Methyl 4-(acetylamino)-5-bromo-o-anisate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

March 20, 2017 - March 22, 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:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Protocol no.154 ECVAM DB ALM

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Test material

Test material information

Constituent 1

Reference substance name:

Methyl 4-(acetylamino)-5-bromo-o-anisate

EC Number:

223-841-7

EC Name:

Methyl 4-(acetylamino)-5-bromo-o-anisate

Cas Number:

4093-34-9

Molecular formula:

C11H12BrNO4

IUPAC Name:

methyl 4-(acetylamino)-5-bromo-o-anisate

Test material form:

solid

Details on test material:

CAS number: 4093-34-9
EC Number: 223-841-7
Other name: Ester Bromopride

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: yes

In chemico test system

Details on the study design:

- Positive control: 100 mM cinnamaldehyde (CAS 104-55-2, Sigma Aldrich batch no. MKBT8955V) (i.e. 5 or 25 mM as final concentration in the reaction mixtures, respectively with cysteine and lysine).
- Co-elution control: 100 mM test item in the appropiate buffer
- 4 references made with 0.500 mM (final concentration in the reaction mixtures) peptides solutions in acetonitrile or in the test item solvent were included in the analysis:
1) Reference control A: prepared with acetonitrile in order to check the calibration curve accuracy,
2) Reference control B: prepared with acetonitrile and included at the beginning and at the end of the sequence in order to check the stability of peptide over time.
3) Reference control C: prepared with acetonitrile, the positive control solvent, in order to check its influence on the peptide stability.
4) Reference control C': prepared with 10% DMSO / 90% acetronitrile, the test item solvent, in order to check its influence on the peptide stability.
- Test system: Cysteine peptide (RS Synthesis, LLC; ref. Ac RFAACAA-COOH; batch no. 160801; purity 94.82%) and Lysine peptide (RS Synthesis, LLC; ref. Ac RFAAKAA-COOH; batch no. 160801; purity 94.19%).

Analytical method:
- Waters e2695 HPLC (Waters 2489 UV detector), cortecs column C18 2.7 µm, dimensions 2.1 x 100 mm
The HPLC column was installed and equilibrated at 30ºC with 50 % of phase A and 50% of phase B, for at least 20 minutes before use. The gradient was performed at least one time before to use the column.
- Conditions:
Mobile phase A: trifluoroacetic acid at 0.1% (v/v) in water; flow rate: 0.21 mL/min; injection volume: 7 μL; wavelength: 220 nm; sequence: 0, 10, 11, 13, 13.5 and 20 minutes.
Mobile phase B: trifluoroacetic acid at 0.085% (v/v) in acetonitrile; flow rate: 0.21 mL/min; injection volume: 7 μL; wavelength: 220 nm; sequence: 0, 10, 11, 13, 13.5 and 20 minutes.
- Method validation parameters: Linearity (R2 ≥ 0.999928) meet the criterion of approval (R2 ≥ 0.99).
A complete sequence was performed for each sample.
The column was re-equilibrated to initial conditions (90% Phase A and 10% of phase B) at least 4 minutes between each injection.
Sequence of the analysis:
The analysis was programmed according to the following principles:
- The reference controls B were placed at the beginning and the end of the analysis (3 repetitions).
- The reference controls C were placed at the beginning of each repetition.
- The standards of the calibration curve and the reference controls A were placed in order to be analyzed progressively throughout the sequence.

Interpretation of the results:
- 0.00 % ≤ mean of cysteine % depletion ≤ 13.89 % = No or minimal reactivity = Negative DPRA Prediction
- 13.89 % ≤ mean of cysteine % depletion ≤ 23.09 % = Low reactivity = Positive DPRA Prediction
- 23.09 % ≤ mean of cysteine % depletion ≤ 98.24 % = Moderate reactivity = Positive DPRA Prediction
- 98.24 % ≤ mean of cysteine % depletion ≤ 100 % = High reactivity = Positive DPRA Prediction

Results and discussion

Positive control results:

The depletion mean of cinnamaldehyde was 56.74 for Lysine and 75.36 for Cystine.

In vitro / in chemico

Results

Results 1

Run / experiment:

mean

Parameter:

other: % Lysine peptide depletion

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 2

Run / experiment:

mean

Parameter:

other: % Cystine peptide depletion

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 3

Key result

Run / experiment:

mean

Parameter:

other: % depletion

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes, the expected DPRA prediction for the 10 proficiency substances was obtained. The resulted cysteine and lysine depletion values fall within the respective reference range for 9 out of the 10 proficiency substances for each peptide (8 out of 10 expected in the OECD guide line).

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for reference control: The mean concentration of the peptides for the Reference control A and for the Reference control C met the concentration validity criteria mM: 0.500 ± 0.05 mM.
Reference C': does not meet the validity criteria. The mean concentration of cysteine is slightly lower than 0.450 mM. The decrease of concentration may be due to an oxidation of the peptide by the DMSO which leads to peptide dimerisation. Given the slight gap with the criteria, depletion percentage of the test item has nevertheless been calculated.
Reference control B/C the CV of the 9 controls B and C were less than 15% (%CV lysine= 0.59, %CV cystine= 2.51).
- Acceptance criteria met for positive control: yes, the depletion mean was 56.74 for Lysine (validity criteria between 40.2% and 69.4%) and 75.36 for Cystine (validity criteria between 60.8% and 100%)
- Acceptance criteria met for variability between replicate measurements: the SD of measurements was 0 for Cysteine (SD validity criteria <14.9%)

Any other information on results incl. tables

The depletion rate was calculated for each type of peptide according to the following formula:

Depletion % = [1- (Peptide peak area with the replicate injection/mean peptide peak area with the C base control)] x 100

Table 1. Reference control results.

	Lysine Peptide	Cysteine Peptide
	Concentration (nM)	Concentration (nM)	Concentration validity criteria (mM)
Reference A	0.510	0.512	0.500±0.050
* Reference C	0.516	0.493
* Reference C’	0.513	0.448

* Reference C: positive control diluant, i.e. acetonitrile

* Reference C’: test item diluent, i.e. = DMSO / Acetronitrile 1:9

 

Table 2. Coefficient of variation between reference controls B and C.

	CV %	CV %	CV validity criteria
Reference B/C	0.59	2.51	< 15%

 

Table 3. Positive control results.

Cinnamaldehyde	Depletion in Lysine Peptide %	Depletion in Cysteine Peptide %
Repetition 1	58.90	75.22
Repetition 2	56.53	75.51
Repetition 3	54.78	-
Mean	56.74	75.36
Depletion validity criteria	40.2 < Depletion > 69.4	60.8 < Depletion > 100

 

Table 4. Test item results.

	Depletion in Lysine Peptide %	Depletion in Cysteine Peptide %	Mean Depletion %
Repetition 1	Co-elution	0
Repetition 2	Co-elution	0
Repetition 3	Co-elution	0
Mean	-	0	0
SD	-	0
SD Validity criteria	< 11.6%	< 14.9%

 

A coelution of the test item with the Lysine peptide has been highlighted.

The sensitization of the test item was assessed from the mean depletion percentage of Cysteine only, according to the OECD GL 442C.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item showed mean depletion of 0% for Cysteine, reflecting no or minimal reactivity, therefore a negative prediction of DPRA. Under these conditions, the test item has to be considered as non-sensitising to the skin.

Executive summary:

To support the identification of the sensitizatin potential of the test item the method DPRA has been performed according to OECD 442C, following GLP. The method is based on the interaction between the test item and lysine or cysteine rich peptides detected with a high performance liquid chromatography (HPLC). The remaining concentration of peptides is measured after 24 hours of incubating with the test item at 25ºC. It is measured with the UV detector of the HPLC system, after gradient elution, at 220 nm. A coelution of the test item with the Lysine peptide has been highlighted and the sensitization of the test item was assessed from the mean depletion percentage of Cysteine only. The test item showed mean depletion of 0% for Cysteine, reflecting no or minimal reactivity, therefore a negative prediction of DPRA. Under these conditions, the test item has to be considered as non-sensitising to the skin.