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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:
Aug-Sep 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
according to OECD429 and under GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
Source: Envigo RMS B.V., Inc (Postbus 6174; 5960 AD Horst / The Netherlands)
Age (beginning of treatment): 1st and 3rd pre-test: 9 - 10 weeks
2nd pre-test: 11 - 12 weeks
Main study: 10 - 11 weeks

Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Concentration:
0.1, 0.25, and 0.5% (w/w)
No. of animals per dose:
5
Details on study design:
Each test group of mice was treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 0.1, 0.25, and 0.5% in DMF. The application volume, 25 µL/ear/day, was spread over the entire dorsal surface of each ear once daily for three consecutive days. A further group of mice (control animals) was treated with an equivalent volume of the relevant vehicle alone (control animals). Five days after the first topical application (day 6) 250 µL of phosphate-buffered saline containing 20.5 µCi of 3H-methyl thymidine (equivalent to 81.8 µCi/mL 3HTdR) were injected into each test and control mouse via the tail vein. Approximately five hours after treatment with 3HTdR all mice were euthanized by using CO2, which was, after harvesting of the lymph nodes, followed by cervical dislocation to ensure death. The draining lymph nodes were rapidly excised and pooled per animal (2 nodes per animal). Single cell suspensions (in phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 µm mesh size). After washing two times with phosphate buffered saline (approx. 10 mL) the lymph node cells were resuspended in 5 % trichloroacetic acid (approx. 3 mL) and incubated at approximately +4 °C for approximately 18 hours for precipitation of macromolecules. The precipitates were then resuspended in 5 % trichloroacetic acid (1 mL) and transferred to scintillation vials with 10 mL of scintillation liquid and thoroughly mixed. The level of 3HTdR incorporation was then measured in a β-scintillation counter. Similarly, background 3HTdR levels were also measured in two 1 mL-aliquots of 5 % trichloroacetic acid. The β-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
All calculations conducted on the DPM values and the ear weights were performed with a validated test script of “R”, a language and environment for statistical computing and graphics.

Results and discussion

Positive control results:
The sensitivity and reliability of the experimental technique employed was assessed by use of α-hexyl cinnamaldehyde dissolved in acetone/olive oil (4+1 v/v) (compound listed in OECD 429 Guideline) which is known to have skin sensitisation properties in mice. The periodic positive control experiment was performed using CBA/CaOlaHsd mice in April 2017
Result: 10 % α-hexyl cinnamaldehyde gave an S.I. of 3.43

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
0.9
Test group / Remarks:
0.1% (w/w)
Key result
Parameter:
SI
Value:
0.9
Test group / Remarks:
0.25% (w/w)
Key result
Parameter:
SI
Value:
1.1
Test group / Remarks:
0.5% (w/w)
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: mean DPM (per animal) of 2443.4, 2320.4, 2299.8, and 2591.0 were determined for the test item at concentrations of 0 (vehicle control), 0.1%, 0.25% and 0.5%, respectively

Any other information on results incl. tables

Results of pre-tests: To determine the highest non-irritant test concentration that at the same time did not induce signs of systemic toxicity, a pre-test was performed in two animals. Two mice were treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 10 and 25% once daily each on three consecutive days. Prior to the first application of the test item and before sacrifice the body weight was determined. Clinical signs were recorded at least once daily. Eventual signs of local irritation were documented and a score was used to grade a possible erythema of the ear skin. Furthermore, prior to the first application of the test item (day 1), on day 3 and before sacrifice (day 6) the ear thickness was determined using a micrometer. Additionally, for both animals, the ears were punched after sacrifice (day 6) at the apical area using a biopsy punch (Ø 8 mm corresponding to 0.5 cm2) and were immediately pooled per animal and weighed using an analytical balance. Eventual ear irritation was considered to be excessive if an erythema of the ear skin of a score value ≥3 was observed at any observation time and/or if an increase in ear thickness of ≥25% was recorded on day 3 or day 6.

At the tested concentrations the animals did not show any signs of systemic toxicity. From day 1 to 6, the animals showed an erythema of the ear skin (day 1 to 5: Score 1, day 6 Score 4 (=eschar formation, detected during preparation)).

Therefore, a second pre-test was performed using test item concentrations of 2.5 and 5%. From day 1 to 6, the animals showed an erythema of the ear skin (Score 1 to 2). On day 6, upon preparation, both animals showed slight eschar formation.

Therefore, a third pre-test was performed using test item concentrations of 0.5 and 1%. From day 2 to 5, the animals showed an erythema of the ear skin (Score 1 to 2). On day 6 the animal treated with 1% of the test substance showed slight eschar

formation.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The test item Ascorbyl Palmitate is not a skin sensitiser.
Executive summary:

In the study the test item Ascorbyl Palmitate formulated in DMF (dimethylformamide) was assessed for its possible skin sensitising potential.

For this purpose a local lymph node assay was performed using test item concentrations of 0.1, 0.25, and 0.5% (w/w). The highest concentration tested was the highest concentration that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed by three pre-experiments. The animals did not show any signs of systemic toxicity during the course of the study and no cases of mortality were observed. From days 2 to 4, the animals treated with a test item

concentration of 0.25 and 0.5% showed an erythema of the ear skin (Score 1). Animals treated with 0.1% test item concentration did not show any signs of local skin irritation. A relevant increase in ear weights was not observed.

In this study Stimulation Indices (S.I.) of 0.9, 0.9, and 1.1 were determined with the test item at concentrations of 0.1, 0.25, and 0.5% (w/w) in DMF, respectively.

The test item Ascorbyl Palmitate was not a skin sensitiser under the test conditions of this study.