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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:
weight of evidence
Study period:
28 June 2017 - 15 August 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2010
Deviations:
yes
Remarks:
The relative humidity in the animal room was between approximately 29-65 % instead of 45–65% and the temperature was between 20-27°C instead of 20-24°C for few hours. This deviation to the study plan, however, did not affect the validity of the study.
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS B.V., Inc
- Age at study initiation: 1st Pre-test: 11 - 12 weeks. 2nd pre-test and Main study: 10 - 11 weeks
- Weight at study initiation: pre-tests and main study: 18.4 - 22.5 g
- Assigned to test groups randomly: yes
- Housing: The animals were distributed into the test groups at random. All animals belonging to the same experimental group were kept in one cage. In the main experiment, the animals were identified by tail tags. In the pre-experiment, animals were identified by cage number.
- Diet: 2018C Teklad Global 18 % protein rodent diet (certified), ad libitum
- Water: tap water ad libitum
- Acclimation period: At least 5 days prior to the start of dosing under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 45-65
- Photoperiod (hrs dark / hrs light): 12 / 12

Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Concentration:
1, 2.5, and 5%
No. of animals per dose:
5
Details on study design:
ANIMAL ASSIGNMENT: The animals were distributed into the test groups at random.

TREATMENT PREPARATION AND ADMINISTRATION:
- Test Item Administration: Each test group of mice was treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 1, 2.5, and 5% in water-free dimethylformamide (DMF). The application volume, 25 μL/ear/day, was spread over the entire dorsal surface (Ø ~ 8 mm) 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).
- Administration of 3H-methyl-thymidine: 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 82 μCi/mL 3HTdR) were injected into each test and control mouse via the tail vein.
- Terminal Procedure: 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.
- Preparation of Single Cell Suspensions: 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.
- Determination of cellular proliferation (incorporation of 3HTdR): 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.

OBSERVATIONS
- Clinical observations: All animals were observed on a daily basis, including pre- and post-dose observations on days 1, 2 and 3. Any clinical signs of systemic toxicity, local skin irritation or signs of ill health during the study were recorded.
- Determination of ear thickness: In the pre-test, the ear thickness was determined prior to the first application of the test item (day 1), on day 3, and on day 6 prior to sacrifice using a micrometer.
- Determination of ear weights: In the pre-test and main experiment, after the lymph nodes have been excised, both ears of mice were punched at the apical area using a biopsy punch (Ø 8 mm corresponding to 0.5 cm2). For each animal both punches were immediately weighed per animal using an analytical balance. The values obtained were taken down manually. The results are described in the report.
- Determination of body weights: The body weights was recorded on day 1 (prior to dosing) and prior to sacrifice (pre-test) or prior to treatment with 3HTdR (main experiment)

INTERPRETATION OF RAW DATA
The proliferative response of the lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph nodes of each animal (DPM/animal) and as the ratio of 3HTdR incorporated into lymph node cells of test animals relative to that recorded for lymph nodes of control animals (Stimulation Index; S.I.). Before DPM/animal values were determined, mean scintillation-background DPM was subtracted from test and control raw data.
A test item is regarded as a sensitiser in the LLNA if the following criteria are fulfilled. 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.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
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 and the ear weights were performed with a validated test script of “R”, a language and environment for statistical computing and graphics.

Within the program a statistical analysis was conducted on the the ear weights to assess whether the difference was statistically significant between the test item groups and negative control group. Statistical significance was set at the five per cent level (p < 0.05). Additionally, the Dean-Dixon-Test and Grubb’s Test were used for identification of possible outliers. An outlier (DPM value determined for animal 16) was detected in the Grubb’s, but not in the Dean-Dixon-Test, and was therefore not excluded from calculations.

However, both biological and statistical significance were considered together.

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 for 25% α-Hexylcinnamaldehyde (in acetone/olive oil, 4+1 v/v): S.I. = 10.1

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
EC3
Remarks on result:
not determinable
Parameter:
SI
Value:
1.9
Test group / Remarks:
5% test substance
Parameter:
SI
Value:
1.7
Test group / Remarks:
2.5% test substance
Parameter:
SI
Value:
1.6
Test group / Remarks:
1% test substance
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
Mean DPM/animal was determined by dividing the sum of the measured values from lymph nodes of all animals within a group by the number of animals in that group (5 animals). The Mean DPM per animal (2 lymph nodes) was 1593.7, 1709.3, and 1900.1 in the 1, 2.5, and 5% dose groups, respectively.

DETAILS ON STIMULATION INDEX CALCULATION
The stimulation index was calculated by dividing the mean DPM per animal for the dose groups by the mean DPM per animal for the vehicle control group.

EC3 CALCULATION
The EC3 value could not be calculated, since all S.I.´s are below the threshold value of 3.

CLINICAL OBSERVATIONS:
No deaths occurred during the study period. No signs of systemic toxicity were observed during the study period. From day 2 to 5, the animals showed an erythema of the ear skin.
The measured ear weight of all animals treated was recorded on test day 6 (after necropsy). A relevant increase in ear weights was not observed.

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:
other: Not skin sensitising
Remarks:
based on CLP criteria
Conclusions:
Under the conditions of this study, the substance is not considered to induce a stimulation index >3 and therefore no sensitisation is expected. Based on this result, the substance does not need to be classified as a skin sensitizer according to criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).
Executive summary:

In this study Tetrahydrolinalyl acetate formulated in water-free dimethylformamide (DMF) was assessed for its possible skin sensitising potential. For this purpose a local lymph node assay was performed according to OECD TG 429, using test item concentrations of 1, 2.5, and 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 two 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 day 2 to 5, the animals showed erythema of the ear skin. A relevant increase of ear weight was not observed. In this study, Stimulation Indices (S.I.) of 1.6, 1.7, and 1.9 were determined with the test item at concentrations of 1, 2.5, and 5% (w/w) in water-free dimethylformamide (DMF), respectively. An EC3 value could not be determined and therefore Tetrahydrolinalyl acetate was found to be not skin sensitising under the test conditions of this study. The substance does not need to be classified as a skin sensitizer according to criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).