Ammonium formate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-06-14 to 2019-12-12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

According to Regulation (EC) No. 1907/2006 (REACH) Annex VII section 8.3.1, as a first step in chemico/in vitro tests have to be performed in order to address skin sensitisation. Only in the case that the in chemico/in vitro methods are not applicable for the substance,or the results are not adequate for classification and risk assessment, can an in vivo skin sensitisation study (preferably Local Lymph Node Assay, EU B.42/OECDTG429) be performed (section 8.3.2). The present test was performed as one of the required in vitro tests to determine whether the substance needs to be classified or not.

Test material

Test material information

Constituent 1

Reference substance name:

Ammonium formate

EC Number:

208-753-9

EC Name:

Ammonium formate

Cas Number:

540-69-2

Molecular formula:

CH2O2.H3N

IUPAC Name:

ammonium formate

Test material form:

solid: crystalline

In chemico test system

Details on the study design:

Skin sensitisation (In chemico test system)
- Details on study design:
A solubility test in ultrapure water was performed before initiation of the test.
100 mM solutions of the test chemical in the appropriate solvent were prepared just before use. The needed amount of test chemical was calculated (0.0324 g ± 10 %) based on the molecular weight and purity 0.0336 g test chemical was weighted for the stock solution used for the cysteine peptide depletion determination and 0.0330 g test chemical was weighted for the stock solution used for lysine peptide depletion determination in the valid runs.
In a 5 mL volumetric glass:
(molecular weight)/(% purity) ×50=target weight of test chemical (mg)

Controls used for the test:
Reference control A: Peptide stock solutions are combined with acetonitrile. System suitability is checked by the use of the three replicates of reference control A.
Reference control B: Peptide stock solutions are combined with acetonitrile. Stability of the peptides are checked by the use of the three replicates of reference control B, measured before and after of the reaction samples.
Reference control C: Peptide stock solutions are combined with the respective solvent of the test item (and acetonitrile in case of cysteine peptide). Three replicates of reference control C are used as a solvent control to which the peptide concentration/depletion of the reaction samples is compared. Also, if the solvent of the test item is not acetonitrile, another reference control C with acetonitrile is prepared to which the peptide concentration/depletion of the positive control samples is compared.
Co-elution controls: Test item stock solution (and acetonitrile in case of cysteine peptide) is combined with the respective buffer solutions in each run. Co-elution controls are used to check for test item and peptide co-elution.
Assembly of reaction samples and controls: See any other information on material and methods.

Relative concentrations of the peptide following the 24 hour reaction time were determined by high performance liquid chromatography with gradient elution and UV detection at 220 nm. Reaction samples, reference controls A, B and C, co-elution controls and positive controls are prepared and analyzed in triplicates in batches of up to 26 chemicals (including controls) to keep the total HPLC analysis time less than 30 hours.
HPLC conditions:
HPLC system: SHIMADZU LC2030 (Prominence-i LC-2030C)
Serial number: L21445402951AE
Column: Zorbax SB-C18 (2.1 x 100 mm, 3.5 µm)
Serial number: USRY003976
Column temperature: 30°C
Sample temperature: 25°C
Detector: UV at 220 nm (D2 lamp)
Injection volume: 7µL
System equilibration: running mobile phase A and mobile phase B in a ratio of 1:1 for 2 hours at 30°C column temperature and running the gradient twice before injecting the first sample
Run time: 20 min
Flow conditions: gradient flow


Prediction model:
The mean percent cysteine and percent lysine depletion value is calculated for each test chemical. Negative depletion is considered as “0” when calculating the mean. By using the cysteine 1:10/lysine 1:50 prediction model (Table 3 (any other information on material and methods incl. tables)), the threshold of 6.38 % average peptide depletion is used to support the discrimination between skin sensitisers and non-sensitisers).
Before applying the cysteine 1:10 lysine/1:50 or the cysteine 1:10 prediction model, the experimental data regarding possible co-elution was evaluated and the appropriate approach was selected based on the below mentioned scenarios in Table 2 (any other information on material and methods incl. tables).
Application of the prediction models assigns a test chemical to a reactivity class (minimal, low, moderate or high reactivity). Chemicals assigned to the minimal reactivity category should be classified as non-sensitisers whereas chemicals assigned to the low, moderate or high reactivity categories should be classified as sensitisers under these test conditions.

Acceptance criteria

The following criteria should be met for the assay to be considered valid:
- the standard calibration curve should have an r² > 0.99
- the mean peptide concentration of reference controls A should be 0.50 ± 0.05 mM and the coefficient of variation (CV) of peptide peak areas for the nine reference controls B and C in acetonitrile should be ≤ 15.0%.
- the mean percent peptide depletion value of the three replicates for the positive control cinnamaldehyde should be between 60.8% and 100% for the cysteine peptide and between 40.2% and 69.0% for the lysine peptide and the maximum standard deviation (SD) for the positive control replicates should be < 14.9% for the percent cysteine depletion and < 11.6% for the percent lysine depletion [3]
If one or more of these criteria is not met the run will be repeated.
The following criteria should be met for a test chemical’s results to be considered valid:
- the maximum standard deviation for the test chemical replicates should be less than 14.9 % for the percent cysteine depletion and less than 11.6 % for the percent lysine depletion
- the mean peptide concentration of the three reference controls C in the appropriate solvent should be 0.50 ± 0.05 mM.
If these criteria are not met these criteria the data will be rejected and the run will be repeated for that specific test chemical.

Results and discussion

Positive control results:

The mean peptide depletion value for the positive control was 61.21 % and mean peptide depletion value for the test chemical was 1.05 %.

In vitro / in chemico

Results

Results 1

Key result

Parameter:

other: mean percent cysteine and lysine depletions

Value:

1.05

Negative controls validity:

not applicable

Positive controls validity:

not applicable

Remarks on result:

other: no/minimal peptide reactivity

Results 2

Key result

Parameter:

other: mean percent depletion values

Remarks:

cysteine peptide

Value:

1.33

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Key result

Parameter:

other: mean percent depletion values

Remarks:

lysine peptide

Value:

0.76

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No precipitates were reported

DEMONSTRATION OF TECHNICAL PROFICIENCY: Prior to routine use of the method, the testing facility demonstrated technical proficiency in a separate study by correctly obtaining the expected DPRA prediction for 10 proficiency substances as recommended in the OECD TG 442C guideline.

ACCEPTANCE OF RESULTS:
- the standard calibration curve should have an r² > 0.99
- the mean peptide concentration of reference controls A should be 0.50 ± 0.05 mM and the coefficient of variation (CV) of peptide peak areas for the nine reference controls B and C in acetonitrile should be ≤ 15.0%.
- the mean percent peptide depletion value of the three replicates for the positive control cinnamaldehyde should be between 60.8% and 100% for the cysteine peptide and between 40.2% and 69.0% for the lysine peptide and the maximum standard deviation (SD) for the positive control replicates should be < 14.9% for the percent cysteine depletion and < 11.6% for the percent lysine depletion

- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Applicant's summary and conclusion

Interpretation of results:

other: not sensitising

Remarks:

in the first part of the adverse outcome pathway

Conclusions:

In the course of this study the skin sensitization potential of the test item “Ammonium Formate” was studied using the Direct Peptide Reactivity Assay (DPRA). The percent cysteine peptide depletion value of the test item was 1.33 % while the percent lysine peptide depletion was 0.76 %. The mean depletion value of the peptides being 1.05 % was used to categorize the test chemical in one of the four classes of reactivity. No co-elution was observed with either cysteine or lysine peptides; therefore the cysteine 1:10 / lysine 1:50 prediction model was used for the discrimination between sensitizers and non sensitizers. The mean peptide depletion of the test item was 1.05 %, which did not exceed the 6.38 % threshold of the applicable prediction model for being positive. Based on these results and the cysteine 1:10 / lysine 1:50 prediction model, the test item “Ammonium Formate” was concluded to have no or minimal reactivity towards the synthetic peptides thus is not a potential skin sensitizer under the experimental conditions of the In chemico Direct Peptide Reactivity Assay (DPRA) method.

Executive summary:

In an in chemico skin sensitisation study performed according to the OECD draft proposal for a new test guideline442C (Direct Peptide Reactivity Assay) the reactivity of the test item Ammonium Formate was evaluated by monitoring peptide depletion following a 24-hour contact between the test item and synthetic cysteine and lysine peptides.

The test item was dissolved at 100 mM in a 1:1 mixture of water:acetonitrile. The positive control was cinnamaldehyde, and for each peptide, the analytical batch included co-elution control samples and three reference control samples.

Reactivity (%depletion) was determined following 24-hour contact between test item and peptide in acetonitrile at the ratios 1:10 cysteine:test item and 1:50 lysine:test item by liquid chromatography with UV-detection.

Peptide reactivity was reported as percent depletion based on the peptide peak area of the replicate injection and the mean peptide peak area in the three relevant reference control C samples. The acceptance criteria for the calibration curve samples, the reference and positive controls as well as for the study samples were satisfied and thus demonstrated the validity of the study.

 Analysis of the chromatograms of the co-elution samples indicated that the test item did not co-elute with either the lysine or the cysteine peptides. As a result, the mean percent depletion values were calculated for each peptide. For the cysteine peptide, the mean depletion value was 1.33% and for the lysine peptide, the mean depletion value was 0.76%.

Since the mean of the percent cysteine and percent lysine depletions was equal to 1.05%, the test item was considered to have no/minimal peptide reactivity. Therefore, the DPRA prediction would be considered as negative and the test item may have no potential to cause skin sensitisation.