Registration Dossier

Administrative data

Description of key information

In the key local lymph node assay, conducted according to OECD 429 Test Guideline and in compliance with GLP, the test substance, Cyrene™, was reported to be not sensitising to skin (Harlan, 2014d).

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:
30 Apr - 02 Jun 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
adopted 22 July 2010
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Hess. Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
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 Laboratories B. B., Horst, Netherlands
- Age at study initiation: 1st pre-test: 8 - 9 weeks; 2nd pre-test: 10 - 11 weeks; main study: 10 - 11 weeks
- Weight at study initiation: 1st pre-test: 19.8 - 21.3 g; 2nd pre-test: 20.5 - 20.1 g; main study 19.1. - 22.5 g
- Housing: group of 2 (pre-studies)/ 4 (main study) animals per cage in Makrolon Type II (pre-test)/ III (main study), with wire mesh top, granulated soft wood bedding
- Diet: 2018C Teklad Global 18% protein rodent diet (certified), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days prior start of dosing
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2- Humidity (%): 45 - 65, except for several hours on four non consecutive days (28 - 65%)
- Photoperiod (hrs dark / hrs light): 12/12
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
5, 10 and 25%
No. of animals per dose:
4
Details on study design:
RANGE FINDING TESTS:The first pre-test was performed with 50 and 100% test item concentration. Animals treated with 50% test item concentration showed an erythema of the ear skin (Score 1) on day 1 to 4. Animals treated with 100% test item concentration showed an erythema of the ear skin (Score 1) between day 1 and 5. The ear thickness for animals treated with 50% test item concentration increased by 27.2% which is considered as an excessive ear irritation because the increase in ear weight is ≥ 25%.The second pre-test was performed with 10 and 25% test item concentration. Animals treated with 10% test item concentration showed an erythema of the ear skin (Score 1) on day 3. Animals treated with 25% test item concentration showed an erythema of the ear skin (Score 1) between day 2 and 3.Due to these results the test item concentration in the main study was assayed at 5, 10 and 25%, where the highest concentration tested was the highest concentration level that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation.
MAIN STUDYANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: 3H-methyl thymidine incorporation determined by β-scintillation
- Criteria used to consider a positive response:
A) the 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,
B) 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: 25 µL of the test item was spread over the entire dorsal surface of each ear of each mouse once daily for three consecutive days. Local irritation reactions were assessed. On day 6 250 µL of phosphate-buffered saline containing 20.4 µCi of 3H-methyl thymidine were injected into each test and control mouse via the tail vein. Approximately 5 h later the draining lymph nodes were excised and pooled for each treatment group. A single cell suspension of lymph node cells of pooled lymph nodes was prepared by gentle mechanical disaggregation through stainless steel gauze (200 µm mesh size). Macromolecules were precipitated with 5% trichloroacetic acid at 4 °C for at least 18 h.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Positive control results:
The positive control substance α-hexyl cinnamic aldehyde with a concentration of 25% in acetone/olive oil (4 + 1; v/v) induced a positive reaction with a stimulation index of 6.79.
Key result
Parameter:
SI
Value:
0.49
Test group / Remarks:
5% test substance in acetone/olive oil (4 + 1; v/v)
Key result
Parameter:
SI
Value:
0.76
Test group / Remarks:
10% test substance in acetone/olive oil (4 + 1; v/v)
Key result
Parameter:
SI
Value:
1.03
Test group / Remarks:
25%test substance in acetone/olive oil (4 + 1; v/v)
Key result
Parameter:
other: disintegrations per minute (DPM)
Value:
1 027.5
Test group / Remarks:
Negative control group
Key result
Parameter:
other: disintegrations per minute (DPM)
Value:
504.1
Test group / Remarks:
5% test substance in acetone/olive oil (4 + 1; v/v)
Key result
Parameter:
other: disintegrations per minute (DPM)
Value:
775.9
Test group / Remarks:
10% test substance in acetone/olive oil (4 + 1; v/v)
Key result
Parameter:
other: disintegrations per minute (DPM)
Value:
1 063
Test group / Remarks:
25% test substance in acetone/olive oil (4 + 1; v/v)

No signs of systemic toxicity were observed during the study period. Animals treated with 25% test item concentration showed an erythema of the ear skin with a score of 1 on day 3 and 4, whereas animals treated with 5 and 10% test item concentration did not show any signs of local skin irritation.

The body weights of the animals were recorded prior the first application and before treatment with 3H-methyl-thymidine. The weights were within the range commonly recorded for animals of this strain and age.

Interpretation of results:
GHS criteria not met
Conclusions:
In the key local lymph node assay, conducted according to OECD 429 Test Guideline and in compliance with GLP, the test substance, (1S,5R)-6,8-dioxabicyclo[3.2.1]octan-4-one, was reported to be not sensitising to skin.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

In the key local lymph node assay, conducted according to OECD 429 Test Guideline and in compliance with GLP, the test substance, Cyrene™, was reported to be not sensitising to skin. No signs of systemic toxicity were observed during the study period (Harlan, 2014d).

At induction, 25 µL of the test item was spread over the entire dorsal surface of each ear of each mouse once daily for three consecutive days. Local irritation reactions were assessed. On day 6 at challenge 250 µL of phosphate-buffered saline containing 20.4 µCi of 3H-methyl thymidine were injected into each test and control mouse via the tail vein. Approximately 5 h later the draining lymph nodes were excised and pooled for each treatment group. A single cell suspension of lymph node cells of pooled lymph nodes was prepared by gentle mechanical disaggregation through stainless steel gauze (200 µm mesh size). Macromolecules were precipitated with 5% trichloroacetic acid at 4 °C for at least 18 h.

No signs of systemic toxicity were observed during the study period. Animals treated with 25% test item concentration showed an erythema of the ear skin with a score of 1 on day 3 and 4, whereas animals treated with 5 and 10% test item concentration did not show any signs of local skin irritation. The body weights of the animals were recorded prior the first application and before treatment with 3H-methyl-thymidine. The weights were within the range commonly recorded for animals of this strain and age. The stimulation indices (SI) of 0.49, 0.76 and 1.03 were determined with the test item concentrations of 5, 10 and 25% in acetone/olive oil (4 + 1; v/v). The EC3 value could not be calculated, since none of the tested concentrations induced a SI greater than the threshold value of 3.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the available data for Cyrene™, no classification for skin sensitisation is required according to Regulation (EC) No 1272/2008.