Triammonium citrate

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 1.49 % ± 2.93 % while the percent lysine peptide depletion was 0.46 % ± 0.79 %. The mean depletion value of the peptides being 0.98 % 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 0.98 %, 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, thirs key event in AOP, the increase of CD86 marker expression (RFI) was 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 positive. Also, effective concentration for CD86 expression (EC150) was not determined, since no clear dose response could be observed. 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 3204 µg/mL, since clear dose response could be observed.

Since all both for CD86 and CD54 marker expression the predominant result was positive, the overall h-CLAT prediction was concluded positive, as well.

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:

to 2019-09-30

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:
Cysteine peptide: Ac-RFAACAA-OH; Molecular weight: 750.88 g/mol
Lysine peptide: Ac-RFAAKAA-OH; Molecular weight: 775.91 g/mol

Cysteine peptide stock solution, 0.667 mM, 0.501 mg/mL:
The needed amount of peptide was calculated based on the equation below (0.00977 g ± 10%). The previously calculated amount of the peptide was pre-weighted (0.01133 g) and 20.87 mL of pH 7.5 phosphate buffer was added right before beginning the assay in the valid run.
"pH=7.5 phosphate buffer" (mL)×(0.501 mg/mL)/(purity of the peptide)= peptide target weight (mg)

Lysine peptide stock solution, 0.667 mM, 0.518 mg/mL:
The needed amount of peptide was calculated based on the equation below (0.00958 g ± 10%). The previously calculated amount of the peptide was pre-weighted (0.01118 g) and 21.006 mL of pH 10.2 acetate buffer was added right before beginning of the assay in the valid run.
"pH=10.2 acetate buffer" (mL)×(0.518 mg/mL)/(purity of the peptide)= peptide target weight (mg)

Positive Control Stock Solutions:
100 mM solutions of the positive control chemical in acetonitrile were prepared just before use. The needed amount of test chemical was calculated (0.0664 g ± 10%) based on the molecular weight and purity of the substance with the equation below. 0.0673 g cinnamaldehyde was weighted for the positive stock solution used for the cysteine peptide depletion determination and 0.0717 g cinnamaldehyde 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 cinnamaldehyde (mg)

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 (0.1228 g ± 10 %) based on the molecular weight and purity of the test item with the equation below. 0.1240 g test chemical was weighted for the stock solution used for the cysteine peptide depletion determination and 0.1240 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:

Mean depletion rate (%) of cinnamaldehyde: mean cysteine peptide depletion 67.59 % ± 0.73 % and mean lysine peptide depletion 54.82 % ± 1.52 %. The overall obtained peptide depletion is 61.12 %

Key result

Parameter:

other: mean percent cysteine depletion

Value:

1.49

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: no/minimal peptide activity

Key result

Parameter:

other: mean percent lysine depletion

Value:

0.46

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: no/minimal peptide activity

Key result

Parameter:

other: overall obtained peptide depletion

Value:

0.98

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

not applicable

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: no precipitates were observed

DEMONSTRATION OF TECHNICAL PROFICIENCY: Prior to routine use of the method, the test 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:
- Acceptance criteria met for negative control: Reference control B replicates were included in the sequence to verify the stability of the peptide over time and reference control C replicates were used to verify that the solvent of the test item did not impact the percent peptide depletion. Both the mean cysteine and lysine peptide concentration of the reference control C (solvent – ultrapure water - upw) replicates were 0.49 mM, and thus being within the acceptable 0.50 ± 0.05 mM range.
- Acceptance criteria met for positive control: The acceptance criteria were met for the positive control with a mean cysteine peptide depletion value of 67.59 % ± 0.73 % and a mean lysine peptide depletion value of 54.82 % ± 1.52 % .
- Acceptance criteria met for variability between replicate measurements: The CV % for the nine reference control B and C replicates in acetonitrile (acn) were well below the acceptable max. of 15 % for both peptides, since it was 2.4 % for cysteine and 0.3 % for lysine peptides.

Interpretation of results:

GHS criteria not met

Conclusions:

In the course of this study the skin sensitization potential of the test item Triammonium Citrate was studied using the Direct Peptide Reactivity Assay (DPRA). The percent cysteine peptide depletion value of the test item was 1.49 % ± 2.93 % while the percent lysine peptide depletion was 0.46 % ± 0.79 %. The mean depletion value of the peptides being 0.98 % 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 0.98 %, 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 “Triammonium Citrate” 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 guideline 442C (Direct Peptide Reactivity Assay) the reactivity of the test item Triammonium citrate 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 ultrapure water. 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.

The concentration of the peptide was determined in each reaction sample and control, from absorbance at 220 nm measuring the peak area of the appropriate peaks and calculating the concentration of the peptide using the linear calibration curves derived from the calibration standards. 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 1.49% and for the lysine peptide, the mean depletion value was 0.46%.

Since the mean of the percent cysteine and percent lysine depletions was equal to 0.98%, 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:

to 2019-09-30

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD/OCDE 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:
Test item concentrations for the main tests were determined in the dose finding (preliminary) tests. First, the test item was tried to be formulated in sodium chloride solution (saline). At the concentration of 100 mg/mL a total dissolution of the test item was observed with approximately 2 minutes of vortexing and a clear homogenous solution was formed. Since saline or medium is the first solvent/vehicle option according to the test guideline and the formulation with saline was suitable for the study no more vehicles were tried and saline was chosen as the appropriate solvent of the test item.
Also, the test item was tried to be formulated in the maximum allowed concentration of 500 mg/mL with saline and clear homogenous solution was gained.

Preliminary tests (Dose finding assays)
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 1004 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 5013 µg/mL was the highest final concentration on the plate in the second run and a 2-fold dilution was used. Since no cytotoxicity was observed, 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.

Negative and positive control
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. The DMSO controls had negative outcomes compared to the medium control for both markers in all valid runs, meaning that the RFI values of CD86 and CD54 marker expression was never over the positive criteria. The cell viabilities of medium and DMSO controls were higher than 90 % in all valid runs (taken cell viabilities of the IgG1 isotypic control). For medium and DMSO controls, the MFI ratio of both CD86 and CD54 to isotype control was over 105 % in all three runs.

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.

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 %.

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:

Exposure date: 09 July, 2019
DNCB; 4.2 µg/mL; CD86: 403; CD54 1734; viability: 61.8%
Exposure date: 15 July, 2019
DNCB; 4.2 µg/mL; CD86: 639; CD54: 534; viability: 73.7 %
Exposure date: 16 July, 2019
DNCB; 4.4 µg/mL; CD86: 404; CD54: 211; viability 81 %

Key result

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:

positive indication of skin sensitisation

Remarks:

in two of three valid runs

Key result

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

Remarks:

in all three runs

Other effects / acceptance of results:

DEMONSTRATION OF TECHNICAL PROFICIENCY: Prior to routine use of the test method described in Annex I of the Test Guideline 442E, our 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:
Exposure date: 09 July, 2019
Medium; CD86: 100; CD54 100; viability: 91.5%
DMSO; 0.2 %; CD86: 126; CD54 34; viability: 91.1%
Exposure date: 15 July, 2019
Medium; CD86: 100; CD54 100; viability: 92.3%
DMSO; 0.2 %; CD86: 53; CD54 65; viability: 91.8%
Exposure date: 16 July, 2019
Medium; CD86: 100; CD54 100; viability: 91.7%
DMSO; 0.2 %; CD86: 73; CD54 59; viability: 93.1%

- Acceptance criteria met for positive control:yes:
Exposure date: 09 July, 2019
DNCB; 4.2 µg/mL; CD86: 403; CD54 1734; viability: 61.8%
Exposure date: 15 July, 2019
DNCB; 4.2 µg/mL; CD86: 639; CD54: 534; viability: 73.7 %
Exposure date: 16 July, 2019
DNCB; 4.4 µg/mL; CD86: 404; CD54: 211; viability 81 %
- Acceptance criteria met for variability between replicate measurements: not applicable

Reactivity check of THP-1 (ATCC) MC2 20180205-20190614

	Concentration (µg/mL)	RFI values of CD86	RFI values of CD54
DNCB	4.4	549	770
NiSO4	101	349	322
LA	1010	122	62
DMSO	0.2 %	138	58
Medium	-	100	100

Dose finding test results

Date	Test dose (µg/mL)	7.8	15.7	31.4	62.8	125.5	251	502	1004
01-02 July 2019	Viability (%)	93.3	92.5	93.1	92.6	93.3	94.7	92.9	93.9
Date	Test dose (µg/mL)	39.2	78.3	156.7	313.3	626.6	1253.3	2506.5	5013.0
02-03 July 2019	Viability (%)	91.2	91.8	91.6	91.5	89.9	89.9	88.9	87.6

Since no cytotoxicity was observed, 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.

Positive and negative control data

	sample	concentration	RFI		viability (%) ‑ IgG1
			CD86	CD54	IgG
Exposure date:   09 July, 2019	Medium	-	100	100	91.5
	DMSO	0.2%	126	34	91.1
	DNCB	4.2 μg/mL	403	1734	61.8
Exposure date:   15 July, 2019	Medium	-	100	100	92.3
	DMSO	0.2 %	53	65	91.8
	DNCB	4.2 μg/mL	639	534	73.7
Exposure date:   16 July, 2019	medium	-	100	100	91.7
	DMSO	0.2 %	73	59	93.1
	DNCB	4.4 μg/mL	404	211	82.0

Interpretation of results:

other: based on the h-Clat prediction model: positive

Conclusions:

In the present study the skin sensitizing potential of Triammonium citrate was determined using the h-Clat prediction model (OECD guideline 442E). The increase of CD86 marker expression (RFI) was 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 positive. Also, effective concentration for CD86 expression (EC150) was not determined, since no clear dose response could be observed. 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 3204 µg/mL, since clear dose response could be observed.
Since all both for CD86 and CD54 marker expression the predominant result was positive, the overall h-CLAT prediction was concluded positive, as well.
Based on these results and the h-CLAT prediction model, the test item “Triammonium Citrate” demonstrated an in vitro sensitizing potential under the experimental conditions of human Cell Line Activation Test.

Executive summary:

In this study conducted according to OECD test guideline 442E (adopted 25 June 2018) THP-1 cells were incubated with Triammonium citrate.

Since no cytotoxicity was observed, 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. Concentration steps in the dose range finding test: 39.2, 78.3, 156.7, 313.3, 626.6, 1253.3, 2506.5, 5013.0 μg/mL, concentration steps in the main test: 1394, 1673, 2007, 2409, 2891, 3469, 4163, 4995 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.

Relative cell viability at the highest test item concentration was reduced to 82.6 % (isotype IgG1 control) in the first experiment to 80.7% (isotype IgG1 control) in the second experiment and to 78.7% (isotype IgG1 control) in the third experiment.

The increase of CD86 marker expression (RFI) was 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 to be positive. Also, effective concentration for CD86 expression (EC150) was not determined, since no clear dose response could be observed. The positive control (DNCB) led to an upregulation of the expression of CD54 and CD86 in both experiments. The threshold of 150% for CD86 (196% experiment 1; 406% experiment 2) and 200% for CD54 (253% experiment 1; 359% experiment 2) were clearly exceeded.

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 as 3204 µg/mL, since a clear dose response could be observed.

Therefore, the test item is considered to be a skin sensitizer under the conditions of the test. The data generated with this method may be not sufficient to conclude on the absence of skin sensitization 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

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:
- 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)

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 luciferase induction > 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 luciferase induction > 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, variation between the values was < 20%

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 “Triammonium citrate” 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 Triammonium citrate 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 Triammonium citrate 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 Triammonium citrate, 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.