2,4-dichlorobenzonitrile

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11.10.2017 - 27.10.2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

According to REACH regulation Annex VII, 8.3.2 column 2 an in vivo test (LNNA is preferred) will only be performed, when the in chemico / in vitro
methods according to 8.3.1, column 1 are not applicable for the test substance or the are not convincing.

The correlation of protein reactivity with skin sensitisation potential of a chemical is well established and represents the first and initial key event in the skin sensitisation process as defined by the AOP. It is therefore a crucial step for the sensitising potential of a chemical.

This test method is able to detect chemicals that cause skin sensitisation and allows for hazard identification in accordance with UN GHS “Category 1”.

Test material

Test material information

Constituent 1

Reference substance name:

2,4-dichlorobenzonitrile

EC Number:

229-493-2

EC Name:

2,4-dichlorobenzonitrile

Cas Number:

6574-98-7

Molecular formula:

C7H3Cl2N

IUPAC Name:

2,4-dichlorobenzonitrile

impurity 1

Reference substance name:

2,4-dichlorotoluene

EC Number:

202-445-8

EC Name:

2,4-dichlorotoluene

Cas Number:

95-73-8

Molecular formula:

C7H6Cl2

IUPAC Name:

2,4-dichloro-1-methylbenzene

impurity 2

Reference substance name:

2,4-dichlorobenzamide

EC Number:

219-505-4

EC Name:

2,4-dichlorobenzamide

Cas Number:

2447-79-2

Molecular formula:

C7H5Cl2NO

IUPAC Name:

2,4-dichlorobenzamide

impurity 3

Reference substance name:

2-chlorobenzonitrile

EC Number:

212-836-5

EC Name:

2-chlorobenzonitrile

Cas Number:

873-32-5

Molecular formula:

C7H4ClN

IUPAC Name:

2-chlorobenzonitrile

impurity 4

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

water

impurity 5

Reference substance name:

unknown

IUPAC Name:

unknown

Test material form:

solid: bulk

Details on test material:

This composition is the usual techical grade of AlzChem AG. It will be used when test substance has this composition (or is very closed) or no specific information is available

In chemico test system

Details on the study design:

HPLC: Agilent, 1200 Series with Chemstation, Rev. B.04.01
Detection: 220 nm (quantification)
258 nm (indicator for co-elution)
Analytical Column: Zorbax SB-C18, 100 mm x 2.1 mm, 3.5 µm
Pre-Column: Phenomenex, AJO-4286, 4.0 x 2.0 mm.

Mobile Phase A: 0.1 % (v/v) trifluoroacetic acid in water
Mobile Phase B: 0.085 % (v/v) trifluoroacetic acid in acetonitrile

Peptides:
Cysteine peptide with an amino acid sequence of Ac-RFAACAA
Lysinepeptide with an amino acid sequence of Ac-RFAAKAA

Positive Control (PC): Cinnamic aldehyde ((2E)-3-phenylprop-2-enal)

Results and discussion

Positive control results:

Mean Cys-Peptide Depletion: 75.35 % (SD: 0.40 %; CV: 0.53 %)
Mean Lys-Peptide Depletion: 56.98 % (SD: 1.07 %; CV: 1.87 %)

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: Cysteine Peptide

Parameter:

other: Peptide Depetion [%]

Value:

0

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 2

Key result

Run / experiment:

other: Lysine Peptide

Parameter:

other: Peptide Depetion [%]

Value:

0

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In this study under the given conditions the test item showed minimal reactivity towards both peptides.
The test item is considered as "non-sensitiser".

Executive summary:

Thein chemico direct peptide reactivity assay (DPRA) enables detection of the sensitising potential of a test item by quantifying the reactivity of test chemicals towards synthetic peptides containing either lysine or cysteine.

In the present study 2,4-Dichlorobenzonitrile was dissolved in acetonitrile based on the results of the pre-experiments.

Based on a molecularweight of 172.06 g/mol a 100 mM stock solution was prepared. The test item solutions were tested by incubating the samples with the peptides containing either cysteine or lysine for 24 ± 2 h at 25 ± 2.5 °C. Subsequently samples were analysed by HPLC.

For the 100 mM solution of the test item no turbidity or precipitation was observed when diluted with the cysteine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. No precipitation, turbidity or phase separation was observed for the any samples. Slight precipitation was observed for the samples of the positive control (including the co-elution control of the positive control). Samples were not centrifuged prior to the HPLC analysis-

For the 100 mM solution of the test item no turbidity or precipitation was observed when diluted with the lysine peptide solution.After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. No precipitation, turbidity or phase separation was observed for the any samples. Phase separation was observed for the samples of the positive control (excluding the co-elution control of the positive control).Samples were not centrifuged prior to the HPLC analysis.

Since the acceptance criteria for the depletion range of the positive control was fulfilled, the observed precipitations and phase separation were regarded as insignificant.

No co-elution of test item with the peptide peaks was observed. Sensitisingpotential of the test item was predicted from the mean peptide depletion of both analysed peptides (cysteine and lysine) by comparing the peptide concentration of the test item treated samples to the corresponding reference control C (RC C_acetonitrile).

The 100 mM stock solution of the test item showed minimal reactivity towards the synthetic peptides. The mean depletion of both peptides was <= 6.38% (0.44%). Based on the prediction model 1 the test item can be considered as non-sensitiser.

The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was 66.16%.