Reaction mass of 3-[(3aS,7aR)-octahydro-1H-4,7-methanoinden-1-yl]propanal and 3-[(2s,3aR,7aS)-octahydro-1H-4,7-methanoinden-2-yl]propanal and 3-[(2r,3aR,7aS)-octahydro-1H-4,7-methanoinden-2-yl]propanal

Endpoint summary

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:

21 January, 2015 - 09 February, 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study is considered to be a reliability 1 as it has been conducted according to OECD Test Guideline 429 using the Skin Sensitisation: Local Lymph Node Assay method and in compliance with GLP.

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 20 to 24 g, and were approx. ten weeks old.

The 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 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 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:

acetone/olive oil (4:1 v/v)

Concentration:

Undiluted test item and the test item at concentrations of 50 % and 25 % v/v in Acetone/Olive oil (4:1 v/v)

No. of animals per dose:

Groups of five mice were treated

Details on study design:

Preliminary Screening Test
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 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)).
Two test substance concentrations were tested; a 50% and 100% concentration. The test system, procedures and techniques were identical to those used in the main study except 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. 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. 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 concentration, at approximately the same time on each day. The concentrations were stirred with a magnetic stirrer immediately prior to dosing. The control animals were treated in the same way as the experimental animals, except that the vehicle was administered instead of the test substance.

The positive control animals were similarly treated to the test animals except that 25 µl of the positive control item, % Alpha- 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 vehicle control group and the positive control group served as common controls with Project number 507448 according to the summary of positive control data for the local lymph node assay.

3H-Methyl Thymidine Administration
Five days following the first topical application of the test item, vehicle control or positive control item (Day 6) all mice were 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).

Observations
Mortality/Viability: Twice daily.
Clinical signs: Once daily on Days 1-6 (on Days 1-3 between 3 and 4 hours after dosing).
Irritation: Once daily on Days 1-6 (on Days 1-3 within 1 hour after dosing) according to the following numerical scoring system. Furthermore, a
description of all other (local) effects was recorded.

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

Terminal Procedures
Termination: Five hours following the administration of 3HTdR all mice 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 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. Radioactive 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).

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 greater than 3 when tested at a concentration of 25 % v/v in acetone/olive oil 4:1.

Parameter:

SI

Remarks on result:

other: The SI values calculated for the substance concentrations 25, 50 and 100% were 3.5, 11.3 and 17.3, respectively.

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Mean DPM/animal values for the experimental groups treated with test substance concentrations 25, 50 and 100% were 1182, 3804 and 5827 DPM, respectively. The mean DPM/animal value for the vehicle control group was 337 DPM.

Interpretation of results:

sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The SI values calculated for the substance concentrations 25, 50 and 100% were 3.5, 11.3 and 17.3, respectively. These results show that the test substance elicits a SI ≥ 3 and an estimated EC3 value of 23.9% was calculated.
The test item was considered to be a sensitiser under the conditions of the test.

Executive summary:

The skin sensitisation potential of FRET 09 -0536 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 3.5, 11.3 and 17.3, respectively. The auricular lymph nodes of the vehicle control animals and the animals treated at 25% were considered normal in size. The nodes of the animals treated at 50% and 100% were considered enlarged. These results show that the test substance elicits a SI ≥ 3. Although, the data do not permit the use of linear interpolation, calculation of EC3 values (the estimated test substance concentration that will give a SI =3) by loglinear extrapolation can provide a reliable estimation of sensitization potency class for use in risk assessment and can avoid the need for repeat animal testing. The data show a clear doseresponse and a slope ratio of 0.38 was calculated indicating that extrapolation can provide a reliable estimation of the EC3 value according to Ryan et al 2007. An estimated EC3 value of 23.9% was calculated. The test item was considered to be a sensitiser and should be classified as skin sensitizer (Category 1B) and labeled as H317: May cause an allergic skin reaction. A NOAEL of xx is derived.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

Migrated from Short description of key information:
The skin sensitisation potential of FRET 09 -0536 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 3.5, 11.3 and 17.3, respectively. The auricular lymph nodes of the vehicle control animals and the animals treated at 25% were considered normal in size. The nodes of the animals treated at 50% and 100% were considered enlarged. These results show that the test substance elicits a SI ≥ 3. Although, the data do not permit the use of linear interpolation, calculation of EC3 values (the estimated test substance concentration that will give a SI =3) by loglinear extrapolation can provide a reliable estimation of sensitization potency class for use in risk assessment and can avoid the need for repeat animal testing. The data show a clear doseresponse and a slope ratio of 0.38 was calculated indicating that extrapolation can provide a reliable estimation of the EC3 value according to Ryan et al 2007. An estimated EC3 value of 23.9% was calculated and the test item was considered to be a sensitiser. A NOAEL of xx is derived.

Justification for selection of skin sensitisation endpoint:
The result of this study is reliable and adequate for covering this endpoint.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the EC3 value of 23.9% in the LLNA study, the substance has to be classified for skin sensitisation. According to EU Classification, Labeling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008, it has to be classified for skin sensitisation,Category 1B, H317: May cause an allergic skin reaction. According to EU Directive 67/548/EEC this results in: Xi-R43: May cause sensitisation by skin contact.