[3R-(3α,3aβ,6α,7β,8aα)]-octahydro-6-methoxy-3,6,8,8-tetramethyl-1H-3a,7-methanoazulene

Endpoint summary

Description of key information

Skin sensitisation:

Animal information: Sensitiser with an EC3 of 32% (OECD TG 429)

Human information: not sensitising at =<5% (HRIPT).

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 09 September and 28 September 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(2010)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

(2008)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

(2003)

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

other: CBA/J strain, inbred, SPF-Quality.

Sex:

female

Details on test animals and environmental conditions:

Female CBA/J strain mice were supplied by Janvier, Le Genest-Saint-Isle, France. On receipt the animals were randomly allocated to cages. The animals were nulliparous and non pregnant. After an acclimatisation period of at least five days the animals were selected at random and given a tail mark with marker pen. At the start of the study the animals were in the weight range of 19.8 to 23.5 g, and were eight to ten weeks old.

Animals were group housed in labeled Makrolon cages containing sterilised sawdust as bedding material. Paper and shelters were supplied as cage-enrichment. Free access to tap water and food (pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany)) was allowed throughout the study.
The temperature and relative humidity were controlled to remain within target ranges of 18 to 24°C and 40 to 70 %, respectively. Any occasional deviations from these targets were considered not to have affected the purpose or integrity of the study. The rate of air exchange was at least 10 changes per hour and the lighting was controlled by a time switch to give twelve hours continuous light and twelve hours darkness.

The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.

Vehicle:

other: Acetone/Olive oil (4:1 v/v)

Concentration:

Undiluted test item or the test item at concentrations of 25 % or 50 % v/v in vehicle.

No. of animals per dose:

Groups of five mice were treated

Details on study design:

Weight of evidence analysis
In the interest of animal welfare and to minimize any testing likely to produce severe responses in animals, a weight of evidence analysis was performed prior to the start of this study. All available information was evaluated (e.g. existing human and animal data, literature, substance data supplied by the Sponsor, analysis of structure activity relationships (SAR), physicochemical properties and reactivity (pH, buffering capacity)). It was concluded by the Study Director that no severe effects were to be expected.

Preliminary Screening Test
A pre-screen test was conducted in order to select the highest test item concentration to be used in the main study. In principle, this highest concentration should cause no systemic toxicity, may give well-defined irritation as the most pronounced response (maximum grade 2 and/or an increase in ear thickness < 25%) and/or is the highest possible concentration that can technically be applied.
Two test substance concentrations were tested; a 50% and 100% concentration. The highest concentration was the maximum possible concentration as required in the test guidelines.
The test system, procedures and techniques were identical as those used in the main study except that the application method may have been different and that the assessment of lymph node proliferation and necropsy were not performed. Two young adult animals per concentration were selected. Each animal was treated with one concentration on three consecutive days. Animals were group housed in labeled Makrolon cages (MII type, height 14 cm). Ear thickness measurements were conducted using a digital thickness gauge (Kroeplin C110T-K) prior to dosing on Days 1 and 3, and on Day 6.
Animals were sacrificed after the final observation.

Main Test
Test Item Administration
Groups of five mice were treated with the undiluted test item or the test item at concentrations of 50 % or 25 % v/v in acetone/olive oil 4:1. The vehicle was selected on the basis of maximizing the solubility using the test substance data provided by the Sponsor and trial preparation results performed at WIL Research Europe. The vehicle was chosen from the vehicles specified in the test guideline: Acetone/Olive oil (4:1 v/v), N,N-dimethylformamide, methylethylketone, propylene glycol and dimethylsulfoxide. There was no information available regarding the solubility or stability in vehicle. No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement. The preliminary screening test suggested that the test item would not produce systemic toxicity or excessive local irritation at the highest suitable concentration. The dorsal surface of both ears was topically treated (25 μL/ear) with the test substance, at approximately the same time on each day. The concentrations were stirred with a magnetic stirrer immediately prior to dosing.
A further group of five mice received the vehicle alone in the same manner.

The positive control animals were similarly treated to the test animals except that 25 µl of the positive control item, α Hexylcinnamaldehyde, technical grade, at a concentration of 5, 10 and 25 % v/v in acetone/olive oil 4:1 was applied to the dorsal surface of each ear.
The positive control group served as common control with Project number 509768 according to the summary of positive control data for the local lymph node assay.

Induction - Days 1, 2 and 3
The dorsal surface of both ears was topically treated (25 μL/ear) with the test substance, at approximately the same time on each day. The concentrations were stirred with a magnetic stirrer immediately prior to dosing.

3H-Methyl Thymidine Administration
Five days following the first topical application of the test item, each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline (PBS) (Merck, Darmstadt, Germany) containing 20 μCi of 3H-methyl thymidine (PerkinElmer Life and Analytical Sciences, Boston, MA, US).

Terminal Procedures - Day 6
Termination: After five hours, all animals were killed by intraperitoneal injection (0.2 mL/animal) of Euthasol® 20% (AST Farma BV, Oudewater, The Netherlands). The draining (auricular) lymph node of each ear was excised. The relative size of the nodes (as compared to normal) was estimated by visual examination and abnormalities of the nodes and surrounding area were recorded. The nodes were pooled for each animal in approximately 3 mL PBS.

Preparation of Single Cell Suspension: Following excision of the nodes, a single cell suspension of lymph node cells (LNC) was prepared in PBS by gentle separation through stainless steel gauze (diameter: 125 μm). LNC were washed twice with an excess of PBS by centrifugation at 200g for 10 minutes at 4ºC. To precipitate the DNA, the LNC were exposed to 5% trichloroacetic acid (TCA) (Merck, Darmstadt, Germany) and then stored in the refrigerator until the next day.

Determination of 3HTdR Incorporation: Precipitates were recovered by centrifugation, resuspended in 1 mL TCA and transferred to 10 mL of Ultima Gold cocktail (PerkinElmer Life and Analytical Sciences, Boston, MA, US) as the scintillation fluid. Radioactivity measurements were performed using a Packard scintillation counter (2800TR). Counting time was to a statistical precision of ± 0.2% or a maximum of 5 minutes whichever came first. The scintillation counter was programmed to automatically subtract background and convert Counts Per Minute (CPM) to Disintegrations Per Minute (DPM).

Observations
Mortality/Viability Twice daily.

Clinical Observations: Once daily on Days 1-6 (on Days 1-3 between 3 and 4 hours after dosing). Any signs of toxicity or signs of ill health during the test were recorded.

Bodyweights: The bodyweight of each mouse was recorded on Day 1 (prior to dosing) and on Day 6 (prior to termination).

Irritation: Once daily on Days 1-6 (on Days 1-3 within 1 hour after dosing) according to the numerical scoring system (see any other information on materials and methods incl. tables). In addition, a description of all other (local) effects was recorded.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

Not performed.

Positive control results:

The positive control item, α-Hexylcinnamaldehyde, technical grade gave a Stimulation Index of 9.1 when tested at a concentration of 25 % v/v in acetone/olive oil 4:1.

Key result

Parameter:

EC3

Value:

32

Pre-screen test:

Scaliness was noted for all animals on Day 6. Very slight erythema (Days 2-5) and scabs (Day 6) were noted for the animals treated at 100%. Variations in ear thickness during the observation period were less than 25% compared to Day 1 pre-dose values. No signs of systemic toxicity were observed in any of the pre-screen animals. Based on these results, the highest test substance concentration selected for the main study was a 100% concentration.

Interpretation of results:

other: sensitising (Cat 1B)

Remarks:

EU CLP 1272/2008 and its amendments

Conclusions:

The SI values calculated for Cedramber concentrations 25, 50 and 100% were 2.3, 4.8 and 13.1, respectively. These results show that the test substance could elicit a SI ≥ 3 and an estimated EC3 value of 32.0% was calculated. The test item was considered to be a sensitiser (Cat 1B) according to EU CLP and GHS criteria.

Executive summary:

The skin sensitisation potential of Cedramber has been tested according to OECD TG 429: Local Lymph Node Assay" method and GLP principles. At 25, 50 and 100% the substance showed SI values of 2.3, 4.8 and 13.1, respectively. The auricular lymph nodes of the animals treated at 25% and 50% and of the control animals were considered normal in size. The lymph nodes of the animals treated at 100% were considered enlarged. No macroscopic abnormalities of the surrounding area were noted for any of the animals. These results show that the test substance could elicit a SI ≥ 3 and an estimated EC3 value of 32.0% was calculated. Reliable negative and positive controls were included. Cedramber was considered to be a sensitiser.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

LLNA:

The skin sensitisation potential of Cedramber has been tested according to OECD TG 429: Local Lymph Node Assay" method and GLP principles. At 25, 50 and 100% the substance showed SI values of 2.3, 4.8 and 13.1, respectively. The auricular lymph nodes of the animals treated at 25% and 50% and of the control animals were considered normal in size. The lymph nodes of the animals treated at 100% were considered enlarged. No macroscopic abnormalities of the surrounding area were noted for any of the animals. These results show that the test substance could elicit a SI ≥ 3 and an estimated EC3 value of 32.0% was calculated. Reliable negative and positive controls were included. Cedramber was considered to be a sensitiser.

HRIPT:

Three HRIPT were performed with 5, 2.5, and 1% test substance in which 37, 41, and 37 subjects were exposed, respectively. In these tests, no sensitisation was observed in all the participants. Under the conditions of the test the substance was not sensitising.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Respiratory sensitisation can be assessed using human data such as indicated in R7.3.5.2 of the ECHA guidance (2014) that indicate respiratory reactions e.g. from consumer experience or occupational exposure. In the absence of such data, the respiratory sensitisation was assessed using the integrated evaluation strategy for respiratory sensitisation data in the ECHA guidance (R7A, Fig. 7.3-2, 2014). The substance is a skin sensitizer, but does not belong to the di-isocyanates. In addition, the substancehas not 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 Cedramber is not considered to be a respiratory sensitizer.

Justification for classification or non-classification

Sensitisation was observed in the LLNA according to OECD 429 with an estimated EC3 of 32.0%. Therefore the substance should be classified as skin sensitizer (Category 1B) and labeled as H317 according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008 and its amendments.