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Toxicological information

Skin sensitisation

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Administrative data

skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
other: EC Guideline 2004/73/EC (29th Adaption of Guideline 67/548/EEC. B.42
Modified LLNA (IMDS). Cell proliferation was measured by cell counting instead of radioactive labelling.
according to guideline
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Modified LLNA (IMDS). Cell proliferation was measured by cell counting instead of radioactive labelling.
according to guideline
OECD Guideline 406 (Skin Sensitisation)
according to guideline
other: OPPTS 870.2600 (EPA)
Principles of method if other than guideline:
The modified Local Lymph Node Assay (IMDS) was performed 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 1,3-dichlorobenzene.
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:
Type of study:
other: LLNA (IMDS)
Justification for non-LLNA method:
Justification attached

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Details on test material:
- Name of test material (as cited in study report): m-dichlorobenzene
- Physical state: liquid
- Analytical purity: 99.4%
- Storage condition of test material: room temperature

In vivo test system

Test animals

other: SPF-bred female NMRI mice of the strain Hsd Win:NMRI
Details on test animals and environmental conditions:
- Source: Harlan Nederland, Kreuzelweg 53, 5960 AD Horst, Netherland
- Age at study initiation: 9 weeks
- Weight at study initiation: 27 ¿ 35 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

- 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 illumi¬nation

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

Study design: in vivo (LLNA)

acetone/olive oil (4:1 v/v)
0 (vehicle control), 2%, 10% or 50%.
No. of animals per dose:
6 animals
Details on study design:
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)
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 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

In vivo (LLNA)

Resultsopen allclose all
Key result
Cell count index
+/- 14.76 %
Test group / Remarks:
Group 4 (50%)
Key result
Cell count Index
+/- 38.34 %
Test group / Remarks:
Group 3 (10%)
Key result
Cell count index
+/- 30.22%
Test group / Remarks:
Group 2 ( 2%)
Key result
Cell count index
+/- 26.13
Test group / Remarks:
Group 1 - Control (0%)
Cellular proliferation data / Observations:
Results are attached to the ESR.


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).

EC3 CALCULATION - Not applicable


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): none reported

The Local Lymph Node Assay Test methodology was checked for reliability in a test on female NMRI mice using Alpha Hexyl Cinnamic Aldehyde formulated in different vehicles (PEG 400, DAE 433, DMP, MEK, acetone/olive oil (4:1) and Cremophor EL/ physiological saline solution 2% v/v) at concentrations of 3%, 10%, 30%. The sensitivity as well as the reliability of the experimental technique is thus confirmed by this study. A similar check is done in regular intervals using one of the above 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% clearly showed the sensitizing potential of the test item.

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% m-dichlorobenzene in A/OO.


After treatment with 1,3-dichlorobenzene there was a clear increase compared to control animals regarding the weights of the draining lymph nodes, which is of statistical significance in the high dose group. The "positive level", which is 1.4 for cell counts, has been exceeded in the high dose group.

A sensitizing potential can be assumed from the increases in cell proliferation in the draining lymph nodes. On the basis of the experiences using this method the "positive level" had been set to an increase in cell count index by 0.4 (i.e. index > 1.4), which has been exceeded in the high dose group.

The "positive level" of ear swelling which is 2 x l0E-2 mm increase, i.e. more than 10% increase in index, has not been reached or exceeded.

The EC 1.4 value calculated is 26.30% for this test item. In accordance with the classification proposed in the Technical Report No. 78 of the ECETOC this value corresponds to a weak skin sensitizer.


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:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Under the conditions of the study, the test item m-dichlorobenzene is a weak sensitiser to mice following dermal application of up to and including 50% concentration.
Executive summary:

A modified Local Lymph Node Assay (IMDS) was performed on 24 female NMRI mice of the strain HsdWin: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 m-dichlorobenzene. A concurrent control of 6 animals treated with Alpha Hexyl Cinnamic Aldehyde was included.

Materials and Methods

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), using the modified LLNA-IMDS approach, with the following test item concentrations:

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

The test item was formulated in dimethylformamide (DMF) to yield a suspension.

The positive control was formulated in Acetone/Olive oil (4:1) (A/OO) to yield a solution.

The modified LLNA-IMDS (Integrated Model for Differentiation of Skin Reactions)

The standard LLNA has been criticised because it uses radioactive thymidine (which is both limiting as it requires an isotope laboratory, because the i.v. application of [3H] thymidine leads to relatively high individual variance), and due to the incidence of false-positive findings due to irritation and other mechanisms.

The IMDS modification measures cell proliferation by cell counting instead of radioactive labeling. In addition, the acute inflammatory skin reaction is determined to discriminate specific from non-specific activation of immune competent cells in the draining lymph nodes. A position paper has been produced on the modified LLNA (referred to as LLNA-IMDS), and is attached to this endpoint study record (Vohr et al, 2009). 

In brief, the modified LLNA-IMDS was investigated in depth in an intra-laboratory validation, published in 2000 (Vohr, HW. et al., 2000). This modified LLNA (as LLNA-IMDS) was subsequently carried out in-house under full GLP conditions as an alternative to guinea pig testing. The reliability of LLNA- IMDS was also demonstrated in CBA mice one year later in an inter-laboratory study (Suda, A. et al., 2001).

As recognised by both ICCVAM and the regulators in the interim, the specific cut-off value (ECt) must be determined for each endpoint to establish cell proliferation and for the murine strain used. The cut-off values are dictated by two parameters: i) individual endpoint variance in the group of individual animals, and ii) the possible maximum stimulation (maximum index) that can be achieved for the relevant parameter. Relatively high stimulation indices (SI) are normally achieved with the radioactive method but the individual variances obtained from individual animal measurements are also rather high. The cut-off that determines a positive result was therefore set at a proliferation index of factor 3 (EC3) for this method. Based on historical LLNA-IMDS data and the assessments of all intra- and inter-laboratory validation studies, the cut-off for the cell count index was set at 1.4 for NMRI (outbred) mice. 

In 2004, the reliability of the modified LLNA (LLNA-IMDS) was investigated in an international catch-up validation study. Extremely convincing results were published in two papers in 2005 (Ehling, G. et al., 2005a; 2005b). In this international study, the cut-off values were confirmed as 1.4 and 1.5 for NMRI (outbred) and BALB/c, respectively. This minor difference is due to the fact that BALB/c mice react slightly more violently overall to non-specific activations – hence the individual variance is somewhat greater than in NMRI (outbred) mice.

Based on copious data and detailed rationale, the modified LLNA (LLNA-IMDS) should be considered as reliable, robust and at least as sensitive as the standard LLNA. The IMDS modification of the LLNA has been published several times in various recognised journals. Moreover, the method has been repeatedly described in meticulous detail in these journals. All validation studies were performed under full GLP conditions. The LLNA-IMDS protocol therefore complies with OECD 429 requirements in every respect.

An English language copy of the full position paper is attached (Vohr, 2009). 


Compared to vehicle-treated animals, the NMRI mice showed 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 after application of the test item m-dichlorobenzene. The ‘positive level’ which is 1.4 for cell count indices has been exceeded in the high dose group. These results show that there is an indication for a skin sensitizing effect after administration of a concentration up to 50 % m-dichlorobenzene in this test system.

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.

No substance specific effects were determined for ear weight either.



In conclusion, these results show that the test item m-dichlorobenzene has 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.