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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:
2011-07-20 to 2012-06-20
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant study according to guideline.
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
(July 22, 2010)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
(2003)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
other: Pre-test: CBA/CaOlaHsd; main study: CBA/CaCrl
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories B.V., Horst, The Netherlands
- Age at study initiation: 8 - 9 (pre-test and main study)
- Weight at study initiation: 16.8 g to 20.1 g
- Housing: grouped in Makrolon Type II / III, with wire mesh top
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 + 2°C
- Humidity: 45-66%
- Photoperiod: 12h light / 12h darkness; 6.00 a.m. - 6.00 p.m.

IN-LIFE DATES: not specified
Vehicle:
acetone/olive oil (4:1 v/v)
Remarks:
25% (w/v)
Concentration:
0, 25, 50 and 100%
No. of animals per dose:
five females
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: good solubility in acetone/olive oil; as a liquid, it is also applicable undiluted (100%).
- Irritation: From day 3 to day 6, both animals treated with either 75 or 100% of the test item showed an erythema of the ear skin (75% test item concentration: Score 1 on day 3 to 6; 100% test item concentration: Score 2 on day 3 and Score 1 from day 4 to 6). However, the thresholsd value for the ear weight or ear thickness values of 25% for excessive local skin irritation mentioned in OECD guideline 429 was not exceeded at any concentration.
- Lymph node proliferation response: not determined.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay (LLNA) in Mice
- Criteria used to consider a positive response: First, that exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the Stimulation Index. Second, that the data are compatible 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 test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear with test item concentrations of 25 and 50% (w/v) in acetone:olive oil (4+1 v/v), and 100% (undiluted test item). The application volume, 25 µL/ear/day, was spread over the entire dorsal surface (appr. 8 mm on average) of each ear once daily for three consecutive days. Two further groups of mice (a vehicle control group and a positive control group) were treated with an equivalent volume of the vehicle alone or with the positive control item at 25% (w/v).
Five days after the first topical application (day 6) 250 µL of phosphate-buffered saline (PBS) containing 19.9 µCi of 3HTdR (equivalent to 79.6 µCi/mL 3HTdR) were injected into each test and control mouse via the tail vein.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
One-Way-Analysis-of-Variance (SigmaStat for Windows, Version 2.0): DPM values, ear weights, lymph node weights and lymph node cell count. If significant: Dunnett test.
Outliers: Dean-Dixon-Test and Grubb’s test (performed using Microsoft Excel 2003).
Positive control results:
When compared to the vehicle control group, the positive control (HCA) resulted in a stimulation index (SI) of 7.97, providing evidence for the validity of the assay.
Parameter:
SI
Remarks on result:
other: SI Test Group / Treatment / Stimulation Index (mean) 1 / 0% / 1.00 2 / 25% / 2.91 3 / 50% / 2.30 4 / 100% / 4.01 5 / pos. contr. / 7.97 EC3 value of 70.5% (w/v) (based on 50 % and 100%)
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Test Group / Treatment / [DPM/Lymph Node Pair (mean)] 1 / 0% / 369.3 2 / 25% / 1074.1 3 / 50% / 847.7 4 / 100% / 1479.7 5 / pos.contr. / 2942.1 Calculation: test group x / test group 1 (vehicle control)

A statistically significant (p<0.05) and biologically relevant increase in the DPM value and lymph node weight was observed at the highest dose group in comparison to the vehicle control group; referring to the lymph node weight, this was also the case for the low dose group. A statistically significant increase in lymph node cell count was not observed in any treated group in comparison to the vehicle control group. For BALB/c mice, a cut off-value for the lymph node cell count index of 1.55 was reported for a positive response. The indices determined for the lymph node cell count did not exceed this threshold. Still, based on the Stimulation Index obtained for the high dose group, which exceeded the threshold value of 3 for a positive response, the test item has to be regarded as a skin sensitizer. Based on the S.I.s obtained with 50% and 100% test item concentration, an EC3 value of 70.5% (w/v) was calculated.

An outlier was identified in the high dose group (DPM value determined for animal number 17). However, as the corresponding lymph node weight value confirmed the result of the DPM value and as exclusion of the outlier did not change the overall test result, the value in question was not excluded from calculation.

Interpretation of results:
sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

The skin sensitising potential of 4 -acetylmorpholine was studied in the LLNA (OECD 429 and GLP). The tested dose levels of 25%, 50% and 100% in acetone/olive oil (4:1 v/v) caused SI's of 2.91, 2.30 and 4.01 respectively. A statistically significant (p<0.05) and biologically relevant increase in the DPM value and lymph node weight was observed in the highest dose group in comparison to the vehicle control group; referring to the lymph node weight, this was also the case for the low dose group. A statistically significant increase in lymph node cell count was not observed in any treated group in comparison to the vehicle control group. For BALB/c mice, a cut off-value for the lymph node cell count index of 1.55 was reported for a positive response. The indices determined for the lymph node cell count did not exceed this threshold. Still, based on the Stimulation Index obtained for the high dose group, which exceeded the threshold value of 3 for a positive response, the test item has to be regarded as a skin sensitizer. Based on the S.I.s obtained with 50% and 100% test item concentration, an EC3 value of 70.5% (w/v) was calculated.


Migrated from Short description of key information:
The test item induced delayed contact hypersensitivity in the murine Local Lymph Node Assay and is thus regarded as skin sensitiser (study: Harlan CCR 2011).

Justification for selection of skin sensitisation endpoint:
The key stusy was selected (GLP compliant study according to guideline).

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available
Additional information:

The test item has no structural alert indicative for respiratory sensitisation. In addition, no human data on respiratory sensitisation have been reported. The test item is considered not to induce respiratory sensitisation.


Migrated from Short description of key information:
Considered not to induce respiratory sensitisation.

Justification for classification or non-classification

Skin sensitisation

Based on the available LLNA study, the test item is clasified a skin sensitiser according to 67/548/EEC and Regulation (EC) No 1272/2008 (GHS, CLP; Class 1B, H317: May cause an allergic skin reaction)

Respiratory sensitisation

Considered not to induce respiratory sensitisation.