1.3, 2.5 and 5.2 respectively. As a SI of 3 or more was recorded for one of the concentrations tested (50% v/v), FRET 08-0338 was considered to have the potential to cause skin sensitization. Based on the results of this study the EC3 value is calculated to be 29.6% v/v. The SI for the positive control substance hexyl cinnamic aldehyde (HCA), was 9.8 which demonstrates the validity of this study.

Any other information on results incl. tables

Preliminary investigation

Mortality and clinical signs

There were no deaths and no signs of ill health or toxicity were observed during this study. Greasy fur on the head was noted following each dosing occasion, this was related to unoccluded dermal administration of a liquid formulation/vehicle and not an effect of the test substance.

 

Dermal reactions

Slight erythema was observed on the ears of both mice dosed at 50% v/v on Days 2 and 3. Slight to moderate or severe erythema was observed on the ears of both mice dosed with the test substance as supplied on Days 2 and 3. Recovery for all animals was seen by Day 4.

 

Measurement of ear thickness

There was no evidence of an effect of treatment on ear thickness.

 

Body weight

There was no indication of an overt effect of treatment on body weight gain. A loss in body weight was noted for one mouse (No. L1) over the study period, however, a small loss in body weight is not uncommon in young laboratory mice and this is not considered to be an effect of treatment.

On the basis of the results from the preliminary investigation, 50% v/v was considered a suitable high concentration for administration in the main phase of the study.

 

Main phase

Mortality and clinical signs

There were no deaths and no signs of ill health or toxicity observed during this study. Wet/greasy fur on the head was noted following each dosing occasion, this was related to unoccluded dermal administration of a liquid formulation/vehicle and not an effect of the test substance.

 

Dermal reactions

No signs of dermal irritation were seen on the ear during the study.

 

Body weight

There was no indication of an effect of treatment on body weight gain.

A loss in body weight was noted for a few mice, over several dose levels including control (Animal Nos. L5, L9, L10, L12, L15, L16, L25, L26 and L29), during the study period, however, a small loss in body weight is not uncommon in young laboratory mice and is not considered to be an effect of treatment.

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
FRET 08-0338 is regarded as a potential skin sensitizer. The EC3 value was calculated to be 29.6% v/v.
Executive summary:

The skin sensitisation potential of the test substance, FRET 08-0338, was assessed according to OECD Test guideline 429 using a local lymph node method. The EC3 value was calculated to be 29.6% v/v. The test substance is regarded as a potential skin sensitizer.