Registration Dossier

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: Guideline study (GLP, QAU)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

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)
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 2,6-Xylidine (BASF Test Item No.: 09/0827-1)
- Physical state: clear, colorless to reddish-yellow liquid
- Analytical purity: concomitantly analysed (BASF study code 10L00003); dose calculation not adjusted to purity
- Lot/batch No.: 000STD77L0
- Expiration date of the lot/batch: December 15, 2011
- Stability under test conditions: verified indirectly by concentration control analysis
- Storage condition of test material: room temperature

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: 8 - 12 weeks
- Weight at study initiation:
- Housing: single in Makrolon Type II, with wire mesh top (EHRET GmbH, 79302 Emmendingen, Germany)
- Diet (e.g. ad libitum): pelleted standard diet, ad libitum (Harlan Laboratories B.V. 5960 AD Horst / Netherlands)
- Water (e.g. ad libitum): tap water
- 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 (%): 25 - 67
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12 / 12

Study design: in vivo (LLNA)

Vehicle:
methyl ethyl ketone
Remarks:
Methyl ethyl ketone (purity 99% from Sigma-Aldrich Chemie GmbH (89555 Steinheim, Germany)
Concentration:
0, 5, 10 or 25% (w/v) in vehicle
No. of animals per dose:
5 (2 for the 2 pre-test groups)
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: the highest test item concentration, which could be technically used, was 100% of the undiluted test item. Upon sponsor’s request, the solutions were formulated in methyl ethyl ketone. Vortexing was necessary to formulate the test item
- Irritation: to previously confirm the appropriateness of the concentrations intended for use in the main experiment, a pre-test was performed using 50 and 100% test substance concentration. During the pre-test, both animals showed signs of systemic toxicity and the animal treated with 100% undiluted test substance was found dead within one hour after the second application. Thus, a second pre-test was performed using test substance concentrations of 10 and 25% (w/v). At these concentrations the animals did not show any signs of systemic toxicity or signs of local skin irritation. Therefore, in this study, three groups each of five female mice were treated with 5, 10 or 25% (w/v) test substance concentration by topical application at the dorsum of each ear on three consecutive days.
- Lymph node proliferation response: only ear thickness and ear weight measurements.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response: a test item is regarded as a sensitiser in the LLNA if the following criteria are fulfilled: (1) 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; and (2) 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.
The estimated concentration of test item required to produce a S.I. of 3 is referred to as the EC3 value.

TREATMENT PREPARATION AND ADMINISTRATION:
- each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear (left and right; entire dorsal surface of 8 mm diameter) of 25 µl test item concentrations in vehicle (methyl ethyl ketone ) or its vehicle alone or with the positive control item at 25% (w/v in acetone:olive oil 4:1), or acetone:olive oil 4:1 alone, once daily for three consecutive days.

- Five days after the first topical application, 250 µl of 79.7 µCi/ml of 3H-methyl thymidine (3HTdR; purchased from Hartmann Analytic, 38124 Braunschweig, Germany; specific activity, 2 Ci/mmol; concentration, 1 mCi/ml) were administered (i.v.) to the test animals via the tail vein.

- Approximately five hours after treatment with 3HTdR all mice were euthanized by intraperitoneal injection of Pentobarbital-Natrium. The draining lymph nodes were rapidly excised and pooled per animal (2 nodes per animal), and single cell suspensions (in phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze. After washing two times, 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 and thoroughly mixed.
The level of 3HTdR incorporation was then measured on a β-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 β-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM).

- After the lymph nodes had been excised, both ears (left and right) of mice were punched at the apical area using a biopsy punch (approx. 0.5 cm2). For each animal both punches were immediately weighed per animal using an analytical balance.
Positive control substance(s):
other: Alpha-Hexylcinnamaldehyde, tech. 85% from (Sigma-Aldrich Chemie GmbH, 82024 Taufkirchen, Germany) diluted in olive oil or acetone
Statistics:
For all statistical calculations SigmaStat for Windows (Version 2.0) was used. A One-Way-Analysis-of-Variance was used as statistical method. In case of significant results of the One-Way-ANOVA, multiple comparisons were performed with the Dunnett test. However, the primary point of consideration was the biological relevance of the results.
A statistical analysis was conducted on the DPM/animal values and on the ear weights to assess whether the difference is statistically significant between test item groups and negative control (vehicle) group and also to assess if there is a dose response relationship.

Results and discussion

Positive control results:
The mean disintegrations per minute was, 858.2 in the test group 5 (control group for the positive control) and 6416.6 in the test group 6 (positive control group). The corresponding mean stimulation index for the test group 6 was 7.5 (see Table 2), thus clearly positive according to the criteria used to consider a positive response (SI > 3).

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: The mean stimulation index was 2.2, 1.78 and 2.74, respectively in the test groups 2, 3 and 4; see Table 2. The EC3 value could not be calculated, since all S.I.´s are below 3.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: The mean disintegrations per minute was 493.0 in the test group 1(control group for the test item) 1089.6, 875.4 and 1356.6, respectively in the test groups 2, 3 and 4 (test item treated groups); see Table 2.

Any other information on results incl. tables

Table 2: Calculation of Stimulation Indices per Dose Group

Test item concentration

Mean DPM per Group (5 animals)a)

SDb)

S.I.

Vehicle for the Test Item (methyl ethyl ketone)

492.5

110.3

1.00

5% 2,6-Xylidine

1089.1S

246.2

2.21

10% 2,6-Xylidine

874.9

280.6

1.78

25% 2,6-Xylidine

1356.1S

486.2

2.75

Vehicle for the Positive Control (acetone:olive oil (4+1))

857.7

196.7

1.00

25% Positive Control

6416.1S

1837.4

7.48

a): mean DPM/Group 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)b): SD= standard deviationS: statistically significant increase in comparison to the relevant control (vehicle) group (p<0.05)

 

 

ADDITIONAL RESULTS

- Viability / Mortality: no deaths occurred during the study period.

- Clinical Signs: no symptoms of local toxicity at the ears of the animals and no systemic findings 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.

- Ear Weights: the measured ear weights of all animals treated were recorded after sacrifice. A statistically significant increase in ear weights was not observed in any of the groups treated with different concentrations of the test item in comparison to the vehicle control group.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
CONCLUSION
The test item 2,6-Xylidine was not a skin sensitiser under the test conditions of this study.