Registration Dossier

Administrative data

Endpoint:
skin sensitisation
Remarks:
in vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Principles of method if other than guideline:
A modification of the assay by measuring the cell counts instead of radioactive labeling provides comparable sensitivity, and has the advantage that the cell suspension can be further analyzed by different methods (flow cytometry, chemiluminescence responses, immunofluorescence) to gain an insight into mechanistic events. A further modification was done by including the measurement of the ear swelling after treatment leading to a much more simplified and reliable assay (Integrated Model for the Differentiation of Skin reactions (IMDS)). By comparing the specific immune reaction induced by the test item in the draining lymph nodes (LN; cell counts / LN weights) with the immediate unspecific acute skin reaction (ear swelling / ear weight) it is possible to discriminate the irritant potential from the sensitizing potential of the compound tested. International standards have been successfully determined using this modification. Such modifications are also authorized in the Note of Guidance SWP/2145/00 of the CPMP (2001) and OECD guideline 429.
With respect to this simple discrimination between sensitizing and irritant local reactions comparable findings have been reported in the human patch test system
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): 1,2,3-trichlorobenzene
- Physical state: white, solid
- Analytical purity: 99.46%
- Lot/batch No.: CHH 180809
- Storage condition of test material: room temperature


In vivo test system

Test animals

Species:
mouse
Strain:
other: SPF-bred female NMRI mice of the strain Hsd Win:NMRI
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Nederland, Kreuzelweg 53, 5960 AD Horst, Netherland
- Age at study initiation: 8 weeks
- Weight at study initiation: 27- 33 grams
- Housing: During the adaptation period up to 8 mice were housed together in conventional Makrolon-type III cages. During the study period the animals were single-housed in type II cages.


- Diet (e.g. ad libitum): The feed, PROVIMI KLIBA SA 3883 maintenance diet for rats and mice (from Provimi Kliba SA, CH-4303 Kaiseraugst, Germany)

- Water (e.g. ad libitum): tap water (drinking bottles) were provided ad libitum.

- Acclimation period: After their arrival, the animals intended for the study were allowed to adapt to the conditions of the animal room for at least 6 days and their state of health was monitored


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


IN-LIFE DATES: From: 19-10-2009 To: 22-10-2009

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:
TREATMENT PREPARATION AND ADMINISTRATION:
The test item was 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.

Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
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) [Mann and Whitney. Ann. Math. Stat. 18 (1947), 50-60; Wilcoxon. Biometrics 1 (1945), 80-83] when the variances are considered homogeneous according to a homogeneity testing like Cochran`s test [Sachs. Springer Verlag, Berlin (1978/2002)]. Alternatively, if the variances are considered to be heterogenous (p< or=0.05), a non-parametric Kruskal-Wallis test has been used (Kruskal-Wallis ANOVA) at significance levels of 5%. Two sided multiple test procedures were done according to Dunnett [Dunett . Ass. J. 50 (1955), 1096-1121; Dunett. Biometrics 20 (1964), 482-491] or Bonferroni-Holm [Holm. Scand. J. Statist. 6 (1979), 65-70], respectively. Outlying values in the LN weights were eliminated at a probability level of 99% by Nalimov's method [Keller, F. Statistik f. naturwissenschaftliche Berufe (1982), 88-89]. In addition, for the LLNA/IMDS the smallest significant differences in the means were calculated by Scheffe's method [Scheffe, H. Biometrica 40 (1953), 87-104 ], which according to Sachs [Sachs. Springer Verlag, Berlin (1978/2002)] 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 values 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 these problems the biological and toxico¬logical relevance is also taken into consideration in the evalua¬tion 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

In vivo (LLNA)

Results
Parameter:
SI
Remarks on result:
other: see "Remarks on results including tables and figures"

Any other information on results incl. tables

Based on results obtained in validation studies and general experiences with this test system groups of mice were treated with vehicle, 2%, 10% or 50% 1,2,3-Trichlorbenzol in A/OO.

 

The NMRI mice showed a clear increase in the weights of the draining lymph nodes and in the stimulation indices for cell counts in the high dose group, which is of statistical significance, compared to control animals after application of the test item 1,2,3-trichlorobenzene. The positive level, which is 1.4 for cell count indices, has been exceeded in the high dose group.

 

The positive level of ear swelling, which is 2x10-2mm increase , i.e. about 10% of the control values, has not been reached or exceeded in any dose group.

A slight statistically significant decrease in ear weight had been determined for group 2. This decrease is within the normal range of variance for this parameter, which means that it is only of statistical significance because the mathematical conditions were favorable.

No substance specific effects were determined for ear swelling.

 

It has to be clarified that the positive levels mentioned above are exclusively defined for the NMRI outbreed mice used for this study . Such positive limits have to be calculated for each strain of mice individually.

The body weights of the animals were not affected by any treatment

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information
Executive summary:

Vohr (2009)

The modified Local Lymph Node Assay (IMDS) was performed in 2009 on 24femaleNMRI mice of the strain Hsd Win:NMRI (6 animals/test item group and 6 control animals) to determine if there is any specific (sensitizing) or non-specific (irritant) stimulating potential of the test item 1,2,3-Trichlorbenzol.

 

The study was conducted according to OECD Guidelines No. 429 and No. 406,
EC Guideline 2004/73/EC (29th Adaptation of Guideline 67/548/EEC, B.42)/Health Effects Test Guideline and OPPTS 870.2600 (EPA) with the following test item concentrations:

 

0 (vehicle control), 2%, 10% and 50%.

 

The test item was formulated in acetone/olive oil (4:1) (A/OO) to yield a solution.

 

Compared to vehicle treated animals there were clear increases regarding the weights of the draining lymph nodes and the cell counts in the high dose group. These increases are of statistical significance. The "positive level" of index 1.4 for the cell counts was clearly exceeded at the high dose group.

 

The positive level of ear swelling, which is 2x10-2mm increase , i.e. about 10% of the control values, has not been reached or exceeded in any dose group.

A slight statistically significant decrease in ear weight had been determined for group 2. This decrease is within the normal range of variance for this parameter, which means that it is only of statistical significance because the mathematical conditions were favorable.

No substance specific effects were determined for ear swelling.

 

In conclusion, these results show that the test item1,2,3-Trichlorbenzolhas a weak sensitizing potential in mice after dermal application of a 50% concentration.

Therefore, the concentration of 10% turned out to be the NOEL for the parameters investigated in this study with respect to skin sensitization.