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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:
05 September 2018 - 18 September 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

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
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
updated 06 July 2012
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
Details on test material:
Batch:102Z5
Purity: 99.9%
white powder
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Sponsor/102Z5
- Purity: 99.9%
- Expiration date of the lot/batch: 31 December 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature, in the dark
(only valid for storage, may be used/formulated in light)
- Stability in Solvent: Not indicated by the Sponsor

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS B.V., Inc
- Females (if applicable) nulliparous and non-pregnant: yes
- Age (at beginning of treatment): Pre-test: 8 - 9 weeks , Main study: 9 - 10 weeks

- Weight at study initiation: Pre-test: 17.6 - 18.3g, Main study: 17.5 - 22.8g
- Housing: In groups; All animals belonging to the same experimental group were kept in one cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: At least 5 days
- Indication of any skin lesions: No

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): approx. 45-65% (except for deviation*)
* The relative humidity in the animal room was between approximately 33-65 % instead of 45–65% for several hours.
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:
propylene glycol
Concentration:
5, 10 and 25%
No. of animals per dose:
4 (main test)
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: 25 suspension in Propylene glycol (PG)
- Irritation: No signs
- Systemic toxicity: No signs
- Ear thickness measurements: Yes
- Erythema scores: No visible erythema

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: B42
- 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:
The highest concentration tested was the highest concentration that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed by a pre-experiment.
The test item was placed into a mortar on a tared balance and PG was added whilst grinding (weight per weight).
The different test item concentrations were prepared individually. Homogeneity of the test item in vehicle was maintained during treatment by stirring.
The preparations were made freshly before each dosing occasion.
Each test group of mice was treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 5, 10, and 25% in PG. 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).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

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 2018.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
1.26
Test group / Remarks:
5%
Key result
Parameter:
SI
Value:
1.11
Test group / Remarks:
10%
Key result
Parameter:
SI
Value:
1.25
Test group / Remarks:
25%
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA :
The precipitates were 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 (DPM).
DETAILS ON STIMULATION INDEX CALCULATION
The mean values and standard deviations were calculated in the body weight tables.
Where appropriate, the EC3 value were calculated according to the equation
EC3 = (a-c) [(3-d)/(b-d)] + c
where EC3 is the estimated concentration of the test item required to produce a 3-fold increase in draining lymph node cell proliferative activity; (a, b) and (c, d) are respectively the co-ordinates of the two pair of data lying immediately above and below the S.I. value of 3 on the local lymph node assay dose response plot.
All calculations conducted on the DPM values were performed with a validated test script of “R”, a language and environment for statistical computing and graphics.
EC3 CALCULATION :
The EC3 value could not be calculated, since all S.I.´s were below the threshold value of 3.
CLINICAL OBSERVATIONS:
No symptoms of local skin irritation at the ears of the animals and no signs of systemic toxicity were observed during the study period.
BODY WEIGHTS:
The body weight of the animals, recorded prior to the first application and prior to treatment with 3HTdR, was 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:
The test item CDA-1 was not a skin sensitiser under the test conditions of this study.