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Toxicological information

Skin sensitisation

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guidelineopen allclose all
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)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Cyrene™- Analytical purity: 99%- Expiration date of the lot/batch: 30 Sep 2015- CAS RN: 53716-82-8

In vivo test system

Test animals

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

Study design: in vivo (LLNA)

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)

Results and discussion

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.

In vivo (LLNA)

Resultsopen allclose all
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)

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.