Ammonium formate

Endpoint summary

Description of key information

OECD guideline study (442C), in chemico direct peptide binding assay, fist key event in AOP, the percent cysteine peptide depletion value of the test item was 1.33 % ± 1.23 % while the percent lysine peptide depletion was 0.76 % ± 0.89 %. 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.

OECD guideline study (442E), hCLAT, activation of dendritic cells, third key event in AOP, the increase of CD86 marker expression (RFI) was not greater than 150 % compared to the negative controls at several concentrations (with >50 % of cell viability) in two out of three valid runs. Based on the 2 out of 3 positive results, CD86 marker expression was concluded negative. The increase of CD54 marker expression (RFI) was greater than 200 % compared to the negative controls at several concentrations (with >50 % of cell viability) in all three independent valid runs. Also, effective concentration for CD54 expression (EC200) was determined as 2237 µg/mL, since clear dose response could be observed.

Despite of the fact that CD86 marker expression was concluded to be negative, since all 3 runs were positive for CD54 marker expressions, the overall h-CLAT prediction was concluded to be positive.

OECD guideline study (442D), KeratinoSens, activation of gene expression in human keratinocytes, second key event in AOP, the luciferase activity induction obtained with the test item was not statistically significant above the threshold of 1.5 at any concentration in either test, meeting all acceptance criteria and the criteria for a negative response. Since the results of the two tests were concordant, no more tests were needed. The overall result was concluded negative for luciferase gene induction.

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:

key study

Study period:

2019-06-14 to 2019-12-12

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))

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.

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.

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 %.

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

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

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

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.

 

Reference 2

Endpoint:

skin sensitisation: in vitro

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

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 442E: IN VITRO SKIN SENSITISATION ASSAYS ADDRESSING THE KEY EVENT ON ACTIVATION OF DENDRITIC CELLS ON THE ADVERSE OUTCOME PATHWAY FOR SKIN SENSITISATION

Version / remarks:

2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of dendritic cells

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.

Details on the study design:

Skin sensitisation (In vitro test system)
- Details on study design:
Preliminary tests
Preparation of the cells
For testing, THP-1 cells were seeded at a density of either 0.1 × E+06 cells/mL or 0.2 × E+06 cells/mL, and precultured in culture flasks for 72 hours or 48 hours respectively. On the day of testing, cells were harvested from the culture flasks and resuspended with fresh maintenance medium at 2 × E+06 cells/mL. Then, cells were distributed into 24 well flat-bottom plate with 500 µL cell supsension / well (1 × E+06 cells/well). The solvent/vehicle control used for the test item was culture medium.
Test item exposure
The culture medium or working solutions described above were mixed 1:1 (v/v) with the cell suspensions prepared in the 24-well. The treated plates were then incubated for
24 ± 0.5 hours at 37° C under 5 % CO2. The plates were sealed with microplate covers prior to the incubation to avoid evaporation of test item.
Propium iodide (PI) Staining
After 24 ± 0.5 hours of exposure, cells were transferred into sample tubes and 600 μL of FACS buffer was added to each sample. Cells were then collected by centrifugation (250 g, 5 min, 4 ºC). The supernatants were discarded and the remaining cells were washed again with 600 μL of FACS buffer. Finally, cells were resuspended in 400 μL of FACS buffer and 20 μL of 1 × PI solution was added for each sample.
Cytotoxicity measurement by flow cytometry and estimation of CV75 value
The PI uptake was analysed using flow cytometry with the acquisition channel FL-3. A total of minimum 10,000 living cells (PI negative) were acquired. When the cell viability was low, up to 30,000 cells including dead cells were acquired or data of one minute after the initiation of the analysis. Cell viability was analyzed by the Apogee Histogram Software by gating out PI positive cells, and the calculated percentage of PI negative cells was displayed on the software.
Results
Two independent runs for the dose finding assay were performed to determine the test item concentration that results in 75 % cell viability (CV75) compared to the solvent/vehicle control chosen in the trial formulation.
In the first run the highest final test item concentration on the plate was 1008 µg/mL and a 2 fold dilution was used when prepping the master solutions. Since no cytotoxicity was observed in any concentrations, the concentration range was shifted in the second run, and the maximum allowed concentration was used (~500 mg/mL stock solution in saline). Thus 5019 µg/mL was the highest final concentration on the plate in the second run and a 2-fold dilution was used.
CD86 and CD54 expression:
Since cytotoxicity was observed only with the highest final concentration on the plate, no CV75 value was determined but the highest allowed concentration was used for setting the dose range for measuring CD86 and CD54 expression in the main test. Eight final concentrations (µg/mL) were used for the test item tested of the main test. These are (nominal concentrations):
Highest allowed concentration - HAC (5000 μg/mL);
1/1.2 × HAC (4167 μg/mL);
1/1.22 × HAC (3472 μg/mL);
1/1.23 × HAC (2894 μg/mL);
1/1.24 × HAC (2411 μg/mL);
1/1.25 × HAC (2009 μg/mL);
1/1.26 × HAC (1675 μg/mL);
and 1/1.27 × HAC (1395 μg/mL).
For testing, THP-1 cells were seeded at a density of either 0.1 × 106 cells/mL or 0.2 × 106 cells/mL, and precultured in culture flasks for 72 hours or 48 hours respectively. On the day of testing, cells were harvested from the culture flasks and resuspended with fresh maintenance medium at 2 × 106 cells/mL. Then, cells were distributed into 24 well flat-bottom plate with 500 µL cell supsension / well (1 × 106 cells/well). Master solutions (MS) were prepared with saline as follows: Eight master solutions (eight concentrations) were prepared of the test item stock solution, by two-fold serial dilutions using saline. These master solutions were then further diluted 50 fold into culture medium to obtain the working solutions (WS).
The working solutions were finally used for exposure by adding an equal volume of working solution (500 µL) to the volume of THP-1 cell suspension (500 µL) in the 24-well plate to achieve a further two-fold dilution as the final concentration of the test item. The solvent/vehicle control used for the test item was culture medium. The culture medium or working solutions described above were mixed 1:1 (v/v) with the cell suspensions prepared in the 24-well. The treated plates were then incubated for 24 ± 0.5 hours at 37° C under 5 % CO2. The plates were sealed with microplate covers prior to the incubation to avoid evaporation of test item. Test item and control substances prepared as working solutions (500 μL) were mixed with 500 μL of suspended cells (1 × 106 cells) at 1:1 ratio in a single replicate, and cells were incubated for 24±0.5 hours.
After 24 ± 0.5 hours of exposure, cells were transferred from the 24-well plate into sample tubes, then 1 mL of FACS buffer was added to each sample and cells were collected by centrifugation (250 g, 5 min, 4 ºC). The washing step was repeated once more with 1 mL of FACS buffer. After washing, cells were blocked with 600 μL of 1 × blocking solution and incubated at 4°C for 15 min. After blocking, cells were split in three aliquots of 200 μL into sample tubes.
After centrifugation (250 g, 5 min, 4 ºC), cells were stained with 50 μL of FITC-labelled anti-CD86, anti-CD54 or mouse IgG1 (isotype) antibodies and incubated at 4° C for 30 min. The antibodies described in the h-CLAT DB-ALM protocol 158° were used. After washing twice with 150 μL of FACS buffer, cells were resuspended in 400 μL of FACS buffer and 20 μL of 1 × PI solution was added to each sample. The expression levels of CD86 and CD54, and cell viability were analysed using flow cytometry. The expression of CD86 and CD54 was analysed with flow cytometry with the acquisition channel FL-1. A total of minimum 10,000 living cells (PI negative) were acquired. When the cell viability was low and 10,000 living cells could not be acquired in 60 seconds, up to 30,000 cells including dead cells were acquired or data of one minute after the initiation of the analysis. Based on the geometric mean fluorescence intensity (MFI), the relative fluorescence intensity (RFI) of CD86 and CD54 for positive control (ctrl) cells and chemical-treated cells were calculated according to the following equation:

RFI = MFI of chemical-treated cells – MFI of chemical- treated isotype control cells × 100/MFI of solvent/vehicle-treated control cells – MFI of solvent/vehicle-treated isotype control cells
The calculated cell viabilities from the isotype control (ctrl) cells (which are stained with mouse IgG1 isotype antibodies) were also noted.
The PI uptake was analysed on channel FL-3. Cell viability was determined by the Apogee Histogram Software by gating out PI positive cells, and the calculated percentage of PI negative cells was displayed on the software.

Controls
Solvent/vehicle controls: Culture medium was used as negative control for the test item and to assess the impact of DMSO. DMSO was tested as a solvent control for the positive control at a single final concentration in the plate of 0.2 %, so it underwent the same dilution as described for the working solutions.
Positive control: DNCB was used as the positive control for CD86/CD54 expression measurement at a final nominal concentration of 4.0 μg/mL in the plate. To obtain a 4.0 μg/mL concentration a 2 mg/mL stock solution of DNCB in DMSO were prepared and further diluted 250-fold with culture medium to a 8 μg/mL working solution. The working solution then was diluted 2-fold when added to the cells.

Prediction model
If the RFI of CD86 is equal to or greater than 150% at any tested dose (>50 % of cell viability) in at least 2 independent runs, AND/OR if the RFI of CD54 is equal to or greater than 200 % at any tested dose (>50 % of cell viability) in at least 2 independent runs, the test item prediction is considered as positive. Otherwise it is considered as a negative.
In case the first two independent runs are not concordant a third run needs to be performed and the final prediction will be based on the mode of conclusions from the three individual runs.
When the item is tested at 5000 µg/mL in saline, 1000 µg/ml in DMSO, or highest soluble dose as the maximal test concentration instead of CV75-based dose and does not meet the positive criteria above without affecting cytotoxicity at all tested doses, the test item prediction should be considered as negative.
Since EC150 and EC200 values are just optional for test items that are found to be sensitisers, the values are calculated only in cases where a firm dose response curve can be constructed.
Up to six runs, meeting requirements for qualified testing are permitted to reach a conclusion for each test item. The six runs may include runs for which the data adoption criteria are not met for this test item. If no prediction can be made after the sixth run, the result is inconclusive.

Acceptance criteria
Requirements for qualified testing
- The cell viabilities of medium and solvent/vehicle controls should be higher than 90 %.
- In the positive control (DNCB), RFI values of both CD86 and CD54 should be over the positive criteria (CD86 ≥ 150 % and CD54 ≥ 200 %) and cell viability should be more than 50 %.
- In the solvent controls, RFI values compared to the medium control of both CD86 and CD54 should not exceed the positive criteria (CD86 > 150 % and CD54 > 200 %) and cell viability should be more than 90 %.
- For both medium and solvent controls, the MFI ratio of both CD86 and CD54 to isotype control should be >105 %.
Abnormal values
- RFI values cannot be less than zero. Regardless of the reason, such values should be omitted from the prediction.
- If an abnormal value is observed, check whether there are abnormal conditions in the run and record them in the reporting section.
Requirement for data acceptance
- For the test chemical, the cell viability should be more than 50% in at least four tested concentrations in each run.
- For the test chemical resulting in negative outcome, the cell viability at the 1.2 x CV75 should be less than 90 %. When the test item is tested at 5000 μg/mL in saline (maintenance medium alternatively) or 1000 μg/mL in DMSO or the highest soluble concentration is used as the maximal test concentration, a negative result is acceptable even if the cell viability is above 90 %.

Positive control results:

The positive control gave expected results for both markers, meaning that the RFI values of both CD86 and CD54 expression was over the positive criteria (CD86 ≥ 150 % and CD54 ≥ 200 %) and the respective cell viabilities were more than 50 % in each valid run.

Key result

Run / experiment:

other: 1-3

Parameter:

other: RFI of CD86

Remarks:

at any tested concentration with cell viability ≥ 50%

Value:

150

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: 1-3

Parameter:

other: RFI of CD54

Remarks:

at any tested concentration with cell viability > 50%

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: not reported

DEMONSTRATION OF TECHNICAL PROFICIENCY:
Prior to routine use of the test method described in Annex I of the Test Guideline 442E, the laboratory demonstrated technical proficiency by correctly obtaining the expected h-CLAT prediction for the 10 proficiency substances recommended in OECD 442E and by obtaining CV75, EC150 and EC200 values that fell within the respective reference ranges. Moreover, a historical database of reactivity check results positive controls and solvent/vehicle controls was generated and has been maintained to confirm the reproducibility of the test method over time.


ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, see table 1
- Acceptance criteria met for positive control: yes, see table 1
- Acceptance criteria met for variability between replicate measurements: not applicable

Table 1: Positive and negative control data:

	sample	concentration	RFI		viability (%) ‑ IgG1
			CD86	CD54	IgG
Exposure date:   08 July, 2019	Medium	-	100	100	91.4
	DMSO	0.2%	125	154	93.8
	DNCB	4.0 μg/mL	254	348	79.6
Exposure date:   17 July, 2019 / 1	Medium	-	100	100	91.1
	DMSO	0.2 %	103	100	92.4
	DNCB	4.4 μg/mL	373	881	63.3
Exposure date:   17 July, 2019 / 2	medium	-	100	100	92.5
	DMSO	0.2 %	104	79	93.2
	DNCB	3.8 μg/mL	676	1381	77.6

Table 2: preliminary test data:

Date	Test dose (µg/mL)	7.9	15.8	31.5	63	126	252	504	1008
01-02 July 2019	Viability (%)	93.9	93.5	94.6	95.0	93.4	94.4	93.9	90.5
Date	Test dose (µg/mL)	39.2	78.4	156.8	313.7	627.4	1254.8	2509.5	5019.0
02-03 July 2019	Viability (%)	92.0	92.5	93.1	91.1	92.2	91.1	87.2	63.5

Table 3: main test data

		RFI for CD86					RFI for CD54
Log-linear extrapolation/ Linear interpolation	EC150/ EC200 (µg/mL)	-	-		-		1260			2434			2237
Median EC150/EC200 (µg/mL)		-					2237

Interpretation of results:

study cannot be used for classification

Remarks:

test is one part of the in vitro testing strategy based on the adverse outcome pathway for sensitisation

Conclusions:

In the present study the skin senitisation potential of Ammonium formate was studied according to OECD guideline 442E (2018). The increase in CD86 marker expression (RFI) was not greater than 150 % at any tested doses (with >50 % of cell viability) compared to the respective negative controls in the three valid runs. Based on the 3 out of 3 negative results, CD86 marker expression was concluded negative.
The increase of CD54 marker expression (RFI) was greater than 200 % compared to the negative controls at several concentrations (with >50 % of cell viability) in all three independent valid runs. Also, effective concentration for CD54 expression (EC200) was determined, since clear dose response could be observed.
Despite of the fact that CD86 marker expression was concluded negative, since all 3 runs were positive for CD54 marker expressions, the overall h-CLAT prediction was concluded positive.

Executive summary:

In this study conducted according to OECD test guideline 442E (adopted 29 July 2016) THP-1 cells were incubated with Ammonium Formate.

The extent of cytotoxicity induced on THP-1 cells by the test item was studied in two dose finding tests. In the first run the highest final test item concentration on the plate was 1008 µg/mL and a 2-fold dilution was used when prepping the master solutions. Since no cytotoxicity was observed in any concentrations, the concentration range was shifted in the second run, and the maximum allowed concentration was used. Thus 5019 µg/mL was the highest final concentration on the plate in the second run and a 2-fold dilution was used. Since cytotoxicity was observed only with the highest final concentration on the plate, no CV75 value was determined but the highest allowed concentration was used for setting the dose range for measuring CD86 and CD54 expression in the main test. Eight doses were used in three independent valid runs: 5000, 4167, 3472, 2894, 2411, 2009, 1675 and 1395 µg/mL.  Cells were incubated with the test item for 24 h at 37°C. After exposure cells were stained and cell surface markers CD54 and CD86 were measured by FACS analysis. Cell viability was assessed in parallel using propidium iodide staining.

Cytotoxic effects were observed for the cells treated with highest concentration of the test item. Relative cell viability at the highest test item concentration was reduced to 42.3% (isotype IgG1 control) in the first experiment, to 40.6% (isotype IgG1 control) in the second experiment and to 61.9% (isotype IgG1 control) in the third experiment.

The increase in CD86 marker expression (RFI) was not greater than 150 % at any tested dose (with >50 % of cell viability) compared to the respective negative controls in the three valid runs. Based on the 3 out of 3 negative results, CD86 marker expression was concluded to be negative. The increase of CD54 marker expression (RFI) was greater than 200 % compared to the negative controls at several concentrations (with >50 % of cell viability) in all three independent valid runs. Also, the effective concentration for CD54 expression (EC200) was determined, since a clear dose response could be observed. Despite of the fact that CD86 marker expression was concluded to be negative, since all 3 runs were positive for CD54 marker expressions, the overall h-CLAT prediction was concluded to be positive.

The positive control (DNCB) led to an upregulation of the expression of CD54 and CD86 in both experiments. The threshold of 150% for CD86 (254% experiment 1; 373% experiment 2 and 676% in experiment 3) and 200% for CD54 (348% experiment 1; 881% experiment 2 and 1381% in experiment 3) were clearly exceeded.

Therefore, the test item is considered to be a skin sensitiser. The data generated with this method may be not sufficient to conclude on the absence or presence of skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-09-30 to 2019-12-12

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)

Version / remarks:

25 June 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Details on the study design:

Skin sensitisation (In vitro test system) ARE-Nrf-2 Luciferase Test Method (KeratinoSens™)
- Details on study design:
Cells were subcultured upon reaching 80 - 90 % confluence and care was taken to ensure that cells were never grown to full confluence. One day prior to testing cells were harvested in thawing medium and distributed into 96-well plates (10 000 cells/well) homogenously. For each individual test in the study, three replicates were used for the luciferase activity measurements, and one parallel replicate for the cell viability assay. One well per plate was left empty to assess background values. Cells were grown for 24 ± 0.5 hours in 96-wells microplates at 37 ± 1 °C in the presence of 5 % CO 2 .
After the 24-hour incubation time, thawing medium was replaced with fresh exposure medium. The 4 × master solutions of the test item and control substances were added to each well in a way that an additional 4-fold dilution was achieved on the plate for the final concentrations to be established (50 µL of 4× master solution to 150 µL of exposure medium). The treated plates were then incubated for about 48 ± 1 hours at 37 ± 1 °C in the presence of
5 % CO 2 . Care was taken to avoid cross-contamination between wells by covering the plates with a foil prior to the incubation with the test item.

Luciferase activity measurements
After the 48-hour exposure time with the test item and control substances, cells were washed with DPBS (270 µL), and 1× lysis buffer (20 µL) for luminescence readings was added to each well for 20 minutes at room temperature (on all three plates). Plates with the cell lysate were then placed in the luminometer for reading. First the luciferase substrate (50 µL) was added to each well and after one second, the luciferase activity was integrated for 2 seconds.

Cytotoxicity
For the cell viability assay, medium was replaced after the 48-hour exposure time with MTT working solution (200 µL) and cells were incubated for 4 hours at 37 ± 1 °C in the presence of 5 % CO 2 . The MTT working solution was then removed and cells were solubilised by the addition of isopropanol (50 µL). After shaking for 30 minutes the absorption was measured at 570 nm with a spectrophotometer.

Acceptance Criteria
For each test item and positive control substance, in order to derive a prediction, at least two independent tests, each containing three replicates for the luminescence measurements and one for viability measurement, were needed. In case of discordant results between the two independent tests, a third test should be performed. Each independent test was to be performed on a different day with fresh stock solution of test items and independently harvested cells. Cells may however have come from the same passage. KeratinoSens™ prediction should be considered in the framework of an IATA and in accordance with the limitations stated in the OECD test guideline.
The luciferase activity induction obtained with the positive control, Trans-Cinnamaldehyde should be statistically significant above the threshold of 1.5 in at least one of the tested concentrations. The EC1.5 value of the positive control should be within two standard deviations of the historical mean of the testing facility or between 7 μM and 30 μM (based on the validation dataset). In addition, the average induction in the parallel plates for Trans-Cinnamaldehyde at 64 μM should be between 2 and 8. If the latter criterion is not fulfilled, the dose-response of Trans-Cinnamaldehyde should be carefully checked, and tests may be accepted only if there is a clear dose-response with increasing luciferase activity induction at increasing concentrations for the positive control. Finally, the average coefficient of variation (CV) of the luminescence reading for the negative
(solvent) control DMSO should be below 20 % in each test which consists of 6 wells tested in triplicate.
Controls:
Negative control
The negative (solvent) control used was DMSO, for which six wells per plate were prepared. It underwent the same dilution as prepared for the master and working solution concentrations for the test item, so that the final negative (solvent) control concentration was 1 % DMSO in exposure medium on the treated plates. This DMSO concentration is known not to affect cell viability and corresponds to the same concentration of DMSO used in the tested chemical and in the positive control.
Positive control
The positive control used was Trans-Cinnamaldehyde for which a series of five 100 × master concentrations ranging from 0.4 to 6.4 mM were prepared in DMSO (from a 200 mM stock solution) and diluted as described for the 4 × master solutions. The final concentration of the positive control on the treated plates ranged from 4 to 64 µM.
Prediction model
A KeratinoSens™ prediction is considered positive if the following 4 conditions are all met in 2 of 2 or in the same 2 of 3 tests, otherwise the KeratinoSens™ prediction is considered
negative:
- the Imax is equal or higher than 1.5-fold and statistically significantly different as compared to the negative/solvent control (as determined by a two-tailed, unpaired Student’s T-test);
- the cellular viability is higher than 70 % at the lowest concentration with induction of luciferase activity ≥ 1.5-fold;
- the EC1.5 value is less than 1000 μM
- there is an apparent overall dose-response for luciferase induction (or a biphasic response).

Positive control results:

The luciferase activity induction obtained with the positive control, Trans-Cinnamaldehyde was statistically significant above the threshold of 1.5 at several concentrations in both tests. The EC1.5 values of the positive control fell between 7 µM and 30 µM (19 µM and 13 µM in the first and second test, respectively).
The average inductions in the parallel plates for Trans-Cinnamaldehyde at 64 μM were 5.49-fold and 3.66-fold in the first and second tests, respectively. In both tests the luciferase activity induction was in the 2 – 8-fold induction range and there was a clear doseresponse relationship in the luciferase activity induction for the positive control. In any of the tests, there was no cytotoxicity (cell viability lower than 70 %) induced by the positive control at any of the tested concentrations.

Key result

Run / experiment:

other: first run

Parameter:

other: EC1.5

Remarks:

µM

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

not determinable

Remarks:

No EC1.5 value was obtained; no induction of luciferase activity above 1.5-fold

Key result

Run / experiment:

other: second run

Parameter:

other: EC1.5

Remarks:

µM

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

not determinable

Remarks:

No EC1.5 value was obtained; no induction of luciferase activity above 1.5-fold

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes, The coefficient of variation (CV%) of the luminescence reading for the negative control DMSO was below 20 % in both tests (12.61 % and 11.76 % respectively).
- Acceptance criteria met for positive control: Yes, The average inductions in the parallel plates for Trans-Cinnamaldehyde at 64 μM were 5.49-fold and 3.66-fold in the first and second tests, respectively. In both tests the luciferase activity induction was in the 2 – 8-fold induction range and there was a clear dose response relationship in the luciferase activity induction for the positive control. In any of the tests, there was no cytotoxicity (cell viability lower than 70 %) induced by the positive control at any of the tested concentrations.
- Acceptance criteria met for variability between replicate measurements: Yes

Individual and average fold induction: 1st run:

		Test item
Concentration (uM)		0.98	1.95	3.91	7.81	15.63	31.25	62.50	125	250	500	1000	2000
Plate ID	20191003-1110-2	1.04	1.11	1.04	0.93	0.96	0.89	1.14	0.90	0.96	0.94	0.96	0.83
	20191003-1110-3	0.93	1.00	0.96	1.10	1.02	0.96	0.93	0.79	0.91	0.82	0.84	0.99
	20191003-1110-4	0.90	1.08	0.97	0.87	1.01	0.99	0.75	0.92	0.91	0.91	0.88	0.67
average induction		0.96	1.06	0.99	0.97	1.00	0.95	0.94	0.87	0.93	0.89	0.89	0.83
significance		0.585	0.605	0.881	0.708	0.993	0.708	0.450	0.343	0.464	0.351	0.305	0.143
viability		101%	105%	103%	105%	108%	108%	98%	101%	105%	101%	104%	101%

Positive control 1st run:

		Trans-Cinnamaldehyde
Concentration (uM)		4	8	16	32	64
Plate ID	20191003-1110-2	1.26	1.42	1.49	2.12	6.17
	20191003-1110-3	1.10	1.16	1.36	1.96	6.10
	20191003-1110-4	0.95	1.25	1.33	1.70	4.21
average induction		1.10	1.28	1.39	1.92	5.49
significance		0.304	0.075	0.016	0.000	0.000
viability		108%	117%	115%	119%	102%

Vehicle control 1st run:

Average DMSO luminescence values for each plate
20191003-1110-2	581117
20191003-1110-3	618000
20191003-1110-4	735400
Average DMSO absorbance value	0.891

Individual and average fold induction: 2nd run:

		Test item
Concentration (uM)		0.98	1.95	3.91	7.81	15.63	31.25	62.50	125	250	500	1000	2000
Plate ID	20191014-1038-2	1.27	1.07	1.11	1.35	1.14	1.07	1.16	1.13	0.92	0.92	0.93	0.70
	20191014-1038-3	0.93	1.10	1.16	0.94	1.07	0.82	0.85	0.87	0.69	0.97	0.97	0.77
	20191014-1038-4	1.02	0.98	1.09	1.09	1.03	0.93	1.06	0.82	0.79	0.83	0.79	0.75
average induction		1.07	1.05	1.12	1.13	1.08	0.94	1.02	0.94	0.80	0.91	0.90	0.74
significance		0.423	0.428	0.112	0.267	0.099	0.291	0.837	0.433	0.018	0.210	0.195	0.007
viability		103%	107%	109%	101%	88%	89%	99%	99%	106%	105%	101%	125%

Positive control 2nd run:

		Trans-Cinnamaldehyde
Concentration (uM)		4	8	16	32	64
Plate ID	20191014-1038-2	1.30	1.50	1.56	2.24	3.86
	20191014-1038-3	1.01	1.14	1.63	1.49	3.48
	20191014-1038-4	1.28	1.26	1.70	1.49	3.64
average induction		1.20	1.30	1.63	1.74	3.66
significance		0.066	0.016	0.002	0.020	0.000
viability		93%	120%	113%	133%	131%

Vehicle control 2nd run:

Average DMSO luminescence values for each plate
20191014-1038-2	471150
20191014-1038-3	522417
20191014-1038-4	506817
Average DMSO absorbance value	0.830

Interpretation of results:

study cannot be used for classification

Conclusions:

In the present study the skin sensitization potential of the test item “Ammonium Formate” was examined using the KeratinoSens™ method (ARE-Nrf2 Luciferase Test Method). or the test item and positive control substance, in order to derive a prediction two independent tests were conducted, in which the concluded results were concordant.
The luciferase activity induction obtained with the test item was not statistically significant above the threshold of 1.5 at any concentration in either test, meeting all acceptance criteria and the criteria for a negative response.
Since the results of the two tests were concordant, no more tests were needed. The overall result was concluded negative for luciferase gene induction.
Based on these results and the KeratinoSens™ prediction model, the test item “Ammonium Formate” was concluded negative under the experimental conditions of KeratinoSens™ method (ARE-Nrf2 Luciferase Test Method).

Executive summary:

In this study conducted according to OECD test guideline 442d (adopted February 04, 2015) transgenic keratinocytes constitutively expressing an ARE-reporter gene were incubated with Ammonium Formate with concentrations of 0.98, 1.95, 3.91, 7.81, 15.63, 31.25, 62.50, 125, 250, 500, 1000, 2000 µM

and 0 µM (solvent control) for 48 h at 37°C. Afterwards the test substance containing medium was removed and the cells lysed and luminescence subsequently measured with a plate reader for 2.000 ms. Beside the luminescence the cell viability was measured using the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium  bromide) assay method. The KeratinoSens assay is considered to provide positive results if the following conditions are all met in two of two independent experimental repetitions:

- the  I max   is  higher  than  (>)  1.5  fold  and  statistically  significant  different  as  compared  to  the solvent (negative) control (as determined by a two-tailed, unpaired Student’s T-test);

- the  cellular  viability  is  higher  than  (>)  70%  at  the  lowest  concentration  with  induction  of luciferase activity above 1.5 fold (i.e. at the EC 1.5  determining concentration);

- the EC 1.5  value is less than (<) 1000 µM (or < 200  µg/mL for test chemicals with no defined MW);

- there  is  an  apparent  overall  dose-response  for  luciferase  induction.

Neither in the first experiment nor in the second experiment a more than 1.5 -fold induction of luciferase activity was observed. The cell viability was >70% in both experiments at all concentrations. An EC1.5 value could not be determined.

The controls confirmed the validity of the study. The luciferase activity induced by the positive control at a concentration of 64 μM was between 2 and 8 (5.49 experiment 1; 3.66 experiment 2). The calculated EC1.5 was between 7 µM and 30 µM (19 µM and 13 µM in

the first and second test, respectively). The average coefficient of variation (CV) of the luminescence reading for the negative (solvent) control DMSO was < 20% ( (12.61 % and 11.76 % respectively).

In this study under the given conditions the test item did not induce the luciferase activity in the transgenic KeratinoSens™ cell line in at least two independent experiment runs. Therefore, the test item can be considered as non-sensitiser. However, the data generated with this method may be not sufficient to conclude definitely on the absence of skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

For the determination of skin sensitization three in vitro test were performed with the test item Ammonium formate describing the three key events in the adverse outcome pathway established for the identification of skin sensitizers.

The Direct Peptide Binding assay showed a negative result for skin sensitization, whereas the hClat assay (activation of dendritic cells) revealed a positive result. Based on the two disconcordant results the third in vitro assay KeratinoSens was conducted with the test item. The result obtained from the last in vitro test was negative for skin sensitization.

According to ‘ANNEX I: Case studies to the Guidance document on the reporting of defined approaches and individual information sources to be used within integrated approaches to testing and assessment (IATA) for skin sensitization’(see also Urbisch et al.[1]) and the Data interpretation procedure (DIP), ‘the prediction model of the DIP then defines that two concordant results addressing two different Key Events (KE) indicate the sensitizing potential, i.e. two positive results indicate a sensitizer, two negative results indicate a non-sensitizer. This also implies that if the first two tests conducted yield a) concordant results, the third does not need to be performed, or b) discordant results in the first two tests necessitates conducting a third test addressing a third KE’. Moreover, it was described that ‘there is no differential weighting of the individual test methods used and no predefined sequential order of testing, the order and information source from which data is obtained is not defined. Due to the higher complexity and resources needed (e.g. flow cytometer needed) to conduct the tests used for KE 3, the DPRA (KE1) and Nrf2-ARE-based tests (KE2) will usually be conducted first. The “2 out of 3 - Sens ITS” is quantitative in that the values from measurements are obtained in each of the individual test methods used. It is qualitative as these values play a secondary role as only binary (positive/negative) results drive the final prediction. The results from the “third” test can then be useful, e.g. to obtain a two out of three prediction when the first two tests are not concordant, or to further corroborate a prediction when assessing when borderline results are achieved’. 

Based on this approach and the obtained results form the conducted in vitro tests with Ammonium formate, the test item is not classified as skin sensitizer according to Regulation (EC) No. 1272/2008 (CLP) and the Globally Harmonized System for Classification and Labelling of chemicals (GHS).

[1]Urbisch, Daniel, et al. "Assessing skin sensitization hazard in mice and men using non-animal test methods."Regulatory Toxicology and Pharmacology 71.2 (2015): 337-351.