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Description of key information

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
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: modern guideline study conducted under GLP
Qualifier:
according to
Guideline:
OECD Guideline 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
other: CBA/CaOlaHsd
Sex:
female
Vehicle:
propylene glycol
Concentration:
2, 5, and 10%
No. of animals per dose:
5
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Parameter:
SI
Value:
1
Test group / Remarks:
vehicle control
Key result
Parameter:
SI
Value:
0.9
Test group / Remarks:
2% test substance
Key result
Parameter:
SI
Value:
1
Test group / Remarks:
5% test substance
Key result
Parameter:
SI
Value:
1.1
Test group / Remarks:
10% test substance
Parameter:
SI
Value:
2.8
Test group / Remarks:
positive control hexyl cinnamic aldehyde
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: mean BrdU laeling index: vehicle control: 0.105 2% MDACH in PG: 0.091 5% MDACH in PG: 0.108 10% MDACH in PG: 0.113 positive control (hexyl cinnamic aldehyde): 0.295
Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
The test item Reaction product of 2,4-Dinitrotoluene and 2,6-Dinitrotoluene and hydrogen was thus not a skin sensitiser under the test conditions of this study.
Executive summary:

The study was conducted to assess the skin sensitizing potential of the test item Reaction product of 2,4-Dinitrotoluene and 2,6-Dinitrotoluene and hydrogen using the Local Lymph Node Assay (LLNA) in mice. Test item solutions at different concentrations were prepared in the vehicle propylene glycol (PG).

The local lymph node assay is recommended by international test guidelines (e.g. OECD) as an animal test for predicting skin sensitization in humans and provides a rational basis for risk assessment. The basic principle underlying the LLNA is that sensitizers induce a primary proliferation of lymphocytes in the lymph node draining the application site. The ratio of proliferation in test item treated groups compared to that in vehicle controls is termed the Stimulation Index (S.I.). BrdU labeling is used to measure cell proliferation.

For this purpose a local lymph node assay was performed using test item concentrations of 2, 5 and 10% (w/w; weight per weight). Doses based on previously performed two pre-tests in the same animal strain. Two mice per concentration were treated epicutaneously with test item concentrations of 2.5, 5, 10 and 25 % (w/w) each on three consecutive days. Signs of systemic toxicity were not observed in any of the pre-tests. At the tested concentration of 25% the animals showed signs of local irritation as confirmed by the ear weight measurements. Both animals in this dose group showed increased ear weights > 25%. In the 10% dose group both animals showed body weight loss > 5%. Due to the fact that the animals in the 25% dose group did not show a body weight loss beyond the threshold, it is likely and assumed that body weight loss in the 10% group is an incidental finding without any effects for the study. In the 2.5 and 5% group the measured values were below the cut-off values.

The following dose levels were selected for the main study: 2%, 5% and 10% (w/w) in propylene glycol. Additionally, a positive control group (25% hexyl cinnamaldehyde in propylene glycol, w/w) was also applied.

In the main study the animals did not show any relevant signs of systemic toxicity during the course of the study and no cases of mortality were observed. A statistically significant or biologically relevant increase in ear weights was not observed in any treated group in comparison to the vehicle control group. Furthermore, the cut-off-value for a positive response (irritation) regarding the ear weight index of 1.25 was not exceeded in any dose group.

A test item is regarded as a sensitizer in the LLNA if exposure to one or more test item concentration results in a 1.6-fold or greater increase in incorporation of BrdU compared with concurrent controls, as indicated by the Stimulation Index (S.I.). The estimated test item concentration required to produce a S.I. of 1.6 is referred to as the EC1.6 value.

In this study Stimulation Indices (S.I.) of 0.9, 1.0 and 1.1 were determined with the test item at concentrations of 2, 5 and 10 % (w/w) in propylene glycol. An EC1.6 value could not be determined as all S.I.s obtained were below the threshold of 1.6.

A statistically significant or biologically relevant increase in the BrdU value and also in lymph node weight and cell count was not observed in any of the tested dose groups in comparison to the vehicle control group. Furthermore, the cutoff-value for a positive response regarding the lymph node cell count index of 1.55 reported for BALB/c mice was not exceeded in any of the tested dose groups.

As expected, a statistical and biological relevant increase in BrdU labelling, lymph node weight, lymph node cell count and ear weight measurement was determined in the positive control. For the positive control a S.I of 2.8 was determined.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

In order to study a possible skin sensitization potential of reaction product of 2,4-dinitrotoluene and 2,6-dinitrotoluene and hydrogen, three groups each of five female mice were treated once daily with the test item at concentrations of 2, 5, and 10% propylene glycol by topical application to the dorsum of each ear for three consecutive days. The appropriateness of the used concentrations was previously assessed by a pre-experiment. A control group of five mice was treated with the vehicle propylene glycol only. A positive control group of five mice was treated with 25 % (w/w)α-hexyl cinnamaldehyde dissolved in propylene glycol. Four days after the first topical application the mice were intraperitoneally injected with BrdU. Approximately 24 hours after intraperitoneally injection, the mice were sacrificed and the draining auricular lymph nodes excised, pooled per animal and immediately weighed. Afterwards, single cell suspensions of lymph node cells were prepared from lymph nodes pooled per animal. An aliquot of each cell suspension was used for determination of lymph node cell count. The proliferative capacity of pooled lymph node cells was determined by the incorporation of BrdU measured in a photometer.In this study S.I. of 0.9, 1.0 and 1.1 were determined with the test item at concentrations of 2, 5, and 10% (w/w) in propylene glycol, respectively. Therefore, the test itemreaction product of 2,4-dinitrotoluene and 2,6-dinitrotoluene and hydrogenwas notfound to be a skin sensitizerunder the test conditions of this study. [BASF SE, 2014]

Sensitization involves a number of key steps in order to take place, and can be described in terms of an adverse outcome pathway (AOP). These include reactivity with skin proteins (peptide reactivity), activation of skin cells (keratinocyte activation) and immune cells (dendritic cell activation). The supporting study carried out for this substance address the first of these key steps.The ECVAM opinion on the direct peptide binding assay has been published on Dec 13, 2013.The results are then used in a supporting manner to underline the hazard classification as a non-sensitizer.The test applicability is limited when testing substances insoluble in the commonly used vehicles and highly volatile substances. Substances susceptible to base-catalyzed hydrolysis cannot be evaluated reliably for binding to lysine as the incubation is performed at pH 10.2. Also substances that interfere with the experimental measurements (e.g., co-elution with the peptide in the DPRA-assay) are not or only partially suitable for testing using in vitro methods. The substance under evaluation did not have any of the above mentioned limitations and the outcome of this assay is therefore considered adequate to support hazard identification for the endpoint of skin sensitization.

Based on the observed results and applying the prediction model proposed in Gerberick et. al (2007) it was concluded that reaction product of 2,4-dinitrotoluene and 2,6-dinitrotoluene and hydrogen shows a minimal chemical reactivity in the DPRA under the test conditions chosen. Thus according to the classification tree model proposed by Gerberick et al. the test substance is predicted to be a non-sensitizer, which is also in line with the results observed in the LLNA. [BASF, 2013]


Migrated from Short description of key information:
Reaction product of 2,4-dinitrotoluene and 2,6-dinitrotoluene and hydrogen was not skin sensitizing in the LLNA (OECD 442B) [BASF SE, 2014] and was proven to be non-reactive in the DPRA [BASF, 2013].

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Classification,Labelling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is not considered to be classified for skin sensitization under Regulation (EC) No. 1272/2008, as amended for the third time in Directive (EC 618/2012).

There is also no information available, that reaction product of 2,4-dinitrotoluene and 2,6-dinitrotoluene and hydrogen is a sensitizer of the respiratory tract.