rac-(1R,4S,4aR,8R,8aS)-9-(dichloromethylidene)-8-hydroxyoctahydro-1,4-methanonaphthalen-5(1H)-one

Endpoint summary

Description of key information

- Skin sensitisation, not sensitising, in vivo, female, mouse, OECD 429, Pooles 2014

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Remarks:

This study was conducted solely to comply with a non-EU national registration requirement.

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 Apr 2014 to 24 Apr 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP compliant, guideline study, available as unpublished report, no restrictions, fully adequate for assessment.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Adopted 22 July 2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

2003

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA/Ca

Remarks:

CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 15-23 g
- Housing: Individually housed in suspended solid floor polypropylene cages furnished with softwood woodflakes.
- Diet: Rodent diet, ad libitum
- Water: Mains water, ad libitum
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25
- Humidity (%): 30-70
- Air changes (per hr): approximately 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
07 Apr 2014 to 24 Apr 2014

Vehicle:

other: 1% pluronic L92 in distilled water

Concentration:

50%, 25%, 10% test item and vehicle only (controls)

No. of animals per dose:

4

Details on study design:

RANGE FINDING 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 dose levels selected for the main test were 50%, 25% and 10% w/w in 1% pluronic L92 in distilled water.

MAIN STUDY
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.
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) giving a total of 20 µCi to each mouse.
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.
The body weight of each mouse was recorded on Day 1 (prior to dosing) and Day 6 (prior to 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. A single cell suspension of the lymph node cells for each individual animal was prepared. After approximately 18 hours incubation at approximately 4°C, the precipitates were recovered by centrifugation, resuspended in 1 mL of 5% trichloroacetic acid (TCA) and transferred to 10 mL of scintillation fluid. 3HTdR incorporation was measured by β-scintillation counting. The number of radioactive disintegrations per minute was then measured using a scintillation system.

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local lymph node assay
- Criteria used to consider a positive response: The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per animal and as the ratio of 3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index). The test item was regarded as a sensitizer if at least one concentration of the test item resulted in a threefold or greater increase in 3HTdR incorporation compared to control values. Any test item failing to produce a threefold or greater increase in 3HTdR incorporation will be classified as a "non-sensitizer." See Table 1 in 'Any other information on materials and methods incl. tables'.

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 by analysis of homogeneity of variance followed by one way analysis of variance (ANOVA). In the event of a significant result from the ANOVA, pairwise comparisons were performed between control and treated groups. For homogenous datasets Dunnett’s Multiple Comparison test was used and for non-homogenous datasets Dunnett’s T3 Multiple Comparison Method was used.

Parameter:

SI

Value:

0.89

Test group / Remarks:

10% test item

Parameter:

SI

Value:

0.87

Test group / Remarks:

25% test item

Key result

Parameter:

SI

Value:

1.04

Test group / Remarks:

50% test item

Cellular proliferation data / Observations:

PRELIMINARY 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 dose levels selected for the main test were 50%, 25% and 10% w/w in 1% pluronic L92 in distilled water.

CELLULAR PROLIFERATION DATA
See Table 1 in ‘Any other information on results incl. tables’.

DETAILS ON STIMULATION INDEX CALCULATION
The stimulation index was lower than 3 for the test item at any tested concentration.
See Table 1 in ‘Any other information on results incl. tables’.

EC3 CALCULATION
Not applicable.

CLINICAL OBSERVATIONS:
No mortality or signs of systemic toxicity was observed for the test item treated animals during the study.

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

Table 1: Skin sensitisation potential

Concentration (% v/v) in 1% pluronic L92 in distilled water	Animal Number	dpm/ Animal^a	Mean dpm/Animal (Standard Deviation)	Stimulation Index^b	Result
Vehicle	1-1	2646.77	2799.46 (±501.11)	na	na
	1-2	2340.33
	1-3	2697.91
	1-4	3512.83
10	2-1	2455.21	2478.49 (±260.30)	0.89	Negative
	2-2	2186.58
	1	2451.90
	2-4	2820.27
25	3-1	3354.34	2429.84 (±697.47)	0.87	Negative
	3-2	2575.16
	3-3	1819.71
	3-4	1970.14
50	4-1	3714.67	2905.99 (±999.17)	1.04	Negative
	4-2	3636.67
	4-3	2694.16
	4-4	1578.47

N/A = not applicable

Interpretation of results:

GHS criteria not met

Conclusions:

The test substance was considered to be a non-sensitizer under the conditions of the test.

Executive summary:

A GLP compliant OECD 429 study was performed to assess the skin sensitization potential of the test item in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear. Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 50% w/w, this concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay (LLNA). Three groups, each of four animals, were treated with 50 µL (25 µL per ear) of the test item as a solution in 1% pluronic L92 in distilled water at concentrations of 50%, 25% or 10% w/w. A further group of four animals was treated with 1% pluronic L92 in distilled water alone.

No mortality or signs of systemic toxicity was observed for the test item treated animals during the study. No treatment related effects were observed on body weight. The Stimulation Indices (expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group) were 0.89, 0.87 and 1.04 (all negative results) for test item concentrations of 10, 25 and 50% w/w, respectively. Therefore, no EC3 value could be determined.

Based on the results, the test substance was considered to be a non-sensitizer under the conditions of the test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Skin sensitisation

A GLP compliant OECD 429 study was performed to assess the skin sensitization potential of the test item in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear. Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 50% w/w, this concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay (LLNA). Three groups, each of four animals, were treated with 50 µL (25 µL per ear) of the test item as a solution in 1% pluronic L92 in distilled water at concentrations of 50%, 25% or 10%w/w. A further group of four animals was treated with 1% pluronic L92 in distilled water alone.

No mortality or signs of systemic toxicity was observed for the test item treated animals during the study. No treatment related effects were observed on body weight.The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group.The Stimulation Indices (expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group) were 0.89, 0.87 and 1.04 (all negative results) for test item concentrations of 10, 25 and 50% w/w, respectively, Therefore,no EC3 value could be determined.

Based on the results, the test substance was considered to be a non-sensitizer under the conditions of the test.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the mouse LLNA study, classification of the test substance as sensitizing is not warranted in accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.