1,2,3-trichlorobenzene

Skin sensitisation

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:

guideline study

Remarks:

GLP study performed according to the OECD TG 429, with IMDS modification. Although not a validated method, the IMDS modification is scientifically accepted. A position paper justifying the use of this modified LLNA is attached to this ESR, and summarised in brief within the executive summary section.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

2002

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

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. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

1,2,3-trichlorobenzene

EC Number:

201-757-1

EC Name:

1,2,3-trichlorobenzene

Cas Number:

87-61-6

Molecular formula:

C6H3Cl3

IUPAC Name:

1,2,3-trichlorobenzene

Test material form:

solid

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

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: analytically verified for up to 4 days
- Reactivity of the test material with the incubation material used (e.g. plastic ware): not reported

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): formulated in AOO
- Final concentration of a dissolved solid, stock liquid or gel: not reported

In vivo test system

Test animals

Species:

mouse

Strain:

NMRI

Remarks:

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:

PRE-SCREEN TESTS:
- Compound solubility: solid, tested at the maximum recommended concentration in vehicle (50%)
- Irritation: none reported
- Systemic toxicity: none reported
- Ear thickness measurements: none reported
- Erythema scores: not reported

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: simulation indice > 1.4

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

Results 1

Key result

Parameter:

other: EC1.4

Value:

25.09

Results 2

Key result

Parameter:

SI

Value:

1

Variability:

+/- 34,68%

Test group / Remarks:

0 % (control)

Results 3

Key result

Parameter:

SI

Value:

0.94

Variability:

+/- 17.01 %

Test group / Remarks:

2%

Results 4

Key result

Parameter:

SI

Value:

1.2

Variability:

+/- 27.84%

Test group / Remarks:

10%

Results 5

Key result

Parameter:

SI

Value:

1.73

Variability:

+/- 16.40%

Test group / Remarks:

50%

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
See Result tables below.

DETAILS ON STIMULATION INDEX CALCULATION
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 counts 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). Means, indices and standard deviations were calculated by an Excel data sheet.
A special BASIC program (GWBASIC compiler) was used to calculate means and standard deviations of the Lymphnodes’ weights. Indices were calculated manually.
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.

EC1.4 CALCULATION
The so-called stimulation (or LLN-) index is calculated by dividing the absolute number of weight or cell counts of the substance treated lymph nodes by the vehicle treated ones. Thus, in case of no stimulating effect the index is always about 1.00 (+/- standard deviation), and the indices of vehicle treated animals are set to 1.00 (+/- standard deviation).

CLINICAL OBSERVATIONS:
None reported

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

SIGNS OF TOXICITY (including dermal irritation at the site of administration, if any, e.g. increased ear thickness).
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.

OTHER
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.

Any other information on results incl. tables

Table 7.4.1 / Tabular summary

Dose (%)	Weight Index (index of mean +/- SD in %)	Cell count index (index of mean +/- SD in %)
0	1.00 +/- 21,29	1.00 +/- 34,68
2	0.89 +/- 16.62	0.94 +/- 17.01
10	0.94 +/- 16.15	1.20 +/- 27.84
50	1.38* +/- 15.09	1.73* +/- 16.40

* statistically significant increase (p <= 0.05)

 

 

Table 7.4.2 / Ear swelling

Dose (%)	Day 1 (mean +/- SD in %)	Day 4 (mean +/- SD in %)	Index day 4
0	17.75 +/- 2.55	18.33 +/- 2.69	1.00
2	17.50 +/- 2.98	17.75 +/- 4.88	0.97
10	17.67 +/- 3.69	18.08 +/- 2.85	0.99
50	17.50 +/- 2.98	18.00 +/- 4.10	0.98

 

Table 7.4.3 / Ear weight

Dose (%)	Day 4 (mean +/- SD in %)	Index day 4
0	13.72 +/- 5.45	1.00
2	12.33* +/- 6.70	0.90
10	13.84 +/- 5.84	1.01
50	13.16 +/- 9.04	0.96

* statistically significant decrease (p <= 0.05)

 

Applicant's summary and conclusion

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

1,2,3-Trichlorbenzolhas a weak sensitizing potential in mice after dermal application of a 50% concentration.

Executive summary:

Vohr (2009)

 

The modified Local Lymph Node Assay (IMDS) was performed in 2009 on 24 female NMRI 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.