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

Endpoint summary

Description of key information

Based on the observed results Reaction product of 2,4-Dinitrotoluene and 2,6-Dinitrotoluene and hydrogen, deaminated is not peptide reactive and does not activate keratinocytes.

Reaction product of 2,4-Dinitrotoluene and 2,6-Dinitrotoluene and hydrogen, deaminated is predicted not to be a skin sensitizer.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Specific details on test material used for the study:

Batch identification: C520/094/2017
CAS No.: mixture of 7003-32-9 + 6321-23-9
Purity: Cyclohexylamine-methyl: 99.763 area-%

Details on the study design:

Synthetic peptides:
Cysteine- (C-) containing peptide: Ac-RFAACAA-COOH (MW=751.9 g/mol)
Lysine- (K-) containing peptide: Ac-RFAAKAA-COOH (MW=776.2 g/mol)

The peptides are custom material (Supplier: GenScript, Piscataway, NJ, USA and JPT Peptide Technologies GmbH, Berlin, Germany)

Positive control results:

The positive control caused depletion of both peptides comparable to historic data

Key result

Parameter:

other: peptide depletion

Remarks:

cystein

Value:

-0.12

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Parameter:

other: peptide depletion

Remarks:

lysine

Value:

-0.48

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Peptide depletion [%]

cystein	sample 1	sample 2	sample 3	mean	SD
NC: ACN	- 0.24	0.30	- 0.06	0.00	0.28
Test substance	- 0.67	- 0.14	0.45	- 0.12	0.56
PC: EGDMA in ACN	60.04	63.78	64.93	62.92	2.55

lysine	sample 1	sample 2	sample 3	mean	SD
NC: ACN	- 0.71	0.04	0.31	0.00	0.62
Test substance	- 0.33	- 0.78	- 0.33	- 0.48	0.26
PC: EGDMA in ACN	9.67	10.22	10.84	10.25	0.59

Interpretation of results:

GHS criteria not met

Conclusions:

The mean C-peptide depletion, caused by the test substance was determined to be -0.12%.
The mean K-peptide depletion, caused by the test substance was determined to be -0.46%.

Executive summary:

Based on the observed results it was concluded that Reaction product of 2,4-Dinitrotoluene and 2,6-Dinitrotoluene and hydrogen, deaminated shows minimal or no chemical reactivity in the DPRA under the test conditions chosen.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Specific details on test material used for the study:

Test-substance No.: 17/0336-1
Batch identification: C520/094/2017
CAS No.: mixture of 7003-32-9 + 6321-23-9
Purity: Cyclohexylamine-methyl: 99.763 area-%

Details on the study design:

Cell line LuSens: Human transgenic keratinocyte cell line derived from HaCaT cells, prepared in collaboration with Christoph J. Wruck, RWTH Aachen, Germany.

Controls:
Negative control (NC): DL-Lactic acid (LA, CAS no.: 50-21-5), 5000 μM (= 450 μg/mL) in 1% DMSO in culture medium 3
Positive control (PC): ethylene glycol dimethacrylate (EGDMA, CAS no.: 97-90-5), 90.8 μM (= 18 μg/mL) in 1% DMSO in culture medium 3
Vehicle control (VC): 1% DMSO in culture medium 3
Blank control: Culture medium 3 without cells
Basal control: Culture medium 3 with cells

Experimental procedure:
LuSens cells from the working cell bank were thawed and cultured using culture medium 1, under standard culture conditions (37°C, ca. 5% CO2, ≥ 90% humidity) for at least passage ≥5 but not longer than 15 passages prior to testing. Before substance incubation, cells were seeded in 96-well microtiter plates (120 μL of 0.83 x 105 cells/mL cell suspensions), using culture medium 2 for incubation for 24 hours. Six independent, valid experiments were performed. In each experiment, three replicates of each test-substance concentration were tested.

After test substance application the plates were sealed with semi-permeable plate sealers in order to prevent evaporation of the test substance. The plates were placed into the incubator under standard culture conditions for the exposure period of 48 hours

After visual inspection of the cells, the supernatant was aspirated from the white assay plate and discarded. The cells were washed twice with 300 μL PBS (with Ca2+/Mg2+). Subsequently 200 μL of One-Glo-preparation (= 100 μL One-Glo- Mix and 100 μL PBS (without Ca2+/Mg2+)) per well was added and cells were shaken on a plate shaker for 10 min at room temperature in darkness. After the incubation the luminescence was measured in the luminometer.

Evaluation of results:
A test substance is concluded to exhibit a keratinocyte activating potential when the luciferase activity exceeds a 1.50 fold-induction of statistical significance with respect to the vehicle control at concentrations that do not reduce viability below 70% in at least two consecutive concentrations of two independent experiments.
A test substance is considered to be negative when the criteria mentioned above are not met up to the maximum concentration (= 2000 μM if molecular weight is applicable or 2000 μg/mL if molecular weight is not applicable) or up to the cytotoxicity limit (at least one concentration displaying viability below 70%).
To be relevant for evaluation, the cell viability must be more than 70% in at least three tested concentrations of an experiment.

Positive control results:

The positive and negative and vehicle control data is comparable to historic data.

Key result

Parameter:

other: keratinocyte activation

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: does not have a keratinocyte activating potential

Other effects / acceptance of results:

A test substance is concluded to exhibit a keratinocyte activating potential when the luciferase activity exceeds a 1.50 fold-induction of statistical significance with respect to the vehicle control at concentrations that do not reduce viability below 70% in at least two consecutive concentrations of two independent experiments.

A test substance is considered to be negative when the criteria mentioned above are not met up to the maximum concentration (= 2000 μM if molecular weight is applicable or 2000 μg/mL if molecular weight is not applicable) or up to the cytotoxicity limit (at least one concentration displaying viability below 70%).

To be relevant for evaluation, the cell viability must be more than 70% in at least three tested concentrations of an experiment.

The CV75 value (= estimated concentration that affords 75% cell viability) was determined by linear regression from the concentration response curve to be 280 μM.

Concentration (final test substance) [µM]	Concentration (test substance) [µM]	mean rel. viability [%]
VC	VC	100
0.5	0.5	91
1	1	99
5	5	92
10	10	99
50	50	101
100	100	88
500	501	59
1000	1002	26
2000	2005	6

The 1st experiment was valid and evaluable with a negative result (see attached background material). However, in the 2nd and 3rd experiment relative viability below 70% was obtained in too many concentrations. Hence, these experiments are not useful for evaluation. In addition, the 2nd experiment was invalid. In order to have more evaluable concentrations (relative viability above 70%), further experiments were carried out using 16 concentrations on two test plates.

As relative viability below 70% was observed only in 1 single concentration of the 4th experiment (see attached background material),and as this concentration was not the highest tested concentration, it is not clearly demonstrated that the concentrations selected were high enough. Also, no luciferase activity above 1.5 fold induction of statistical significance with respect to the vehicle control was observed in two consecutive concentrations. Hence, higher concentrations up to 1202 µM were tested in further experiments 5th until 7th.

Interpretation of results:

GHS criteria not met

Conclusions:

In summary, after 48 hours of exposure to test substance Reaction product of 2,4-Dinitrotoluene and 2,6-Dinitrotoluene and hydrogen, deaminated luciferase activity in LuSens cells was not induced in at least two consecutive concentrations with statistical significance affording at least 70% viability in at least two independent experiments.

Executive summary:

Based on the results observed, it has to be concluded that test substance Reaction product of 2,4-Dinitrotoluene and 2,6-Dinitrotoluene and hydrogen, deaminated does not have a keratinocyte activating potential.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information:

Test Method	Test Result	Test Evaluation
Direct Peptide Reactivity Assay (DPRA)	0.00% mean peptide depletion (-0.12% cysteine peptide depletion; -0.46% lysine peptide depletion).	Negative
Keratinocyte Activation Assay - LuSens	In at least two independent experiments no biologically relevant ARE-dependent luciferase activity induction was observed.	Negative
Dendritic Cell Line Activation Assay Human Cell Line Activation Test (hCLAT)	Not determined	Not determined

Based on the results summarized in that table above, Reaction product of 2,4-Dinitrotoluene and 2,6-Dinitrotoluene and hydrogen, deaminated is not peptide reactive and does not activate keratinocytes. Therefore, Reaction product of 2,4-Dinitrotoluene and 2,6-Dinitrotoluene and hydrogen, deaminated is predicted not to be a skin sensitizer.

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 predicted not to be a skin sensitizer under Regulation (EC) No 1272/2008.