Triammonium citrate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

published data, test in S. typhimurium TA97, TA98, TA100, TA104, with and without metabolic activation, applied in concentrations of 500, 1000 and 2000 µg/plate, negatice, source substance: citric acid

published data,test in S. typhimurium TA98, TA100, TA1535 and TA1537, with metabolic activation, maximum concentration 5000 µg/plate, negative, source substance citric acid

published data,test inS. typhimurium TA98, TA100, TA1535 and TA1537, with metabolic activation, maximum concentration 4000 µg/plate, negative, source substance ammonium sulfate

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Target gene:

his locus

Species / strain / cell type:

S. typhimurium, other: TA 98, TA 100, TA 1535, TA 1537

Metabolic activation:

with

Metabolic activation system:

The liver microsome fraction (S-9) was prepared from the liver of Fischer rats (Charles River Japan Co.) pretreated 5 days before with polychlorinated biphenyls (500 mg/kg body weight of Kanechlor KC-400 in olive oil, ip).

Test concentrations with justification for top dose:

Six different concentrations were used, the highest dose for citric acid was 5000 µg/plate and for Ammonium sulfate 10000 µg/plate. Only the highest dose was reported because up to this dose no cytotoxicity occured and no significant increase of revertants was observed.

Vehicle / solvent:

phosphate buffer

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate

Evaluation criteria:

The result was considered positive if the number of colonies found was twice the number in the control (exposed to the appropriate solvent or untreated).

Key result

Species / strain:

S. typhimurium, other: TA98, TA100, TA1535, TA1537

Remarks:

test item: citric acid

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

not specified

Key result

Species / strain:

S. typhimurium, other: TA98, TA100, TA1535, TA1537

Remarks:

test item:ammonium chloride

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

not specified

Conclusions:

According to the publication of Yamanaka et al., 1990, ammonium chloride and citric acid was negative when test in abacterial reverse mutation assay in S. typhimurium strains TA98, TA100, TA1535, TA1537 up to the top-concentration of 10000 µg/plate and 5000 µg/plate, respectively. Thus, according to Regulation (EC) No 1272/2008 (CLP) and the Globally Harmonized System for Classification and Labelling of Chemicals (GHS) both substances do not need to be classified as mutagenic.

Executive summary:

In a reverse gene mutation assay in bacteria according to OECD guideline 471 (adopted 21 July 1997), Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 were exposed to Ammonium sulfate in concentrations of up to 10000 µg/plate in all strains in the presence of mammalian metabolic activation (rat liver S9 mix). No cytotoxic effects were noted in all strains in the presence of metabolic activation.

No biologically relevant increases were observed in the number of revertant colonies in any of the five tester strains following treatment with Ammonium sulfate at any concentration levels in the presence of S9 Mix in the performed experiments. Based on the results obtained under the experimental conditions applied, the test item did not induce gene mutations.

In conclusion, the test item Ammonium sulfate has no mutagenic activity in the bacterial tester strains under the test conditions used in this study.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

his locus

Species / strain / cell type:

S. typhimurium TA 97

Species / strain / cell type:

S. typhimurium TA 98

Species / strain / cell type:

S. typhimurium TA 100

Species / strain / cell type:

S. typhimurium, other: TA 104

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : Male CD-COBS rats (200-250 g)
- method of preparation of S9 mix 0, 40, 60, 80, 100 mg/kg body weight of phenobarbital (PB) were administered intraperitoneally in 3 doses (1 mL/day). Liver homogenate S9 fraction was prepared as in Ames et al. (1975).
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): The activity of the prepared S9 was evaluated using 2 tester strains (TA100 and TA98) and the positive control mutagen 2-aminoanthracene (2-AA).

Test concentrations with justification for top dose:

0, 500, 1000, 2000 µg/plate, justification for dose selection not reported.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene 10 µg/plate

Details on test system and experimental conditions:

plate incorporation assay

Evaluation criteria:

Evaluated according to Ames et al.1983

Key result

Species / strain:

S. typhimurium TA 97

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium, other: TA 104

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Results for citric acid:

Citric acid conc. (µg/plate)
	Salmonella typhimurium strain
	TA97		TA98		TA100		TA104
	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9
0	68.3±2.51	28.0±2.64	44.3±1.15	30.0±2.00	158.6±5.13	130.6±3.78	440.0±13.22	377.3±7.09
500	61.0±3.60	28.0±4.35	41.6±1.52	25.6±1.15	146.3±4.04	125.6±4.72	492.0±10.58	440.0±2.00
1000	70.3±1.52	25.0±0.57	47.0±1.73	29.3±2.51	145.6±4.04	128.0±3.46	561.3±2.30	517.3±8.32
2000	63.0±1.73	26.0±1.73	39.6±0.57	32.0±2.00	132.6±1.15	124.3±0.57	509.3±9.01	482.0±13.85
2-AA	438.6±11.54	35.0±2.64	435.3±3.05	36.0±2.64	568.3±5.85	136.6±2.51	938.6±4.61	653.3±15.56

Conclusions:

According to the present test for bacterial reverse mutation (according to OECD 471), citric acid does not increase the number of revertants when applied in concentrations up to 2000 µg/plate with and without metabolic activation. Thus, according to Regulation (EC) No 1272/2008 (CLP) and the Globally Harmonized System for Classification and Labelling of Chemicals (GHS) citric acid does not need to be classified as mutagenic.

Executive summary:

In a reverse gene mutation assay in bacteria similar to OECD guideline 471, Salmonella typhimurium strains TA97, TA98, TA100, and TA104 were exposed to citric acid dissolved in water in concentrations of 0, 500, 1000, 2000 µg/plate in all strains in the absence and presence of mammalian metabolic activation (rat liver S9 mix).

The test substance was tested up to the maximum concentration of 2000 µg/plate. No cytotoxic effects were noted in all strains in the presence and absence of metabolic activation.

No biologically relevant increases were observed in the number of revertant colonies in any of the five tester strains following treatment with Citric acid at any concentration levels, either in the presence or absence of S9 Mix in the performed experiments. Based on the results obtained under the experimental conditions applied, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

In conclusion, the test item Citric acid has no mutagenic activity in the bacterial tester strains under the test conditions used in this study.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Please refer to the endpoint summary for Genetic Toxicity for more detailed information.

No genotoxicity data are available for the salt Triammonium citrate. Based on the ionic nature of Triammonium citrate it is expected to dissociate when it is dissolved in aqueous solutions, thereby generating NH4 and citrate ions. Hence, in test systems where the test substance is applied in aqueous solutions the test organisms are mainly in contact with the free ions. Hence, a readacross approach was developed based on the hypothesis that the target substance causes the same toxic effects as compared to its constituents, namely ammonium ions and citrate. Since the target substance is composed of the source substances the breakdown products are considered to be identical.

There are two published studies available which are investigating the mutagenic potential of citric acid in a reverse bacterial mutation assay.
Al-Aani & Al-Lami conducted the study with citric acid in concentrations of 500, 1000 and 2000 µg/plat, with and without metabolic activation in the Salmonella typhimurium strains TA97, TA98, TA100, TA104. Up to 2000 µg/plate no significant increase of revertants was detected in any strain. It was therefore concluded that citric acid has no mutagenic potential.

Ishidate et al conducted also a bacterial reverse mutation assay in the S. typhimurium strains TA98, TA100, TA1535 and TA1537with metabolic activation. They used six concentrations with the maximum concentration of 5.0 mg/plate. According to the authors citric acid was non-mutagenic under the conditions of the test. Furthermore, these authors conducted the test also with ammonium chloride another ammonium salt. Here the maximum concentration applied was 4.0 mg/plate, again without any sign of mutagenicity.

No other data on mutagenicity for free ammonium ions is available but based on its ubiquitous occurrence and its low toxicity ammonium ions are not considered to increase the mutagenic potential of citric acid. According to EFSA ammonia originates from both natural and anthropogenic sources and is an important source of nitrogen for mammals in the synthesis of amino acids, DNA, RNA and proteins. It is naturally present in different foods and is also produced endogenously in all mammalian species. Following oral exposure, exogenous ammonium is rapidly and extensively absorbed and transported to the liver together with endogenous ammonium produced in the gastrointestinal tract (estimated to be 3-4 g/person per day, (43-57 mg/kg body weight (b.w.) per day for a 70 kg adult)). In the liver, ammonium is metabolised to urea and glutamine. The metabolites are either excreted as urea or retained for the synthesis of amino acids or other biomolecules. Based on these information Ammonium is considered to be of no concern for developmental or reproductive effects, or for carcinogenicity and it shows low acute oral toxicity. Following a single exposure to aqueous solutions of ammonium chloride by gavage, LD 50 values of 1630 mg/kg b.w. (equivalent to 548 mg ammonium/kg b.w.) and 1220 mg/kg b.w. (equivalent to 410 mg ammonium/kg b.w.) were observed in male and female rats, respectively (OECD, 2003). Oral LD 50 values for ammonium sulphate range from 2000 mg/kg b.w. to 4250 mg/kg b.w. (544-1156 mg ammonium/kg b.w.) (OECD, 2004). Both ammonium chloride and ammonium sulphate gave negative results when tested in bacterial reverse mutation assays (OECD, 2003 , 2004 ).
Overall, based on this information ammonium shows no genotoxic potential.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

S. typhimurium, other: TA 98, TA 100, TA 1535, TA 1537

Remarks:

Ishidate et al. (ammonium chloride and citric acid)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium, other: TA 97, TA 98, TA 100, TA 104

Remarks:

Al-Ani & Al-Lami 1988 (citirc acid)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

valid

Conclusions:

In the present test for bacterial reverse mutation (according to OECD 471), citric acid does not increase the number of revertants when applied in concentrations up to 2000 µg/plate with and without metabolic activation. According to the publication of Yamanaka et al., 1990, ammonium chloride and citric acid was negative when test in abacterial reverse mutation assay in S. typhimurium strains TA98, TA100, TA1535, TA1537 up to the top-concentration of 10000 µg/plate and 5000 µg/plate, respectively.
Thus, according to Regulation (EC) No 1272/2008 (CLP) and the Globally Harmonized System for Classification and Labelling of Chemicals (GHS) citric acid and ammonium chloride do not need to be classified as mutagenic.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

No genotoxicity data are available for the salt Triammonium citrate. Based on the ionic nature of Triammonium citrate it is expected to dissociate when it is dissolved in aqueous solutions, thereby generating NH4 and citrate ions. Hence, in test systems where the test substance is applied in aqueous solutions the test organisms are mainly in contact with the free ions. Hence, a readacross approach was developed based on the hypothesis that the target substance causes the same toxic effects as compared to its constituents, namely ammonium ions and citrate. Since the target substance is composed of the source substances the breakdown products are considered to be identical.

There are two published studies available which are investigating the mutagenic potential of citric acid in a reverse bacterial mutation assay.

Al-Aani & Al-Lami conducted the study with citric acid in concentrations of 500, 1000 and 2000 µg/plat, with and without metabolic activation in the Salmonella typhimurium strains TA97, TA98, TA100, TA104. Up to 2000 µg/plate no significant increase of revertants was detected in any strain. It was therefore concluded that citric acid has no mutagenic potential.

Ishidate et al conducted also a bacterial reverse mutation assay in the S. typhimurium strains TA98, TA100, TA1535 and TA1537with metabolic activation. They used six concentrations with the maximum concentration of 5.0 mg/plate. According to the authors citric acid was non-mutagenic under the conditions of the test. Furthermore, these authors conducted the test also with ammonium chloride another ammonium salt. Here the maximum concentration applied was 4.0 mg/plate, again without any sign of mutagenicity.

No other data on mutagenicity for free ammonium ions is available but based on its ubiquitous occurrence and its low toxicity ammonium ions are not considered to increase the mutagenic potential of citric acid. According to EFSA[1]ammonia originates from both natural and anthropogenic sources and is an important source of nitrogen for mammals in the synthesis of amino acids, DNA, RNA and proteins. It is naturally present in different foods and is also produced endogenously in all mammalian species. Following oral exposure, exogenous ammonium is rapidly and extensively absorbed and transported to the liver together with endogenous ammonium produced in the gastrointestinal tract (estimated to be 3-4 g/person per day, (43-57 mg/kg body weight (b.w.) per day for a 70 kg adult)). In the liver, ammonium is metabolised to urea and glutamine. The metabolites are either excreted as urea or retained for the synthesis of amino acids or other biomolecules. Based on these information Ammonium is considered to be of no concern for developmental or reproductive effects, or for carcinogenicity and it shows low acute oral toxicity. Following a single exposure to aqueous solutions of ammonium chloride by gavage, LD 50 values of 1630 mg/kg b.w. (equivalent to 548 mg ammonium/kg b.w.) and 1220 mg/kg b.w. (equivalent to 410 mg ammonium/kg b.w.) were observed in male and female rats, respectively (OECD, 2003). Oral LD 50 values for ammonium sulphate range from 2000 mg/kg b.w. to 4250 mg/kg b.w. (544-1156 mg ammonium/kg b.w.) (OECD, 2004). Both ammonium chloride and ammonium sulphate gave negative results when tested in bacterial reverse mutation assays (OECD, 2003[2], 2004[3]). Overall, based on this information ammonium shows no genotoxic potential.

[1]EFSA Journal 2012;10(10):2918

[2]OECD (Organisation for Economic Co-operation and Development), 2003. Ammonium chloride – CAS N°: 12125-02-9.

[3]OECD (Organisation for Economic Co-operation and Development), 2004.Ammonium sulfate – CAS N°: 7783-20-2.

Justification for classification or non-classification

Based on the available infomation and the results from mutagenicity tests with the source substances citric acid and ammonium sulfate Triammonium citrate is not classified as mutagenic according to Regulation (EC) No. 1272/2008 (CLP) and the Globally Harmonized System for Classification and Labelling of Chemicals (GHS).