Triammonium citrate

Endpoint summary

Description of key information

Dermal irritation study according to OECD Guideline 439 (In Vitro Skin Irritation) (adopted July 28, 2015), 10 mg Triammonium citrate applied to a three-dimensional human epidermis model tissue for 15 min, post-incubation time 42 h, viability measurement, result 97% viability of control, no indication for skinn irritation

Eye irritation study according to OECD Guideline 438 (2018), 30 mg Triammonium citrate each were applied to 3 corneas for 240 min, measurement of corneal opacity, corneal swelling and Fluorescein retention, overall ICE classes were once II (based on the fluorescein retention of 1.3) and twice III (based on the corneal swelling of 13% within 75 minutes and corneal opacity score of 1.7), no prediction can be made.

Eye irritationn study according to OECD Guideline 492 (2019), EpiOcular test method, 50 mg Triammonium citrate were applied to 0.6cm³ of tissue for 6h, postincubation time 18h, viability measurement, result 2% viability compared to control, no prediction can be made.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

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

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 system:

human skin model

Source species:

human

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: EpiSkin™Small Model
- Tissue batch number: 19-EKIN-025
- Date of initiation of testing: 2019-06-19

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

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: approx. 25mL 1 x PBS
- Observable damage in the tissue due to washing: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3h
- Spectrophotometer: Thermo Scientific; Multiscan FC
- Wavelength: 570 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 granular 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%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TTEST 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): 10 µL
- Concentration (if solution): 5% SDS (aq.)

Duration of treatment / exposure:

15min ± 0.5 min

Duration of post-treatment incubation (if applicable):

42h ± 1h

Number of replicates:

Triplicates

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean of 3 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

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean of 3 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

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean of 3 replicates

Value:

97

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 treatment 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

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 Triammonium citrate 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), Triammonium citrate 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 Triammonium citrate compared to the negative control tissues was 97%. Since the mean relative tissue viability for the test substance was above 50%, Triammonium citrate is identified to be not irritating.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Justification for classification or non-classification

Justification for Classification of Triammonium citrate (eye irritation potential)

In accordance with Regulation (EC) No. 1907/2006 (REACH) Annex VII section 8.2 and 8.2.1 two in vitro tests were conducted to identify Triammonium citrate as serious eye irritant/corrosive or for no classification. The first in vitro test used was an organotypic in vitro test, the ICE assay (Isolated Chicken Eye, OECD guideline 438, 2018). The endpoints evaluated are corneal opacity, swelling, fluorescein retention, and morphological effects (e.g., pitting or loosening of the epithelium). The scores for determination of each parameter observed are:

Corneal opacity:

Score	Observation
0	No opacity
0.5	Very faint opacity
1	Scattered or diffuse areas; details of the iris are clearly visible
2	Easily discernible translucent area; details of the iris are slightly obscured
3	Severe corneal opacity; no specific details of the iris are visible; size of the pupil is barely discernible
4	Complete corneal opacity; iris invisible

Corneal swelling:

For the calculation of Maximum Swelling, small negative numbers for swelling (0 to -5%) following application are counted as zero. Large negative numbers (> -12 %) are probably due to erosion and indicate a severe effect (scored as class IV). Cases of values of -5 % to -12 % are evaluated on a case by case basis but in the absence of other findings do not indicate a severe effect.

Fluorescein retention:

Score	Observation
0	No fluorescein retention
0.5	Very minor single cell staining
1	Single cell staining scattered throughout the treated area of the cornea
2	Focal or confluent dense single cell staining
3	Confluent large areas of the cornea retaining fluorescein

The results for Triammonium citrate from this assay are:

Observation	Value	ICE Class^1
Mean maximum corneal swelling at up to 75 min	13%	III
Mean maximum corneal swelling at up to 240 min	13%	II
Mean maximum corneal opacity	1.7	III
Mean fluorescein retention	1.3	II
Other Observations	None
Overall ICE Class^1	1xII, 2xIII

 

Based on the results from this assay, Triammonium citrate could neither be classified as eye corrosive (Category I) nor as not classified according to Regulation (EC) No 1272/2008 (CLP) and the Globally Harmonized System for Classification and Labelling of Chemicals (GHS).

For this reason a second in vitro assay was performed based on the recommendations of REACH Guidance: ‘How to use new or revised in vitro test methods to address Serious eye damage/Eye irritation’, Feb 2018 and according to Regulation (EC) No. 1907/2006 (REACH) Annex VII section 8.2.1.

The second in vitro test used was the EpiOcular™ assay (OECD guideline 492): The eye irritation potential of a chemical may be predicted by measuring its cytotoxic effect, as reflected in the MTT assay, on the EpiOcular™ (OCL-200-EIT) tissue. The EpiOcular™ model can be used to identify chemicals that do not require classification for eye irritation or serious eye damage according to the UN GHS classification system. EpiOcular™ OCL-200, a three-dimensional RhCE tissue model, is produced using primary human epidermal keratinocytes. Relative tissue viability for tissue exposed to the test item is determined against tissues treated with the negative control by the reduction of the vital dye MTT (3-[4,5 - dimethylthiazol-2-yl] - 2,5 - diphenyltetrazolium bromide).

The evaluation criteria for this assay are:

Depending on the regulatory framework in member countries, the test item is identified as not requiring classification and labelling according to UN GHS (No Category) if the mean percent tissue viability after exposure and post-exposure incubation is more than
(>) 60%. In this case no further testing with the help of other test methods is required.

The prediction model (PM) is described below:

Criteria forIn Vitro interpretation	Classification
Mean tissue viability % is ≤ 60 %	Category 2 or Category 1
Mean tissue viability % is > 60 %	No Category

 

The results for Triammonium citrate of this assay are:

Treament with the test item Triammonium Citrate showed significantly reduced cell viability in comparison to the negative control (mean tissue viability: 2.2 %). All obtained test item viability results were below 60% when compared to the viability values obtained from the negative control.

 

Based on the results obtained from the second assay conducted with Triammonium citrate also revealed no clear classification according to Regulation (EC) No 1272/2008 (CLP) and the Globally Harmonized System for Classification and Labelling of Chemicals (GHS).

In order meet the requirements for registration of Triammonium citrate according to Regulation (EC) No. 1907/2006 (REACH) Annex VII and due to the recommendations outlined in REACH Guidance: ‘How to use new or revised in vitro test methods to address Serious eye damage/Eye irritation’, Feb 2018 stating that an ‘in vivo eye irritation study shall only be considered at Annex VIII level’ and . ‘at Annex VIII, if neither of these conclusions can be made, other in vitro study(ies) for this endpoint shall be considered. If the in vitro studies are not suitable for the substance, or the results are not adequate for classification and risk assessment, a further test conducted in vivo to assess the eye irritation potential shall be considered, i.e. none of the in vitro methods described above can be used for the direct identification of eye irritants (Cat. 2 of CLP)’. Furthermore. it is recommended that in case of inconclusive results an assessment of all available information, e.g. from QSAR(s) or skin corrosion studies should take place in order to adequately classify the substance.

 

Other available information:

In the ‘Guidance Document on an Integrated Approach on Testing and Assessment (IATA) for Serious Eye’, 2017 an additional test is mentioned to conclude that a substance does not need to be classified as Category I (Causes serious eye damage), the Fluorescein Leakage Test (OECD guideline 460). Based on the previously conducted tests and their results and due to the false-positive results which were reported in the Guidance document, this test was not considered to classification purposes. In vivo testing is not considered in terms of animal welfare and because according to REACH and the respective guidances it is not foreseen for registrations in the tonnage band of 1-10 t/a (Annex VII).

For the test item Triammonium citrate a skin irritation in vitro study is available conducted according to OECD guideline 439 (Reconstructed Human Epidermis Test Method). Based on the test results Triammonium citrate was not classified as skin irritant according to Regulation (EC) No 1272/2008 (CLP) and the Globally Harmonized System for Classification and Labelling of Chemicals (GHS). Like the EpiOcular Test method the RhE Test is based on the measurement of cell viability after treatment with the test item. After a 1 5min treatment with the test item and subsequently post-incubation time of 42h the cell viability was 97% of the cell viability of the control, thus Triammonium citrate was not classified as skin irritant.

The Performance of a QSAR method is not recommended for ionic structures because the estimations are based on neutral organics equations using the minimum toxicity principle assuming a simple non-polar narcosis model. This might underestimate the actual toxicity of the test item if used without comparison to empirical data from structurally similar substances. However, only the skin irritation potential could be estimated using a battery of QSAR estimation models, i.e. Case Ultra, Leadscope and SciQSAR via the Danish QSAR webpage (http://qsar.food.dtu.dk/). All predictions were performed for citric acid and revealed a negative result for severe skin irritation in rabbit, furthermore all predictions are considered within the applicability domain of the prediction model.

Since Triammonium citrate is a salt constituted of citric acid and Ammonium ions via a ionic bond and it is expected that the substance dissociates when it is dissolved in aqueous solutions, any test conducted in medium, aqueous solutions or water is considered to be exposed only the free ions, thus, it is admissible to use data generated from citric acid and other ammonium salts for the evaluation of the eye irritation potential.

Citric acid in general is considered to be a safe chemical as indicated by its GRAS status (SCOGS database). It is generally used as food supplement and preservative. Although some studies indicate that it is highly irritating to eyes(OECD SIDS, 2001) its salts are not classified as corrosive. Tripotassium citrate and Trisodium citrate are classified as Category II Causes severe eye irritation, Disodium hydrogen citrate is not classified for any hazard. For none of the citric acid salts a harmonized classification is available. This indicates that citrates are in general less irritating than the acid and that the irritancy potential is dependent on the type of counter ion of the salt.

Data for eye irritation for ammonium ions are lacking. However, ammonium ions are basically generated from ammonia following the equilibrium:

NH₃+ H₃O⁺ NH₄⁺+ H₂O

The fraction of ammonia in this equilibrium increases with increasing pH and temperature. Under normal physiological conditions at pH 7.4 and 25°C the predominant chemical species is ammonium.

Ammonia is an integral part of the intermediate metabolism and is endogenously formed by every organism capable of nitrogen fixation. Ammonia is an important source of nitrogen for mammals and plants due to its use in the synthesis of amino acids, DNA, RNA and proteins. It is produced endogenously in all mammalian species. Bacterial degradation of nucleic acids and amino acids in the gut produces ammonia in amounts of 3-4 g/day (ATSDR, 2004[1], EFSA, 2011[2]). Most of this ammonia is transported to the liver for detoxification where it is used in the synthesis of urea and glutamine. This process is an important part of the regulation of systemic pH due to the neutralisation of excess bicarbonate, created from the breakdown of amino acids, by hydrogen ions released from ammonium during the synthesis of urea (Häussinger, 2007[3]). Thus, ammonium is ubiquitously present in organisms and the environment.

Data on eye irritation for Ammonium ions are not available, however, there are some information on the eye irritation potential for ammonium salts like ammonium sulfate or ammonium chloride.

Ammonium chloride was found to be severely irritating to eyes and therefore was classified in Category II ‘Causes serious eye irritation’ Although it was reported that in studies with rabbits according to OECD 405 the effects caused by Ammonium chloride were fully reversible within 8 days and thus ammonium chloride was considered to be moderately irritant to skin and eyes (OECD SIDS, 2003[4]).

Ammonium sulfate was also evaluated by the OECD[5]and it was reported that also in studies conducted with rabbits according to OECD guideline 405, ammonium sulfate only caused slight redness of the eyes which was also found if the rabbits were treated the same way with talcum powder. However, the effects seen were fully reversible within 8 days. Based on these observations it was considered that ammonium sulfate is not irritating to skin and eyes. 

As a consequence, ammonium ions per se are not considered to be irritating in fact the counter ion might be responsible for the irritating effect.

Based on the information presented here, it is assumed that neither citrate nor ammonium are corrosive to the eye. The test item Triammonium citrate showed in two in vitro tests performed inconclusive results, i.e. in two in vitro test which are validated to distinguish between Category I (Causes serious eye damage/ corrosive) and no classification no prediction could be made. Thus, it was concluded that based on the irritating properties which were shown for both constituents to a similar extent, the irritancy potential of Triammonium citrate cannot be increased in comparison to the irritancy potential of the constituents.

Moreover, since the substance Triammonium citrate is currently voluntary classified as Category II’ Causes serious eye irritation’ this classification will be retained.

[1]ATSDR (Agency for Toxic Substances and Disease Registry), 2004. Toxicological profile for ammonia. U.S. Department of Health and Human Services, Atlanta, Georgia, 269 pp.

[2]EFSA Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids, 2011. Scientific Opinion on Flavouring Group Evaluation 46 Revision 1 (FGE.46Rev1). Ammonia and three ammonium salts from chemical group 30. EFSA Journal 2011; 9(2):1925, 35 pp.

[3]Häussinger D, 2007. Ammonia, urea production and pH regulation. In: The Textbook of Hepatology:

from basic science to clinical practice, 3rd Edition. Eds Rodes J, Benhamou J-P, Blei A, Reichen J and Rizzetto M. Wiley-Blackwell, 181-192. 

[4]OECD SIDS, AMMONIUM CHLORIDE CAS N°: 12125-02-9, 2003

[5]OECD SIDA, AMMONIUM SULFATE CAS N° 7783-20-2, 2004