Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010-05-03 - 2010-06-15
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP - Guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
: acceptable modifications refer to the measurement of cell proliferation by cell counting instead of radioactive labeling (according to Vohr, H.-W. et al., 1994; Ikarashi, Y. et al., 1993; Ehling, G. et al., 2005; Gamer, A.O. et al., 2008)
GLP compliance:
yes
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): benzothiazol
- Physical state: clear, light yellowish liquid
- Analytical purity: 98.3 %
- Lot/batch No.: RA004071Y6
- Expiration date of the lot/batch: April 6, 2012
- Stability under test conditions: verified for up to 4 days
- Storage condition of test material: room temperature

In vivo test system

Test animals

Species:
mouse
Strain:
NMRI
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Nederland, Kreuzelweg 53, 5969 AD Horst, The Netherland
- Age at study initiation: 8 weeks
- Weight at study initiation: 25 - 30 g
- Housing: individual
- Diet: ad libitum (PROVIMI KLIBA SA 3883 maintenance diet for rats and mice from Provimi Kliba SA, CH-4303 Kaiser august)
- Water: tap water ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 40 - 70 %
- Air changes (per hr): 10 changes per hour
- Photoperiod (hrs dark / hrs light): 12h / 12h, with artificial illumination

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
0 % (vehicle control), 2 %, 10 % and 50 %
No. of animals per dose:
6
Details on study design:
MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: modified Local Lymph Node Assay (IMDS); the modifications refer to the measurement of cell proliferation by cell counting instead of radioactive labeling and additionally the measurement of the ear swelling and ear weight to determinate between irritant and sensitising potential of the test compound
- Criteria used to consider a positive response: the positive level for cell count index was 1.4 and for ear swelling 2x10E-2 mm increase (for NMRI mice)

TREATMENT PREPARATION AND ADMINISTRATION:
Test item formulation:
The test item in the formulation or the vehicle were applied epicutaneously onto the dorsal part of both ears of the animals. This treatment was repeated on three consecutive days (d1, d2 and d3). The volume administered was 25 µl/ear.

LLN Weight and cell count determinations:
The weight and cell count determinations were carried out by appropriate laboratory procedures. The weights of the lymph nodes were determined on a Mettler semiautomatic balance and stored in an IBM compatible PC. After crushing the lymph nodes through a sieve in a 12-well plate, the cell count per mL were determined using a Multisizer 3 from Coulter Electronics. These data were also directly collected and processed by computer (Multisizer 3 software and Excel).

Ear Swelling:
Before the first treatment and before sacrifice the thickness of the animals was measured using a spring-loaded micrometer (Oditest, Dyer Company or Fa. Kroeplin). Means, indices and standard deviations of the ear swelling were calculated by an Excel data sheet.

Ear weight:
On day 4 of the study the ear weight of the sacrified animals was measured using a punch to take of a piece of every ear with a diameter of 8 mm. The weights were determined on a Mettler semiautomatic balance. Means, indices and standard deviations of the ear weights were calculated by an Excel data sheet.

Body weights:
The body weights of the animals were recorded at the start and the end of the study (day 1 and day 4)
Statistics:
When it was statistically reasonable, the values from treated groups were compared with those from the control group(s; vehicle) by a one-way analysis of variance (ANOVA) when the variances are considered homogeneous according to a homogeneity testing like Cochran´s test. Alternativley, if the variances are considered to be heterogenous (p=0.05), a nonparametric Kruskal-Wallis test has been used (Kruskal-Wallis ANOVA) at significance levels of 5%. Two sided multiple test procedures were done according to Dunnett or Bonferroni-Holm, respectively. Outlying values in the LN weights were eliminated at a propability level of 99% by Nalimov´s method. In addition, for the LLNA/IMDS the smallest significant differences in the means were calculated by Scheffe´s method, which according to Sachs can be used for both equal and unequal sample sizes.
In this method of statistical processing of measurements a large number of comparisons is made, and as a result of the multiple tests the overall probability of error is considerably greater than the p value suggest (increased number of false-positive results). On the other hand, the known methods of adjusting p values lead to an excessive increase in the number of false negatives. In view of the these problems the biological and toxicological relevance is also taken into consideration in the evaluation of statistical significance.
For this reason, in the case of indices only the standard deviations between groups and difference analysis of the mean values were used in the evaluation of the biological relevance.

Results and discussion

Positive control results:
A check is done in regular intervals using one of the mentioned vehicles in order to confirm the reliability of the method. The last reliability test using Alpha Hexyl Cinnamic Aldehyde formulated in acetone/olive oil (4:1) at concentrations of 3%, 10% and 30% clearlyshowed the sensitizing potential of the positive control substance.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: Cell count index: The stimulation index at a concentration of 2 % was 1.25 The stimulation index at a concentration of 10 % was 1.26 The stimulation index at a concentration of 50 % was 1.34
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: n.a.

Any other information on results incl. tables

Direct LLNA (NMRI mice, female, 6 animals/group):

Dose (%)

Weight index (index of mean ± SD in %)

Cell count index (index of mean +/- SD in %)

0 (vehicle = AOO)

1.00 ± 13.56

1.00 ± 17.11

2

1.21 ± 20.06

1.25 ± 37.56

10

1.16 ± 19.37

1.26 ± 24.73

50

1.23 ± 20.57

1.34 ± 25.83

Ear swelling (NMRI mice, female, 6 animals/group, in 0.01 mm):

Dose (%)

Day 1 (mean ± SD in %)

Day 4 (mean ± SD in %)

Index day 4

0 (vehicle = AOO)

16.83 ± 3.43

17.83 ± 4.02

1.00

2

17.42 ± 2.96

18.00 ± 4.74

1.01

10

17.25 ± 4.37

17.75 ± 4.25

1.00

50

17.25 ± 4.37

18.00 ± 3.35

1.01

Ear weight (NMRI mice, female, 6 animals/group, in mg per 8 mm diameter punch):

Dose (%)

Day 4 (mean ± SD in %)

Index day 4

0 (vehicle = AOO)

12.47 ± 5.82

1.00

2

12.27 ± 7.07

0.98

10

12.20 ± 6.88

0.98

50

11.69 ± 5.84

0.94

AOO = acetone/olive oil (4:1)

Cell counts:

Animal group

Mean value - Cell count (thousand cells / mL lymph node suspension)

SD

SD (%)

Cell count index

Vehicle

8530.75

1459.46

17.11

1.00

2 % solution

10656.17

4002.80

37.56

1.25

10 % solution

10778.92

2666.04

24.73

1.26

50 % solution

11454.92

1958.70

25.83

1.34

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Executive summary:

Vohr (Bayer Health Care), 2010

Benzothiazole was assessed for sensitising properties at concentrations of 0% (vehicle only), 2%, 10% and 50%(v/v) in an LLNA according to OECD 429 with acceptable modifications. The modifications refer to the measurement of cell proliferation by cell counting instead of radioactive labelling and additionally the measurement of ear swelling and ear weight. The study was performed on female NMRI mice of the strain Hsd Win:NMRI; 6 animals were used per group. Acetone/olive oil (4:1 (v/v)) was used as vehicle.

None of the three tested concentrations of the test item reached the cell count index of 1.4. The stimulation indices were: at a concentration of 2 % = 1.25; at a concentration of 10 % = 1.26 and at a concentration of 50 % = 1.34. Compared to vehicle-treated animals, none of the parameters measured in the substance-treated groups, i.e. cell counts and weights of the draining lymph nodes, ear weights and ear swelling, reached or exceeded the "positive levels" defined for this assay. The test item is regarded to have no skin sensitising properties.