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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
Study period:
18 October 2011 - 9 February 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed according to OECD 429 and under GLP-conditions.
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymphnode assay (LLNA)
Test material information:
Composition 1
Species:
mouse
Strain:
other: CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories UK Ltd., Oxon, UK
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 15 - 23 g
- Housing: individually, in suspended solid-floor propylene cages furnished with softwood woodflakes
- Diet (e.g. ad libitum): ad libitum, 2014C Teklad Global Rodent Diet
- Water (e.g. ad libitum): ad libitum, tap water
- Acclimation period: at least 5 days

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

IN-LIFE DATES: From: To:
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
25%, 50%, 100%
No. of animals per dose:
5
Details on study design:
RANGE FINDING TESTS:
- Concentration: Daily treatment for 3 consecutive days (application of 25µL undiluted test item to the dorsal surface of each ear).
- Irritation: 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 50% and 25% v/v in acetone/olive oil 4:1 were selected for the main test.
- Lymph node proliferation response:

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay (LLNA) - individual animal approach
- Criteria used to consider a positive response (DSD/CLP): Stimulation Index (SI) > 3

TREATMENT PREPARATION AND ADMINISTRATION:
- The mice were treated by daily application of 25µL of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days.
- The test item was formulated within two hours of being applied to the test system. It is assumed that the formulation was stable for this duration.
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 reponse 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.
Positive control results:
SI expressed as the mean radioactive incorporation for α-Hexylcinnamaldehyde was 7.67, which is a positive result (SI > 3).
Parameter:
SI
Remarks on result:
other: - 25%: 2.69 - 50%: 3.66 - 100%: 3.52
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Mean DPM/animal (Standard Deviation) - Vehicle (0%): 2295.90 (± 789.82) - 25%: 6168.85 (± 2029.92) - 50% 8413.63 (± 1117.15) - 100% 8073.27 (± 3667.10)

- 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.

- Bodyweight: Bodyweight changes of the test animals between Day 1 and Day 6 were comparable to those observed in the corresponding control group animals over the same period.

- An EC3 value of 33% was calculated using this equation: EC3 = c + [[(3-d)/(b-d)] x (a-c)].

Interpretation of results:
sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The skin sensitising potential of Cashmeran was determined using a Local Lymph Node Assay (LLNA).. A maximum Stimulation Index of 3.66, and a calculated EC3 value of 33% indicate that the substances needs to be classified as a sensitiser according to the criteria outlined in Annex I of 1272/2008/EC and Annex VI of 67/548/EEC, under the conditions of this study
Executive summary:

This Local Lymph Node Assay (OECD 429) was performed to determine the sensitising potential of Cashmeran in mice. Groups of 5 mice were treated with the undiluted Cashmeran or Cashmeran at concentrations of 50% or 25% v/v in acetone/olive oil 4:1.α-Hexylcinnamaldehyde, freshly prepared as a 25% v/v dilution in acetone/olive oil 4:1, was used as positive control. Clinical observations and bodyweights were recorded and lymph node proliferation was determined using 3HTdR incorporation.

 

The number of radioactive disintegrations per minute (dpm) reflect the proliferation reponse of lymph node cells, and were 2295.90, 6168.85, 8413.63, and 8073.27 mean dpm/animal for the 0%, 25%, 50%, and 100% concentration groups, respectively. This corresponds with a lymph node proliferation of 2.69, 3.66 and 3.52, respectively, for the Cashmeran-treated groups, calculated as the Stimulation Index (SI). No mortality and no signs of systemic toxicity were observed. No effects on bodyweight (gain) were observed.

An EC3 value (the concentration of test item expected to cause a 3 -fold increase in 3HTdR incorporation) of 33% was calculated. Based on the absence of effects at the lowest concentration tested, a NOEC of 25% could be established.

 

Under the conditions of this study, a maximum SI of 3.66 was calculated. Considering the SI threshold of 3 as stated in Annex I of 1272/2008/EC and Annex VI of 67/548/EEC, the substance needs to be classified as sensitiser for concentrations ≥ 33%.

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

A Local Lymph Node Assay (OECD 429) was performed to determine the sensitising potential of Cashmeran in mice. Groups of 5 mice were treated with the undiluted Cashmeran or Cashmeran at concentrations of 50% or 25% v/v in acetone/olive oil 4:1.α-Hexylcinnamaldehyde, freshly prepared as a 25% v/v dilution in acetone/olive oil 4:1, was used as positive control. Clinical observations and bodyweights were recorded and lymph node proliferation was determined using 3HTdR incorporation.

The number of radioactive disintegrations per minute (dpm) reflect the proliferation response of lymph node cells, and were 2295.90, 6168.85, 8413.63, and 8073.27 mean dpm/animal for the 0%, 25%, 50%, and 100% concentration groups, respectively. This corresponds with a lymph node proliferation of 2.69, 3.66 and 3.52, respectively, for the Cashmeran-treated groups, calculated as the Stimulation Index (SI). A limited dose effect concentration is derived. No mortality and no signs of systemic toxicity were observed. No effects on bodyweight (gain) were observed.

An EC3 value (the concentration of test item expected to cause a 3 -fold increase in 3HTdR incorporation) of 33% was calculated.

Under the conditions of this study, a maximum SI of 3.66 was calculated. Considering the SI threshold of 3 as stated in Annex I of 1272/2008/EC and Annex VI of 67/548/EEC, the substance needs to be classified as sensitiser for concentrations ≥ 33%. Based on the absence of effects at the lowest concentration tested, a NOEC of 25% could be established.

 In a human Repeated Insult Patch test according to a modified Shelanski - Shelanski human patch test method, 120 human subjects (male/female) were treated on the (cleaned) upper back to 10% (w/w) of the test article in EtOH:DEP (1:3) with an occlusive patch for 24 hours (0.2 ml on 3.63 m2 patch). This 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. An extra assessment was done after 96 hours in case of reactions.

Six volunteers discontinued study participation for reasons unrelated to the test material. In the remaining 106 human volunteers no adverse skin reactions were observed after repeated induction (9 times) followed by one challenge patch. Therefore it can be concluded that the test article does not induce sensitization up to that 10% concentration, under the conditions of this test.

Migrated from Short description of key information:
Skin sensitisation: sensitising to skin, EC3 33% (LLNA, OECD429) and a NOAEL in a HRIPT test of 10%.

Justification for selection of skin sensitisation endpoint:
Two adequate studies are available: an LLNA and a HRIPT test. The LLNA shows sensitization and will be used for classification and labelling. For the risk characterisation the NOAEL observed in the HRIPT test will be used because this will give the lowest DNEL.

Respiratory sensitisation

Link to relevant study records
Reference
Endpoint:
respiratory sensitisation
Data waiving:
other justification
Justification for data waiving:
other:
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:
The respiratory sensitization of Cashmeran 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)           Cashmeran 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 Cashmeran 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.

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