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Description of key information

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-compliant OECD TG 429.
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
other: CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Netherlands
- Age at study initiation: 7-12 weeks
- Weight at study initiation: 13.6-21.8 g
- Housing: in groups of 4 in Makrolon type-3 cages with standard softwood bedding
- Diet: pelleted standard Kliba 3433 mouse maintenance diet, ad libitum
- Water: community tap water from Itingen, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12 / 12

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
1, 2.5, 5, 10 and 25% in acetone/olive oil, 4:1
No. of animals per dose:
4 females/dose
Details on study design:
MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response: a test item was regarded as a sensitiser if the following criteria were fulfilled:
-- exposure to at least one concentration of teh test item resulted in an incorporation of 3-H-methl thymidine at least 3-fold or greater than recorded in control mice, as indicated by the stimulation index;
-- the data are comparible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

TREATMENT PREPARATION AND ADMINISTRATION:
Each group of mice was treated by epidermal topical application to the dorsal surface of each ear lobe (left and right) with different test item concentrations. The mice of the positive control group (alpha-hexylcinnamaldehyde) were treated in the same way at a concentration of 25%. The application volume of 25 μL was spread over the entire dorsal surface of each ear lobe once daily for three consecutive days. A further group of mice was treated with an equivalent volume of the relevant vehicle alone. A hair dryer was used to dry the ear's surface as quickly as possible to avoid loss of test item applied.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Mean values and standard deviations were calculated.
Positive control results:
Stimulation index of 7.2 for 25% solution
Parameter:
SI
Remarks on result:
other: 0.6, 0.7, 0.6, 1.3 and 4.9 for 1, 2.5, 5, 10 and 25% solution, respectively
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: 3960, 4687, 4063, 8100 and 31487 for 1, 2.5, 5, 10 and 25% solution, respectively

No test item-related clinical signs were observed in any group. The body weight of the animals, recorded at the start of acclimatisation period and prior to necropsy, was within the range commonly recorded for animals of this strain and age.

Interpretation of results:
sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Lyral is sensitising to skin.
Executive summary:

In a GLP-compliant study, performed according to OECD Guideline 429 (Local Lymph Node Assay in mice), five groups of 4 female mice were treated by topical application to the dorsal surface of each ear lobe with 1, 2.5, 5, 10 and 25% solution of Lyral in acetone/olive oil (4:1 v/v) for three consecutive days. A positive control group of four mice was treated with alpha-hexylcinnamaldehyde (25%) and a vehicle control group was treated with the vehicle alone. Five days after the first topical application the mice were injected intravenously into a tail vein with 3H-thymidine, the draining auricular lymph nodes excised, pooled per group and their proliferative capacity measured by the incorporation of 3H-thymidine in a β-scintillation counter. The stimulation indices were 0.6, 0.7, 0.6, 1.3, and 4.9 for 1, 2.5, 5, 10 and 25% solution of Lyral, respectively. For the positive control a stimulation index of 7.2 was obtained. Based on the results of the study, Lyral is considered to give a positive result in the LLNA test.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

Lyral has been tested in an LLNA test and two well conducted HRIPT test which will be used for the DNEL derivation. In addition, the present risk assessment will also take into account the DNEL derived by the Research Institute for Fragrance Materials (RIFM) and the maximum concentration in the end product established by the International FRagrance Association.

LLNA study: A GLP-compliant study, performed according to OECD Guideline 429 (Local Lymph Node Assay in mice), was available for assessment (RCC Ltd., 2001). Five groups of 4 female mice were treated by topical application to the dorsal surface of each ear lobe with 1, 2.5, 5, 10 and 25% solution of Lyral in acetone/olive oil (4:1 v/v) for three consecutive days. A positive control group of four mice was treated with alpha-hexylcinnamaldehyde (25%) and a vehicle control group was treated with the vehicle alone. Five days after the first topical application the mice were injected intravenously into a tail vein with 3H-thymidine, the draining auricular lymph nodes excised, pooled per group and their proliferative capacity measured by the incorporation of 3H-thymidine in a β-scintillation counter. The stimulation indices were 0.6, 0.7, 0.6, 1.3, and 4.9 for 1, 2.5, 5, 10 and 25% solution of Lyral, respectively. For the positive control a stimulation index of 7.2 was obtained. Based on the results of the study, Lyral is considered to give a positive result in the LLNA test. An EC3 value of 17.1% and a NOEL of 10% were calculated based on the study results. This value can be converted to an EC3 figure expressed as μg/cm2 using EC3 (μg/cm2 ) = 17.1% x 250 x 1 g/mL = 4275 μg/cm2.

Skin sensitising properties of Lyral were extensively investigated in a number of studies with human volunteers. Two most recent human repeated insult patch tests, using the largest numbers of volunteers, will be summarised here.

First HRIPT test in 2003: In the assay conducted by Harrison Research Laboratories, Inc. (2003) 235 volunteers (63 males, 172 females, aged from 18 to 70 years) were challenged occlusively with 0.3 mL of the test material for 24 hours on Monday, Wednesday and Friday, for 3 consecutive weeks, for a total of 9 applications. After a ca. 2 weeks rest period they were challenged at a virgin site for 24 hours and reactions were scored immediately after the patch removal, 24, 48 and 72 hours later. The test substance was applied as a 5.3% solution in 75% DEP:25% ethanol. Vehicle control and normal saline were used as negative controls. 201 subjects completed the study. The applied amount of the test substance corresponded to a total dose of 4000 μg/cm2 (rounded off figure).

During the induction phase, one of the subjects exhibited a 1-level plus edema reaction; the test site was changed. Other subjects exhibited low-level, transient (±/1) reactions. At the challenge, low-level, transient (±) reactions were exhibited. Low-level, transient reactions (±/1) were also observed in several subjects upon exposure to control substances. No effect was observed at a concentration of 5.3%.

Though the application site area was not reported, it is assumed that this area is based on a standard site, which is 3.63 cm2. The NOEL in this study is considered to be 5.3%, corresponding with 4000 ug/cm2.

Second HRIPT test: In the second assay conducted by Consumer Product Testing Co. (1999) a dose of 0.2 mL of a 15% solution of the substance in 75% alcohol SD39C/25% DEP was applied to a 3.63 cm2 area patch (equivalent to 8264 μg/cm2). The patches were applied under occlusion to the upper back of 119 volunteers (18 males, 91 females, 18-77 years) for 24 hours with a 24-48 hours rest period on Monday, Wednesday and Friday for 3 consecutive weeks, for a total of 9 applications. 109 volunteers completed the study. Approximately 2 weeks after the last application the subjects were challenged with 24 hr application of the same patch at a virgin site. Subjects that reacted to a challenge were rechallenged approximately 5-6 weeks after the primary challenge. In case of a positive reaction, a second rechallenge was performed after a 5 weeks rest period.

Scattered, barely perceptible (+) to moderate (2-level) patch test responses were observed in 4/119 test panelists during the induction phase. Positive reactions were observed in two subjects (#27 and #106) in the challenge phase. These subjects were rechallenged after a 5-6 weeks rest period. Subject #106 exhibited no reactions to the treatment and was considered to be not sensitised. Subject #27 exhibited a positive reaction and was rechallenged again after 5 weeks of rest. The reaction was again positive. The subject #27 had psoriasis and had participated in several patch tests a year before the study. He also showed a mild positive response (1-level) to a deodorant product. It was concluded that the subject reactivity appeared to have been elicited by the material itself and was not indicative of pre-sensitisation. Thus no sensitisation reactions were considered to be observed in the study. No effect was observed at a concentration of 15%.

This NOEL value of 15% can be converted to a value expressed as μg/cm2 based on an application volume of 0.2 mL, an application area of 3/4" x 3/4" (3.63 cm2) and the relative density of the substance of ca. 1 (0.9908) g/mL will become ).15 x 200 mg/ 3.63 cm2 = 8264 μg/cm2.

According to the REACH Guidance on information requirements and chemical safety assessment, the EU Expert group on skin sensitization nominated by the Technical Committee on Classification and Labelling concluded that it would not be appropriate to derive a DNEL for skin sensitization based on elicitation (see Chapter R.8 of the REACH Guidance on information requirements and chemical safety assessment, Appendix 8-10, page 128). Therefore data on induction of skin sensitisation are used for DNEL derivation. This also means that information on elicitation from amongst others the study of Schnuch et al. (2009) or Scientific Committee on Consumer Safety (SCCS) (http://ec.europa.eu/health/scientific_committees/consumer_safety/docs/sccs_o_074.pdf) will not be used to establish the NOAEL.

Lyral was only sensitising in the LLNA test and therefore the NOEL established in this test will be used as a starting point to derive a DNEL.

RIFM also has established a DNEL for skin sensitisation, calling it a NESIL using a Weight of Evidence approach:

All data on the induction of sensitisation presented in this submission show that the weight of evidence No Expected Sensitisation Induction Level (NESIL) for induction is 4000μg/cm2. This is supported by the robustness of the scientific data and highlighted by: 1) the murine local lymph node assay showed an EC3 value of 17.1% (4275μg/cm2); 2) a human repeated insult patch test with 201 subjects that confirms that a dose of 4000μg/cm2does not induce sensitisation; 3) HMPCC, which collectively show that doses between 938 and 1592μg/cm2did not induce sensitisation in 749 subjects; and 4) Lyral alone did not induce sensitisation in predictive human sensitisation studies.

This derived NESIL has led to the following maximum concentration in the end products (www.ifraorg.org, 44th Amendment). IFRA has identified several categories for consumer exposure (http://rifm.org/doc/QRAInfoJuly2011.pdf). Only the categories relevant for REACH are presented below:

Category 7: 0.02 % (intimate wipes)

Category 9: 0.2 % (soap type, toilet paper and napkins)

Category 10: 0.2 % (Detergents: all types)

Category 11: (e.g. air fresheners, insecticides, shoe polishes, toilet blocks, fuels, paints), See Note

Note: Category 11 includes all non-skin contact or incidental skin contact products. Due to the negligible skin contact from these types of products there is no justification for a restriction of the concentration of this fragrance ingredient in the finished product.


Migrated from Short description of key information:
In a well conducted LLNA test Lyral showed to be skin sensitising at an EC3 of 17%, a NOEL of 10% was derived in this study. Also two HRIPT studies are available, showing no sensitisation up to 15%.. Lyral has no structural alert for respiratory sensitisation.

Justification for selection of skin sensitisation endpoint:
Skin sensitisation needs to be assessed according to the REACH regulation. The LLNA study according to OECD TG 429 has been selected in which the substance showed to be a sensitiser. The HRIPT test is information is added for the risk assessment.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:
Migrated from Short description of key information:
Lyral is not expected to be of concern for respiratory sensitisation. According to the REACH guidance on respiratory sensitisation (R.7.4) the substance is not expected to be a respiratory sensitser because its structure does not contain (di)isocyanate groups which are known for causing such an effect (Scheme of R.7A, Fig. 7.3-2). Lyral also does not contain any structural alerts mentioned in the document on respiratory effects of the EU Scientific committee e.g. iso-thiocyanates, amines, anhydrides, acrylates, diazonium salts, reactive dyes or metals http://ec.europa.eu/health/scientific_committees/reports/index_en.htm, select Annex VI)

Justification for selection of respiratory sensitisation endpoint:
In an expert statement it has been reasoned that Lyral is not a respiratory sensitiser based on its chemical structure. The substance does not contain respiratory sensitising groups.

Justification for classification or non-classification

Based on the positive results in the LLNA study, Lyral has to be classified as sensitizing to skin (R43) according to EU Directive 67/548/EEC (DSD). According to EU Classification, Labeling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008. Since the EC3 > 2%, Lyral has to be classified as Skin Sensitization Category 1B, H317: May cause an allergic skin reaction. Since Lyral does not have a structural alert for respiratory sensitisation and because of its low volatility, classification and labelling for respiratory sensitisation is not warranted.