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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:
04 July 2012 to 18 July 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes (incl. QA statement)
Remarks:
30 March 2009
Type of study:
mouse local lymph node assay (LLNA)

Test material

1
Reference substance name:
Gum benzoin, Siam
EC Number:
232-556-7
EC Name:
Gum benzoin, Siam
Cas Number:
9000-72-0
IUPAC Name:
Benzoin resinoid obtained from the resin of Styrax tonkinensis by extraction with solvent
Test material form:
solid

In vivo test system

Test animals

Species:
mouse
Strain:
CBA/Ca
Remarks:
CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories B.V.
- Females nulliparous and non-pregnant: yes
- Age at study initiation: pre-test: 9 - 10 weeks (beginning of treatment). Main study: 8 – 9 weeks (beginning of treatment)
- Weight at study initiation: Pre-test 22 and 21.4 g for test animal 1 and 2, respectively. Main study, mean weight 20.9 ± 1.2
- Housing: All animals belonging to the same experimental group were kept in one cage. Makrolon Type II / III, with wire mesh top. Granulated soft wood was used for bedding.
- Diet: 2018C Teklad Global 18% protein rodent diet, 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.
- Indication of any skin lesions: Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 + 2
- Humidity (%): 45-65 (>95% for several hours)
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Concentration:
Pre-study: 25 and 50% (w/w)
Main-study: 10, 25, and 50% (w/w)
No. of animals per dose:
Pre-study: 1
Main-study: 5
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: The highest test item concentration, which could be technically used, was a 50 % solution in dimethylsulfoxide. Vortexing was used to formulate the test item. At higher concentrations, an applicable formulation of the test item was not achieved, neither by the use of other vehicles nor by using additional methods to formulate the test item (e.g. sonicating, warming to 37°C).
- Irritation and systemic toxicity: 2 mice, topical administration to dorsal surface of the ear, conc. 25% and 50%, daily for 3 consecutive days, Body weight was determined prior to treatment and befor sacrifice, clinical signs were recorded once daily. Eventual signs of local skin irritation were documented and a score was used to grade a possible erythema of the ear skin. 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 (S0247 Kroeplin, 36381 Schlüchtern, Germany). After sacrifice both ears (left and right) of mice were punched at the apical area using a biopsy punch (Stiefel, Ø 8 mm corresponding to 0.5 cm2). For each animal both punches were immediately weighed per animal using an analytical balance.


MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymphnode Assay
- Criteria used to consider a positive response:
The proliferative response of the lymph node cells was 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 lymph nodes 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.

TREATMENT PREPARATION AND ADMINISTRATION:
- Test Item Preparation: The test item was placed into an appropriate container on a tared balance and dimethylformamide was added. The different test item concentrations were prepared serially. The preparations were made freshly before each dosing occasion. Concentrations were in terms of material as supplied.
-Topical Application: Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear with different test item concentrations of 10, 25, and 50% (w/w) in dimethylformamide. The application volume, 25 XL/ear/day, was spread over the entire dorsal surface (diameter of ca. 8 mm) of each ear once daily for three consecutive days. A further group of mice was treated with an equivalent volume of the relevant vehicle alone (control animals).
- Administration of 3H-Methyl Thymidine: 3H-methyl thymidine (3HTdR) was purchased from Hartmann Analytic, 38124 Braunschweig, Germany (specific activity, 2 Ci/mmol; concentration, 1 mCi/mL). Five days after the first topical application (day 6) 250 XL of phosphate-buffered saline (PBS) containing 19.5 XCi of 3HTdR (equivalent to 3HTdR 78 XCi/mL) were injected into each test and control mouse via the tail vein.

OBSERVATIONS
In addition to the sensitising reactions the following observations and data were recorded during the test and observation period:
Mortality / Viability: At least once daily from experimental start to necropsy
Body weights: In the pre-test: prior to the first application and prior to sacrifice. In the main experiment: prior to the first application and prior to treatment with 3HTdR.
Ear thickness: In the pre-test prior to the first application of the test item (day 1), on day 3 and before sacrifice (day 6). In the main test, prior to the first application and prior to treatment with 3HTdR.
Ear weights: In the pre-test and main test after sacrifice; biopsy punches were taken from each ear.
Clinical signs (local / systemic): Clinical signs (systemic toxicity or local skin irritation) were recorded at least once daily. Especially the treatment sites were observed carefully.

DETERMINATIONS
- Determination of Ear Thickness: Prior to the first application of the test item and prior to treatment with 3HTdR.the ear thickness was determined using a micrometer (S0247 Kroeplin, 36381 Schlüchtern, Germany)
- Determination of Incorporated 3HTdR: Approximately five hours after treatment with 3HTdR all mice were euthanised by intraperitoneal injection of Pentobarbital-Sodium (Release, WDT, 30827 Garbsen, Germany). 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 Xm 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 at least 18 hours for precipitation of macromolecules. The precipitates were then resuspended in 5 % trichloroacetic acid (1 mL) and transferred to plastic scintillation vials with 10 mL of ‘Ultima Gold’ scintillation liquid (Perkin Elmer (LAS) GmbH, 63110 Rodgau, Germany) and thoroughly mixed. The level of 3HTdR incorporation was then measured on a Beta-scintillation counter (Tricarb 2900 TR, Perkin Elmer (LAS) GmbH, 63110 Rodgau, Germany). Similarly, background 3HTdR levels were also measured in two 1ml-aliquots of 5 % trichloroacetic acid. The Beta- scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM).
- Determination of Ear Weights: After the lymph nodes have been excised, both ears (left and right) of mice were punched at the apical area using a biopsy punch (Stiefel, Ø 8 mm corresponding to 0.5 cm2). For each animal both punches were immediately weighed per animal using an analytical balance.
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 and for the DPM values (group mean DPM ± standard deviation).
- For all statistical calculations SigmaStat for Windows (Version 2.0) was used.
- A One-Way-Analysis-of-Variance (ANOVA) was was conducted on the ear weights and thickness to assess whether the difference was statistically significant between test item groups and negative control (vehicle) group.
- Statistical significance was set at the five per cent level (p < 0.05).
- The Dean-Dixon-Test was used for identification of possible outliers (performed with Microsoft Excel 2003).
- Both biological and statistical significance were considered together.

Results and discussion

Positive control results:
Positive control experiment performed in April 2012 (Harlan study number 1486301) with substance: Alpha-Hexylcinnamaldehyde. Exposure to 0, 5, 10, and 25% test substance resulted in an SI of 1.00, 0.88, 1.51, and 3.73, respectively. The EC3 was estimated to be 20.1% (w/v).

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
EC3
Remarks:
%
Value:
4.8
Remarks on result:
other: An estimated EC3 value was determined by means of extrapolation from the two lowest test item concentrations and their respective Stimulation Indices. This extrapolated EC3 value was calculated as 4.8% (w/w).
Parameter:
SI
Value:
5.28
Test group / Remarks:
10%
Parameter:
SI
Value:
8.22
Test group / Remarks:
25%
Parameter:
SI
Value:
9.02
Test group / Remarks:
50%
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
Mean DPM per animal (two lymph nodes) were 838.3, 4426.5, 6893.1, and 7559.1 in the vehicle control group, 10%, 25%, and 50% dose group respectively.

DETAILS ON STIMULATION INDEX CALCULATION
In this study Stimulation Indices of 5.28, 8.22 and 9.02 were determined with the test item at concentrations of 10, 25, and 50% (w/w) in dimethylformamide. A clear dose response was observed.

EC3 CALCULATION
The EC3 value could not be calculated, since all S.I.´s were above the threshold value of 3. However, upon request, an estimated EC3 value was calculated by means of extrapolation (refer to the "any other information" section).

CLINICAL OBSERVATIONS:
No deaths occurred during the study period. No systemic findings were observed during the study period.
On days 2-6, the animals treated with a test item concentration of 50% showed a very slight to well defined erythema of the ear skin (days 2, 3, 4, 6: Score 1; day 5: Score 2).

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.

EAR THICKNESS / EAR WEIGHT
The measured ear thickness of all animals treated was recorded prior to the 1st application, on study day 3 and prior to necropsy (day 6). A relevant increase in ear thickness was not observed. The measured ear weight of all animals treated was recorded on day 6 after necropsy. A relevant increase in ear thickness was not observed.

Any other information on results incl. tables

Calculation of the EC3 Value by Means of Extrapolation:

      S.I.
 Concentration  25% (a)  8.2 (b)
 Concentration  10% (c)  5.3 (d)
 EC3 = 2(log2 (c) + (3-d) / (b-d) * (log2(a) - log2(c)))      
 EC3  4.8%  3.0

EC3 = Estimated concentration for a S.I. of 3.

a,b,c,d = Co-ordinates of the two pairs of data lying immediately above the S.I. value of 3 on the LLNA dose response plot. The point with the higher S.I. is denoted as (a, b). The point with the lower S.I. is denoted as (c, d).

Applicant's summary and conclusion

Interpretation of results:
other: Skin Sens. 1B
Remarks:
in accordance with EU CLP (EC 1272/2008 and its updates)
Conclusions:
Under the conditions of the test the substance was considered sensitising and resulted in SI values of 5.28, 8.22, and 9.02, in the 10, 25, and 50% dose group, respectively. An estimated EC3 value was determined by means of extrapolation from the two lowest test item concentrations and their respective Stimulation Indices. This extrapolated EC3 value was calculated as 4.8% (w/w). The substance is considered a skin sensitiser category 1B.
Executive summary:

In the study the test item Benzoin Laos Orpur 50%/DPG dissolved in dimethylformamide was assessed for its possible skin sensitising potential in accordance with OECD TG 429. For this purpose a local lymph node assay was performed using test item concentrations of 10, 25, and 50% (w/w). The highest concentration tested was the highest concentration that was technically feasible and which did not cause systemic toxicity and excessive local skin irritation as confirmed by a pre-experiment. The animals did not show any signs of systemic toxicity during the course of the study and no cases of mortality were observed. On days 2-6, the animals treated with a test item concentration of 50 % showed an erythema of the ear skin (days 2, 3, 4, 6: Score 1; day 5: Score 2). No statistically significant increases in ear thickness or ear weights were observed. Under the conditions of the test the substance was considered sensitising and resulted in SI values of 5.28, 8.22, and 9.02, in the 10, 25, and 50% dose group, respectively. An estimated EC3 value was determined by means of extrapolation from the two lowest test item concentrations and their respective Stimulation Indices. This extrapolated EC3 value was calculated as 4.8% (w/w). The substance is therefore considered a skin sensitiser category 1B.