Ammonium formate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-06-19 to 2019-12-18 (provisional draft final report)

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 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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 vitro test system

Test system:

human skin model

Cell type:

non-transformed keratinocytes

Cell source:

other: not specified

Justification for test system used:

The EPISKIN model has been validated for irritation testing in an international trial. After a review of scientific reports and peer reviewed publications on the EPISKIN method, it showed evidence of being a reliable and relevant stand-alone test for predicting rabbit skin irritation, when the endpoint is evaluated by MTT reduction and for being used as a replacement for the Draize Skin Irritation test (OECD TG 404 and Method B.4 of Annex V to Directive 67/548/EEC) for the purposes of distinguishing between skin irritating and
no- skin irritating test substances (STATEMENT OF VALIDITY OF IN-VITRO TESTS FOR SKIN IRRITATION; ECVAM; Institute for Health & Consumer Protection; Joint Research Centre; European Commission; Ispra; 27 April 2007).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkinTM Small Model (EpiSkinTMSM), manufactured by EPISKIN SNC Lyon, France
- Tissue batch number(s):
Supplier:SKINETHIC Laboratories , 4, rue Alexander Fleming, 69366 Lyon Cedex 07 - France
Batch No.:19-EKIN-025
Expiry date: 24 June 2019

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature/ 37°C
- Temperature of post-treatment incubation (if applicable): 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: approximately 25 mL PBS 1x solution to remove all of the test material from the epidermal surface
- Observable damage in the tissue due to washing: No

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 3 mg/mL in saline buffer
- Incubation time: 3h
- Spectrophotometer: Thermo Scientific; Multiscan FC
- Wavelength: 570 ± 10 nm
- Linear OD range of spectrophotometer: 0.2136 – 3.1752

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: Determined by SDS concentration in a MTT test, results should be between 1.5 mg/mL ≤ IC50 ≥ 3.0 mg/mL, results were: 2.1 mg/mL
- Barrier function: Number of cell layers > 4; 7 cell layers were detected
- Morphology: Multi-layered, highly differentiated epidermis consisting of organized basal, spinous and grnular layers, and a multilayered stratum corneum (7 cell layers)
- Contamination: On blood of donors, the absence of HIV-1 and HIV-2 antibodies, hepatitis C antibodies and hepatitis B antigen HBs was verified. On cells from donors, the absence of bacteriafungus and mycoplasma were verified.

NUMBER OF REPLICATE TISSUES: 3 for the treatment cells

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- N. of replicates : 3
- Method of calculation used: Negative control:
OD Negative Control (ODNC) = ODNCraw – ODblank mean
–The corrected mean OD of the 3 negative control values is calculated: this corresponds to 100% viability
Positive control
OD Positive Control (ODPC) = ODPCraw – ODblank mean
– The corrected mean OD of the 3 positive control values is calculated
– The % viability for each positive control replicate is calculated relative to the mean negative control:
% Positive Control 1 = (ODPC1 / mean ODNC) ×100
% Positive Control 2 = (ODPC2 / mean ODNC) ×100
% Positive Control 3 = (ODPC3 / mean ODNC) ×100
– The mean value of the 3 individual viability % for positive control is calculated:
Mean PC % = (%PC1 + %PC2 + %PC3) / 3
OD Treated Tissue (ODTT) = ODTTraw – ODblank mean
– The corrected mean OD of the 3 test item values is calculated
– The % viability for each test item replicate is calculated relative to the mean negative control:
% Treated Tissue 1 = (ODTT1 / mean ODNC) ×100
% Treated Tissue 2 = (ODTT2 / mean ODNC) ×100
% Treated Tissue 3 = (ODTT3 / mean ODNC) ×100
– The mean value of the 3 individual viability % for test item is calculated
Mean TT % = (%TT1 + %TT2 + %TT3) / 3



PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 %.
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50%.
- Justification for the selection of the cut-off point(s) if different than recommended in TG 431 and 439

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 mg

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL
- Concentration (if solution): 1 x PBS

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10µL
- Concentration (if solution):5% SDS (aq.)

Duration of treatment / exposure:

15 min ± 0.5 min

Duration of post-treatment incubation (if applicable):

42h ± 1h

Number of replicates:

Triplicates

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean of three replicates

Value:

100

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Remarks:

determination of viability in the negative control

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean of three replicates

Value:

21

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Remarks:

Determination of viability in the positive control

Results 3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean of three replicates

Value:

100

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Remarks:

Determination of the viability in the treated cells

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: Not reported
- Direct-MTT reduction: No
- Colour interference with MTT: No

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes, the mean OD value of the three negative control tissues should be between 0.6 and 1.5 and the standard deviation value (SD) of the % viability should be ≤ 18.
- Acceptance criteria met for positive control: Yes, the acceptable mean percentage viability range for positive controls is 0-40% and the standard deviation value (SD) of the % viability should be ≤ 18.
- Acceptance criteria met for variability between replicate measurements: For test chemicals, the standard deviation value (SD) of the % viability should be ≤ 18.
- Range of historical values if different from the ones specified in the test guideline: No

Interpretation of test results
According to the United Nations Globally Harmonized System (UN GHS) of Classification and Labelling of Chemicals (7th revised edition; 2017) and as implemented in the European Commission Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures (EU CLP), the irritancy potential of test substances is predicted for distinguishing between irritant or corrosive (Category 2 or Category 1) and non-irritant (No Category) substances.
In the present study, the irritancy potential of test substances is predicted by mean tissue viability of tissues exposed to the test substance. The test chemical is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1), if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control. However, this test method (OECD 439) cannot resolve between UN GHS Categories 1 and 2, further information on skin corrosion (OECD 431) will be required to decide on its final classification. In case the test chemical is found to be non-corrosive, and shows tissue viability after exposure and post-treatment incubation is less than or equal (≤) to 50 %, the test chemical is considered to be irritant to skin in accordance with UN GHS Category 2.
Depending on the regulatory framework in member countries, the test chemical may be considered as non-irritant to skin in accordance with UN GHS No Category if the tissue viability after exposure and post-treatment incubation is more than (>) 50 %.
The prediction model (PM) is described below:

Criteria for In Vitro
interpretation Classification
Mean tissue viability % is ≤ 50 % Category 2 or Category 1
Mean tissue viability % is > 50 % No Category

Any other information on results incl. tables

OD values and viability percentages of the controls and test item:

Substance	Optical Density (OD)		Viability (%)
Negative Control: 1x PBS	1	1.002	105
	2	0.899	94
	3	0.952	100
	mean	0.951	100
	standard deviation (SD)		5.46
Positive Control: SDS (5 % aq.)	1	0.238	25
	2	0.197	21
	3	0.178	19
	mean	0.204	21
	standard deviation (SD)		3.20
Test Item: Ammonium Formate	1	0.966	102
	2	0.890	94
	3	1.002	105
	mean	0.953	100
	standard deviation (SD)		6.05

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The results obtained from this in vitro skin irritation test, using the EPISKIN model, indicated that the test item reveals no skin irritation potential under the utilised testing conditions. The test item Ammonium Formate is considered to be non-irritant to skin and is therefore not classified (UN GHS No Category).

Executive summary:

In a dermal irritation study performed in accordance with OECD Guideline 439 (In Vitro Skin Irritation) (adopted July 28, 2015), Ammonium formate was applied to the three-dimensional human epidermis model tissue for an exposure period of 15 minutes in triplicates. 5μL of deionised water were topically applied to the epidermal surface in order to improve further contact between the solid and the epidermis. Each approximately 10 mg of the test item were applied to the wetted tissues. The test item was spread to match the surface of the tissue. After 15 minutes exposure at room temperature, the tissues were washed with phosphate buffered saline to remove any residual test material. Subsequently the tissue constructs were incubated for 42 h at 37°C. Cytotoxicity (irritancy) was expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

The positive (5% SDS) and negative (deionised water) control gave responses that were within the acceptance criteria and as such demonstrated the validity of the study.

The relative mean tissue viability obtained after 15 minutes treatment with Ammonium Formate compared to the negative control tissues was 100%.

Since the mean relative tissue viability for the test substance was above 50%, Ammonium Formate is identified to be not irritating.