Registration Dossier

Toxicological information

Skin sensitisation

Currently viewing:

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment.

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:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes (incl. certificate)
Remarks:
Harlan Cytotest Cell Research GmbH, ln den Leppsteinswiesen 19, 64380 Roßdorf
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Identity: Metanilsäure
- Other Name: 3-Aminobenzenesulfonic acid (EINECS)
- Batch No.: 10
- Content of Main Component: 97.5% (w/w) dose calculation not adjusted to content
- Certificate of Analysis: AZ 691/Toxe1 (January 17, 2012)
- Storage: At room temperature
- Expiration Date: January 17, 2014 (Statement of producer)

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories B.V., Postbus 6174, 5960 AD Horst / The Netherlands
- Age at study initiation: 1st pre-test: 9 - 10 weeks (beginning of treatment); 2nd pre-test: 11 - 12 weeks (beginning of treatment); 3rd pre-test: 10 – 11 weeks (beginning of treatment); Main study: 11 – 12 weeks (beginning of treatment)
- Weight at study initiation: 1st pre-test: 21.4-22.9g; 2st pre-test: 20.9 - 21.1g; 3st pre-test: 19.5g; main study: 20.3 - 23.7g
- Housing: in group in Makrolon Type II (pre-test) / III (main study), with wire mesh top (EHRET GmbH, 79302 Emmendingen, Germany) with granulated soft wood bedding (Rettenmaier & Söhne GmbH + Co. KG, 73494 Rosenberg, Germany)
- Diet (e.g. ad libitum): 2018C Teklad Global 18% protein rodent diet (certified), ad libitum (Harlan Laboratories B.V., 5960 AD Horst, Netherlands)
- Water (e.g. ad libitum): tap water, ad libitum (Gemeindewerke, 64380 Rossdorf, Germany)
- 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 (%): relative humidity 45-65% (during the pre-tests: for several hours on several days >95%)
- Photoperiod (hrs dark / hrs light): artificial light 6.00 a.m. - 6.00 p.m.

Study design: in vivo (LLNA)

Vehicle:
dimethyl sulphoxide
Concentration:
0.05, 0.1, 0.25 % (w/w)
No. of animals per dose:
4
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: A solubility experiment was performed according to the recommendations given by OECD 429. The highest test item concentration, which could be technically used was a 5 % suspension in dimethylsulfoxide. Vortexing and sonicating 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. warming to 37°C).
- Irritation: 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. Two mice were treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 2.5 and 5% once daily each on three consecutive days. At the tested concentrations, the animals did not show any signs of systemic toxicity. However, in both animals a visible ear swelling of 29.9 and 30.5%, respectively, compared to pre-treatment values was observed on day 6 of treatment, indicating excessive local skin irritation. Ear weights were also increased compared to historical vehicle data. Moreover, both animals developed a slight eschar formation on the ear skin on days 4 and 6.
Therefore, a second pre-test was performed with test item concentrations of 0.5 and 1%. Again, ear swelling of more than 25% compared to pre-treatment values was noted in the high dose animal. Excessive increase in ear weights and erythema formation (score: 1-2) also indicated a significant irritation of the ear skin in both animals.
A third pre-test was performed using concentrations of 0.1 and 0.25%. Although in the animal treated with 0.1% test item increase in ear weights was 35% compared to historical vehicle values (i.e., 27.2 mg/animal for dimethylsulfoxide), all other parameters indicating irritation of the ear skin were within the desired range for this animal. The animal treated with a concentration of 0.25% did not develop any signs of excessive skin irritation.
Thus, it was decided together with the sponsor to assay the test item in the main study at 0.05, 0.1 and 0.25% (w/w). The highest concentration tested was the highest level that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation in the pre-experiment.
- Lymph node proliferation response: not determined

MAIN STUDY
Test Item Preparation:
The test item was placed into an appropriate container on a tared balance and dimethylsulfoxide was added. The different test item concentrations were prepared individually. Homogeneity of the test item in vehicle was maintained during treatment using a magnetic stirrer. 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 test item concentrations of 0.05, 0.1 and 0.25% (w/w) in dimethylsulfoxide. 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 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 μL of phosphate-buffered saline (PBS) containing 20.5 μCi of ³HTdR (equivalent to ³HTdR 81.8 μCi/mL) were injected into each test and control mouse via the tail vein.

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 group (8 nodes per group). 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 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 ß-scintillation counter (Tricarb 2900 TR, Perkin Elmer (LAS) GmbH, 63110 Rodgau, Germany). Similarly, background ³HTdR levels were also measured in two 1 mL-aliquots of 5 % trichloroacetic acid. The ß-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.

Interpretation of Raw Data:
The proliferative response of lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph node (DPM/lymph node) and as the ratio of ³HTdR incorporated into lymph node cells of test lymph nodes relative to that recorded for control lymph nodes (Stimulation Index; S.I.). Before DPM/lymph node 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.

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).
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.

Positive Control Data:
The sensitivity and reliability of the experimental technique employed was assessed by use of a substance which is known to have skin sensitisation properties in CBA/CaOlaHsd mice. The periodic positive control experiment was performed with α-Hexyl cinnamaldehyde in acetone:olive oil (4+1 v/v) using CBA/CaOlaHsd mice in April 2012.
Positive control substance(s):
other: α-Hexyl cinnamaldehyde in acetone:olive oil (4+1 v/v)
Statistics:
Statistical Analysis:
The mean values and standard deviations were calculated in the body weight tables. For all statistical calculations SigmaStat for Windows (Version 2.0) was used. A One-Way-Analysis-of-Variance was used as statistical method on the ear weights to assess whether the difference was statistically significant between test item groups and negative control (vehicle) group. In case of significant results of the One-Way-ANOVA, multiple comparisons were performed with the Dunnett test or the Student Newman Keuls test. However, both biological and statistical significance were considered together.

Results and discussion

Positive control results:
Experiment performed in April 2012 (Harlan study number 1486301) with α-Hexylcinnamaldehyde an EC3=20.1% was observed.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: Test item concentration % (w/w); Stimulation Index 0; 1.00 0.05; 0.87 0.1; 0.97 0.25; 0.78 The EC3 value could not be calculated, since all S.I.´s are below the threshold value of 3.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Remarks:
Test item concentration % (w/w); DPM per lymph node 0; 861.6 0.05; 745.9 0.1; 835.1 0.25; 668.3 Since the lymph nodes of the animals of a dose group were pooled, DPM/node was determined by dividing the measured value by the number of lymph nodes pooled.

Any other information on results incl. tables

Viability / Mortality:

No deaths occurred during the study period.

Clinical Signs:

No systemic findings were observed during the study period. On day 2, a very slight erythema of the ears was observed in the animals treated with 0.25% of the test item (score: 1). On day 4, a very slight erythema of the ears was observed in all treatment groups (score: 1). On day 6, the ears of the animals were scabby in all treatment groups.

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 Weights:

The measured ear weight of all animals treated was recorded on day 6 (after necropsy). In the animals treated with the highest test item dose (i.e., 0.25%), a statistically significant increase (p < 0.05) in ear weight was observed in comparison to the values of the vehicle control group.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU