Indeno[4,5-d]-1,3-dioxin, 4,4a,5,6,7,8,9,9b-octahydro-7,7,8,9,9-pentamethyl-

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:

Between 06 May 2003 and 12 May 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with OECD TG 405 under GLP.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

other: CBA/Ca (CBA/CaBkl)

Sex:

female

Details on test animals and environmental conditions:

Sixteen female CBA/Ca (CBA/CaBkl) strain mice were supplied by B & K Universal Ltd, Hull, UK. On receipt the animals were randomly allocated to cages. The animals were nulliparous and non-pregnant. At the start of the study the animals were in the weight range of 15 to 23 g, and were eight to twelve weeks old. After an acclimatisation period of at least five days, each animal was selected at random and given a number unique within the study by
indelible ink-marking on the tail and a number written on the cage card.

The animals were individually housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes. Free access to mains tap water and food (Certified Rat and Mouse Diet (Code 5LF2) supplied by International Product Supplies Limited, Wellingborough, Northants, UK) was allowed throughout the study.

The temperature and relative humidity were controlled to remain within target ranges of 19 to 25 °C and 30 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 approximately fifteen changes per hour and the lighting was controlled by a time switch to give twelve hours continuous light (06.00 to 18.00) 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:

10 %, 25 % or 50 % v/v in acetone/olive oil 4:1

No. of animals per dose:

4

Details on study design:

Test Material Administration
Groups of four mice were treated with the test material at concentrations of 10 %, 25 % or 50 % v/v in acetone/olive oil 4: 1. Information available suggested that the test material would not produce systemic toxicity or excessive local irritation at the highest suitable concentration. The mice were treated by daily application of 25 µL of the appropriate concentration of the test material to the dorsal surface of each ear for three consecutive days (Days 0, 1, 2). The test material 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 four mice received the vehicle alone in the same manner.

3H-Methyl Thymidine Administration
Five days following the first topical application of the test material (Day 5) all mice were injected via the tail vein with 250 µL of phosphate buffered saline containing 3H-methyl thymidine 3HTdR: 80 µCi/mL, specific activity 2.0 Ci/mmol, Amersham Biosciences UK Ltd) giving a total of 20 µCi to each mouse.

Observations
Clinical Observations: All animals were observed twice daily on Days 0, 1 and 2 and on a daily basis on Days 3, 4 and 5. Any signs of toxicity or signs of ill health during the study were recorded.

Bodyweights: The bodyweight of each mouse was recorded on Day 0 (prior to dosing) and Day 5 (prior to termination).

Terminal Procedures
Termination: Five hours following the administration of 3HTdR all mice were killed by carbon dioxide asphyxiation. The draining auricular lymph nodes from the four mice were excised and pooled for each experimental group. For each group 1 mL of phosphate buffered saline (PBS) was added to the pooled lymph nodes.

Preparation of Single Cell Suspension: A single cell suspension of pooled lymph node cells 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 labelled with the project number and dose concentration. The lymph node cells suspension was transferred to a 10 mL 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 pooled lymph node cells were pelleted at 1400 rpm (approximately 190 g) for ten 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 overnight incubation at 4 °C, the precipitates were recovered by centrifugation at 2 100 rpm (approximately 450 g) for ten minutes, re-suspended in 1 mL of TCA and transferred to 10 mL of scintillation fluid (Optiphase 'Trisafe'). 3HTdR incorporation was measured by beta-scintillation counting. The "Poly QTM" vials containing the samples and scintillation fluid were placed in the sample changer of the scintillator and left for approximately twenty 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 twenty 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).

Positive control substance(s):

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

Parameter:

SI

Remarks on result:

other: see Remark

Remarks:

A stimulation index of greater than 3 was recorded for the two higher concentrations of the test material (25 and 50 % v/v). A stimulation index of less than 3 was recorded for the lower concentration of the test material (10 % v/v). The estimated EC3 is 20% and the NOEC is 10%.

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: The radioactive disintegrations per minute (dpm) per lymph node and the stimulation index (SI) are given in Table 1.

Clinical Observations and Mortality Data There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the study. Bodyweight Bodyweight changes of the test animals between Day 0 and Day 5 were comparable to those observed in the corresponding control group animals over the same period.

Table 1: Dpm, Dpm/Node and Stimulation Index (SI)

Concentration (% v/v) in acetone/olive oil 4:1	Dpm	Dpm/Node^a	Stimulation Index (SI)^b	Result
Vehicle	13920.13	1740.02	N/A	N/A
10	18601.90	2325.24	1.34	Negative
25	44121.08	5515.14	3.17	Positive
50	42621.67	9077.71	5.22	Positive

a = Dpm/node obtained by dividing the Dpm value by 8 (total number of lymph nodes)

b = Stimulation Index of 3.0 or greater indicates a positive result

N/A = Not applicable

Interpretation of results:

sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The skin sensitisation potential of the test material was assessed according to OECD Guideline 429. The test material was considered to be a sensitiser under the conditions of the test. The test material was classified as a sensitiser according to EU labelling regulations Commission Directive 2001/59/EC.

Executive summary:

A study was performed to assess the skin sensitisation potential of the test material according to OECD TG 429 using CBA/Ca strain mouse Three groups, each of four animals, were treated with 50 µL (25 µL per ear) of the test material as a solution in acetone/olive oil 4:1 at concentrations of 10 %, 25 % and 50 % v/v. A further group of four animals was treated with acetone/olive oil 4:1 alone. The Stimulation Index (SI) expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows; at 10% the SI was 1.34, at 25%, 3.17 and at 50%, 5.22.The test material was considered to be a sensitiser under the conditions of the test with an EC3 value of circa 20%.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

Two studies on skin sensitisation are available an LLNA and a HRIPT test. The LLNA results are taken forward to the hazard and risk characterisation because a positive result was found in this test. The HRIPT test is shortly summarised below:

In a human Repeated Insult Patch test according to a modified Shelanski - Shelanski human patch test method, 102 human subjects (male/female) were treated on the (cleaned) upper back to 4% (w/w) of the test article in EtOH: DEP (1:3) with an occlusive patch for 24 hours (0.2 ml on 3.63 cm2 patch). This application results in an exposure of 2360 ug/cm2 (0.2 g x 1.07 (RD) x 0.04 (fraction test substance) x 1000 (conversion to ug) / 3.63 cm2). This application was repeated 9 times on Monday, Wednesday and Friday during the induction period. Sites were graded for dermal irritation 24 hours after removal of the patches on Tuesday and Thursday and 48 hours after removal on Saturday. After a 2-week rest period challenge patches were applied on untreated test sites for 24 hours. Dermal irritation was evaluated 0, 24 and 48 hours after removal of the patches. Hundred subjects completed the study. Challenge reactions were scored at 24, 48 and 72 hours. During inducation and during challenge all scores were zero.This means that the substance did not show skin sensitising properties in human volunteers up to 4% (2360 ug/cm2).

Migrated from Short description of key information:
A study was performed to assess the skin sensitisation potential of the test material according to OECD TG 429 (LLNA) using CBA/Ca strain mouse Three groups, each of four animals, were treated with 50 µL (25 µL per ear) of the test material as a solution in acetone/olive oil 4:1 at concentrations of 10 %, 25 % and 50 % v/v. A further group of four animals was treated with acetone/olive oil 4:1 alone. The Stimulation Index (SI) expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows; at 10% the SI was 1.34, at 25%, 3.17 and at 50%, 5.22.The test material was considered to be a sensitiser under the conditions of the test with an EC3 value of circa 20% and the NOEC is 10%.

Justification for selection of skin sensitisation endpoint:
One reliable LLNA test is available and is adequate for covering this endpoint and it exceeds the reliability of the available HRIPT test.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

The respiratory sensitization of the substance is assessed using the integrated evaluation strategy for respiratory sensitization data in the ECHA guidance (R7A, Fig. 7.3-2); 1) The substance is a weak skin sensitizer; 2) The substance does not belong to the di-isocyanates; 3) The substance has not structural alerts or is structurally related to chemicals causing respiratory sensitization 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. Using this ITS in the ECHA guidance it can be concluded that the substance is not a respiratory sensitiser.

Migrated from Short description of key information:
For respiratory sensitisation the results of the integrated evaluation strategy for respiratory sensitization data in the ECHA guidance (R7A, Fig. 7.3-2) are adequate for assessing this endpoint and result in the absence of respiratory sensitisation.

Justification for selection of respiratory sensitisation endpoint:
For respiratory sensitisation the results of the integrated evaluation strategy for respiratory sensitization data in the ECHA guidance (R7A, Fig. 7.3-2) are adequate for assessing this endpoint.

Justification for classification or non-classification

Based on the available information, Nebulone should be classified as sensitising to the skin in accordance with the criteria outlined in Annex I of 67/548/EEC (R43) (DSD) and Annex VI of 1272/2002/EC (H317 (1B) (CLP). Using the ITS in the ECHA guidance (R.7a) Nebulone is not considered to be a respiratory sensitiser in accordance with the criteria outlined in Annex I of 67/548/EEC and Annex VI of 1272/2002/EC.