Decahydrospiro[furan-2(3H),5'-[4,7]methano[5H]indene]

Endpoint summary

Description of key information

Skin sensitisation (OECD TG 429): sensitising

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vivo (LLNA)

Remarks:

LLNA was initiated before 11 October 2016

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 05 October 2016 and 17 November 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

LLNA was initiated before the REACH regulation came into force requesting in vitro tests first (at 11 October 2016)

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 22 July 2010

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

Animal Information
Female CBA/Ca (CBA/CaOlaHsd) strain mice were supplied by Envigo RMS B.V., Inc., Horst, The Netherlands. On receipt the animals were randomly allocated to cages. The animals were nulliparous and non pregnant. After an acclimatization period of at least 5 days the animals were selected at random and given a number unique within the study by indelible ink marking on the tail and a number written on a cage card. At the start of the study the animals were in the weight range of 15 to 23 g, and were 8 to 12 weeks old.

Animal Care and Husbandry
The animals were housed in suspended solid floor polypropylene cages furnished with softwood woodflakes. Free access to mains tap water and food (2014C Teklad Global Rodent diet supplied by Envigo RMS (UK) Limited, Oxon, UK) was allowed throughout the study.
The temperature and relative humidity were set to achieve limits of 19 to 25 oC and 30 to 70%, respectively. The rate of air exchange was at least fifteen changes per hour and the lighting was controlled by a time switch to give 12 hours continuous light and 12 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 or the test item at concentrations of 30% or 10% v/v in acetone/olive oil 4:1

No. of animals per dose:

Groups of five mice were treated

Details on study design:

Preliminary Screening Test
As no toxicological information was available regarding the systemic toxicity/irritancy potential of the test item, a preliminary screening test was performed using one mouse. The mouse was treated by daily application of 25 µL of the undiluted test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mouse was observed twice daily on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Local skin irritation was scored daily according to the scale included in "Any other information on materials and methods incl. tables". Any clinical signs of toxicity, if present, were also recorded. The body weight of the mouse was recorded on Day 1 (prior to dosing) and on Day 6.
The thickness of each ear was measured using a Mitutoyo 547-300S gauge (Mitutoyo Corporation), pre-dose on Day 1, post dose on Day 3 and on Day 6. Any changes in the ear thickness were noted. Mean ear thickness changes were calculated between time periods Days 1 and 3 and Days 1 and 6. A mean ear thickness increase of equal to or greater than 25% was considered to indicate excessive irritation and limited biological relevance to the endpoint of sensitization.

Main Test
Test Item Administration
Groups of five mice were treated with the undiluted test item or the test item at concentrations of 30 % or 10 % v/v in acetone/olive oil 4:1. This vehicle was chosen as it produced the most suitable formulation at the required concentration according to the vehicle determination record. The test item was formulated within 2 hours of being applied to the test system. It is assumed that the formulation was stable for this duration. 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 skin irritation at the highest suitable concentration. The mice were treated by daily application of 25 µL of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The test item formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette.
A further group of five mice received the vehicle alone in the same manner.

A routine positive control study has been performed. Three groups, each of five animals, were treated with 50 µL (25 µL per ear) of α-Hexylcinnamaldehyde, tech., 85% as a solution in acetone/olive oil 4:1 at concentrations of 5%, 10% or 25% v/v. A further group of five animals was treated with acetone/olive oil 4:1 alone and served as the vehicle control group.

3H-Methyl Thymidine Administration
Five days following the first topical application of the test item or vehicle (Day 6) all mice were injected via the tail vein with 250 µL of phosphate buffered saline (PBS) containing 3H-methyl thymidine (3HTdR: 80 µCi/mL, specific activity 2.0 Ci/mmoL, ARC UK Ltd) giving a total of 20 µCi to each mouse.

Observations
Clinical Observations: All animals were observed twice daily on Days 1, 2 and 3 and on a daily basis on Days 4, 5 and 6. 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 Day 6 (prior to termination).

Terminal Procedures
Termination: Five hours following the administration of 3HTdR all mice were killed by carbon dioxide asphyxiation followed by cervical separation. For each individual animal of each group the draining auricular lymph nodes were excised and processed. For each individual animal 1 mL of PBS was added to the lymph nodes.

Preparation of Single Cell Suspension: A single cell suspension of the lymph node cells for each individual animal was prepared by gentle mechanical disaggregation through a 200-mesh stainless steel gauze. The lymph node cells were rinsed through the gauze with 4 mL of PBS into a petri dish labeled with the study number and dose concentration. The lymph node cells suspension was transferred to a centrifuge tube. The petri dish was washed with an additional 5 mL of PBS to remove all remaining lymph node cells and these were added to the centrifuge tube. The lymph node cells were pelleted at 1400 rpm (approximately 190 g) for 10 minutes. The pellet was re-suspended in 10 mL of PBS and re-pelleted. To precipitate out the radioactive material, the pellet was re-suspended in 3 mL of 5% Trichloroacetic acid (TCA).

Determination of 3HTdR Incorporation: After approximately 18 hours incubation at approximately 4 oC, the precipitates were recovered by centrifugation at 2100 rpm (approximately 450 g) for 10 minutes, re-suspended in 1 mL of TCA and transferred to 10 mL of scintillation fluid. 3HTdR incorporation was measured by beta-scintillation counting. The "Poly Q™" vials containing the samples and scintillation fluid were placed in the sample changer of the scintillator and left to stand in darkness for approximately 20 minutes. The purpose of this period of time in darkness was to reduce the risk of luminescence, which has been shown to affect the reliability of the results. After approximately 20 minutes, the vials were shaken vigorously. The number of radioactive disintegrations per minute was then measured using the Beckman LS6500 scintillation system (Beckman Instruments Inc, Fullerton, CA, USA).

Positive control substance(s):

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

Statistics:

Data was processed to give group mean values for disintegrations per minute and standard deviations where appropriate. Individual and group mean disintegrations per minute values were assessed for dose response relationships. Data was first assessed for suitability by analysis of normality and homogeneity of variance. If the assumptions that the data are both normally distributed and has homogeneity of variances, then parametric one way analysis of variance (ANOVA) and Dunnett’s multiple comparison procedure were used to determine statistical significance. If the assumptions were not met, non-parametric Kruskal-Wallis Rank Sum and Mann-Whitney U test procedures were used.
Probability values (p) are presented as follows:
P<0.001 ***
P<0.01 **
P<0.05 *
P>=0.05 (not significant)

Positive control results:

The positive control item, α-Hexylcinnamaldehyde, tech., 85%, gave a Stimulation Index of greater than 3 when tested at a concentration of 25 % v/v in acetone/olive oil 4:1. The concentration of α-Hexylcinnamaldehyde, tech., 85% expected to cause a 3 fold increase in 3HTdR incorporation (EC3 value) was calculated to be 20%. α-Hexylcinnamaldehyde, tech., 85% was considered to be a sensitizer under the conditions of the test.

Key result

Parameter:

EC3

Remarks:

(%)

Value:

68

Key result

Parameter:

other: NOEC

Remarks:

(%)

Value:

30

Parameter:

SI

Remarks on result:

other: The following SI values were derived at 10, 30 and 100%: 1.27, 1.82 and 3.98, respectively. Based on these results the EC3 is 68%. The NOEC is 30%.

Cellular proliferation data / Observations:

Clinical Observations and Mortality Data:
There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test.

Body Weight:
Body weight change of the test animals between Day 1 and Day 6 was comparable to that observed in the corresponding control group animals over the same period.

Preliminary Screening Test:

No signs of systemic toxicity, visual local skin irritation or irritation indicated by an equal to or greater than 25% increase in mean ear thickness were noted.

Based on this information the undiluted test item and the test item at concentrations of 30% and 10% v/v in acetone/olive oil 4:1were selected for the main test.

Interpretation of results:

other: Skin sensitiser, Category 1B

Remarks:

in accordance with EU CLP 1272/2008 and its amendments

Conclusions:

The test item was considered to be a sensitiser under the conditions of the test and resulted in an EC3 of 68%.

Executive summary:

The skin sensitisation potential of the test substance has been tested according to OECD TG 429: Local Lymph Node Assay" and GLP. Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 100%, this concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay. Three groups, each of five animals, were treated with 50 µL (25 µL per ear) of the undiluted test item or the test item as a solution in acetone/olive oil 4:1 at concentrations of 30% or 10% v/v. A further group of five animals was treated with acetone/olive oil 4:1 alone. At 10, 30 and 100% the substance showed SI values of 1.27, 1.82 and 3.98, respectively. The concentration of test item expected to cause a 3 fold increase in 3HTdR incorporation (EC3 value) was calculated to be 68%. A NOEC of 30% is derived. The test item was considered to be a sensitiser under the conditions of the test.

Reference 2

Endpoint:

skin sensitisation: in vitro

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

LLNA study with Vigoflor

The skin sensitisation potential of the test substance has been tested according to OECD TG 429: Local Lymph Node Assay" and GLP. Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 100%, this concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay. Three groups, each of five animals, were treated with 50 µL (25 µL per ear) of the undiluted test item or the test item as a solution in acetone/olive oil 4:1 at concentrations of 30% or 10% v/v. A further group of five animals was treated with acetone/olive oil 4:1 alone. At 10, 30 and 100% the substance showed SI values of 1.27, 1.82 and 3.98, respectively. The concentration of test item expected to cause a 3 fold increase in 3HTdR incorporation (EC3 value) was calculated to be 68%. A NOEC of 30% is derived. The test item was considered to be a sensitiser under the conditions of the test.

Additional human information:

Five HRIPT studies are available. In the first HRIPT some positive but questionable results (4/10 subjects) were seen. The concentration with which the subjects were tested is not presented.

In additional tests a total 147 subjects were tested. Most subjects were tested with 1% and 71 with 2.5% substance. No skin sensitisation was seen. The substance is therefore a non-human sensitiser up to 2.5%.

Justification for classification or non-classification

Based on the EC3 value of 68% 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 and its amendments, it has to be classified for skin sensitisation, Category 1B, H317: May cause an allergic skin reaction.