Reaction product of 2,4-Dinitrotoluene and 2,6-Dinitrotoluene and hydrogen

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:

other: modern guideline study conducted under GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)

Deviations:

no

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:

Reaction product of 2,4-Dinitrotoluene and 2,6-Dinitrotoluene and hydrogen

EC Number:

939-489-9

Molecular formula:

C7 H16 N2

IUPAC Name:

Reaction product of 2,4-Dinitrotoluene and 2,6-Dinitrotoluene and hydrogen

Test material form:

liquid

Details on test material:

Test Item (as cited in study report): Reaction product of 2,4-Dinitrotoluene and 2,6-Dinitrotoluene and hydrogen
BASF Test Item No.: 10/0117-4
Batch Number: 73583556P0
CAS-No.: 13897-55-7 and 13897-56-8
Purity: Mixture of isomers (in total > 99%)
Physical state, colour: Liquid / yellowish, clear
Homogeneity: The test item was homogeneous by visual inspection.
Storage conditions: Room temperature
Expiration Date: November 14, 2015
Density [g/mL]: 0.929

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/CaOlaHsd

Sex:

female

Study design: in vivo (LLNA)

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)

Results and discussion

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

1

Test group / Remarks:

vehicle control

Results 2

Key result

Parameter:

SI

Value:

0.9

Test group / Remarks:

2% test substance

Results 3

Key result

Parameter:

SI

Value:

1

Test group / Remarks:

5% test substance

Results 4

Key result

Parameter:

SI

Value:

1.1

Test group / Remarks:

10% test substance

Results 5

Parameter:

SI

Value:

2.8

Test group / Remarks:

positive control hexyl cinnamic aldehyde

Results 6

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

Applicant's summary and conclusion

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.