Ammonium dihydrogen citrate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test: not mutagenic

In vitro Chromosome aberration test: not clastogenic

In vitro gene mutation study in mammalian cells: not mutagenic

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 1989

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

no TA102 or WP2uvrA, no information of historical data.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

- S. typhimurium: Histidine gene

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction induced by Aroclor 1254

Test concentrations with justification for top dose:

Experiment 1 and 2 all 4 strains:
With and without S9-mix: 20, 100, 500, 2500, and 5000 μg/plate

Vehicle / solvent:

- Solvent used: aqua dest.
- Justification for choice of solvent: the substance was completely soluble in aqua dest.

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

other: 5 μg N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) (dissolved in DMSO) for the strains TA 100 and TA 1535; 10 μg 4-nitro-o-phenylendiamine (dissolved in DMSO) for the strain TA 98

Remarks:

Without S9

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 10 μg 2-aminoanthracene (dissolved in DMSO) for the strains TA 100, TA 98, TA 1537 and TA 1535

Remarks:

With S9

Details on test system and experimental conditions:

METHOD OF APPLICATION:in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 20 minutes
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: 3 replicates in both experiments.

NUMBER OF CELLS EVALUATED: 10^8 cells

DETERMINATION OF CYTOTOXICITY
- Method: Reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.

Evaluation criteria:

In general, a substance to be characterized as positive in the Ames test has to fulfill the following requirements:
- doubling of the spontaneous mutation rate (control)
- dose-response relationship
- reproducibility of the results .

Statistics:

Not performed.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

but tested up to and including 5000 μg/plate.

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

but tested up to and including 5000 μg/plate.

Vehicle controls validity:

valid

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

Remarks:

but tested up to and including 5000 μg/plate.

Vehicle controls validity:

valid

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

Remarks:

but tested up to and including 5000 μg/plate.

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation was observed up to and including 5000 μg/plate.

HISTORICAL CONTROL DATA: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: No toxicity was observed up to and including 5000 μg/plate.

Conclusions:

The substance is not mutagenic in the Salmonella typhimurium reverse mutation assay performed according to OECD 471 guideline.

Executive summary:

The mutagenic activity of Ammonium sulphate was evaluated in accordance with OECD 471 guideline. The test was performed in two independent experiments, experiment 1 a plate incorporation test and experiment 2 a pre-incubation test using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100. Dose levels up to and including 5000 μg/plate were tested in the absence and presence of S9-mix. Adequate positive controls and solvent controls were included.

No precipitation of the test item and no toxicity was observed up to and including the concentration of 5000 μg/plate.

No increase in revertant colony numbers of any of the four tester strains was observed following treatment with the substance at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). Based on the results, Ammonium sulphate is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

No data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: In this publication an Ames test was conducted using a similar OECDTG 471 protocol without GLP.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

no TA102 or WP2uvrA, no details on positive controls, no evaluation on precipitate and toxicity.

Principles of method if other than guideline:

Method of Ames, McCann & Yamasaki (1975)

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Target gene:

- S. typhimurium: Histidine gene

Species / strain / cell type:

S. typhimurium, other: TA92, TA1535, TA100, TA1537, TA94 and TA98

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by polychlorinated biphenyls (500 mg/kg body weight)

Test concentrations with justification for top dose:

Experiment 1:
TA92, TA1535, TA100, TA1537, TA94 and TA98 (without and with S9): six different concentrations, max dose 5000 μg/plate

Vehicle / solvent:

- Solvent used: Phosphate buffer
- Justification for choice of solvent/vehicle: no data

Negative solvent / vehicle controls:

yes

Remarks:

phosphate buffer

Positive controls:

not specified

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation;

DURATION
- Preincubation period: 20 min.
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: duplicates

NUMBER OF CELLS EVALUATED: no data

DETERMINATION OF CYTOTOXICITY: no data

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).
If no reasonable dose response was detected, additional experiments using different doses or induced mutation frequency assays (Yoshikawa, Nakadate, Watabe et al. 1980) were performed.

Statistics:

Not performed.

Key result

Species / strain:

S. typhimurium, other: TA92, TA1535, TA100, TA1537, TA94 and TA98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: no data

RANGE-FINDING/SCREENING STUDIES: not performed.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
No data..

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No data.

Conclusions:

In an Ames test performed with Citric acid equivalent to OECD 471 guideline without GLP, Citric acid was found to be not mutagenic with and without S9-mix.

Executive summary:

An Ames test with Citric acid was performed equivalent to OECD 471 guideline without GLP.

One single pre-incubation experiment was performed with Salmonella typhimurium strains TA92, TA94, TA1535, TA98, TA100 and TA1537 up to and including 5000 μg/plate with and without S9 -mix.

In the presence and absence of S9 -mix, no significant increases in the numbers of revertant colonies were detected in any Salmonella typhimurium strains at the maximum dose up to and including 5000 μg/plate.

Based on the results of this study it is concluded that Citric acid is not mutagenic in the Salmonella typhimurium assay with and without S9 -mix.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

No data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: In this publication an Ames test was conducted using a similar OECDTG 471 protocol without GLP.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

no TA102 or WP2uvrA, no reliable positive control without S9, no details on precipitate to confirm the highest tested concentration, limited details on test system and conditions.

Principles of method if other than guideline:

According to Maron and Ames, 1983.

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Target gene:

- S. typhimurium: Histidine gene

Species / strain / cell type:

S. typhimurium, other: TA97, TA98, TA100 and TA104

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by phenobarbital

Test concentrations with justification for top dose:

Experiment 1:
TA97, TA98, TA100 and TA104 (without and with S9): 500, 1000 and 2000 µg/plate

Vehicle / solvent:

- Solvent used: distilled water
- Justification for choice of solvent/vehicle: no data

Negative solvent / vehicle controls:

yes

Remarks:

distilled water

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (10 µg/plate)

Remarks:

Without and with S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: no data

NUMBER OF REPLICATIONS: triplicate

NUMBER OF CELLS EVALUATED: no data

DETERMINATION OF CYTOTOXICITY: Routine examination of the bacterial background lawn.

Evaluation criteria:

No data.

Key result

Species / strain:

S. typhimurium, other: TA97, TA98, TA100 and TA104

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

up to and including 2000 µg/plate

Vehicle controls validity:

valid

Positive controls validity:

other: with S9: yes, without S9: no

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: no data

RANGE-FINDING/SCREENING STUDIES: not performed.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
The spontaneous reversions of the tester strains were within the acceptable range (Levin et al., 1982; Marion and Ames, 1983).

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Routine examination of the bacterial background lawn indicates the absence of toxicity.

Conclusions:

In an Ames test performed with Citric acid equivalent to OECD 471 guideline without GLP, Citric acid was found to be not mutagenic with and without S9-mix.

Executive summary:

An Ames test with Citric acid was performed equivalent to OECD 471 guideline without GLP.

One single direct plate experiment was performed with Salmonella typhimurium strains TA97, TA98, TA100 and TA104 up to and including 2000 µg/plate with and without S9 -mix. A reliable positive control with S9 -mix was included. Routine examination of the bacterial background lawn indicates the absence of toxicity. In the presence and absence of S9 -mix, the numbers of revertants in all tester strains for all concentrations tested were not significantly different from the respective negative controls.

Based on the results of this study it is concluded that Citric acid is not mutagenic in the Salmonella typhimurium assay with and without S9 -mix.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 April 2001 - 17 May 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

February 1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ICH Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals (1997).

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction induced by Aroclor 1254

Test concentrations with justification for top dose:

Initial toxicity-mutation assay, all 5 strains:
With and without S9-mix: 2.5, 7.5, 25, 75, 200, 600, 1800 and 5000 μg/plate.
Confirmatory mutagenicity assay, all 5 strains:
With and without S9-mix: 50, 150,500, 1500 and 5000 μg/plate.

Vehicle / solvent:

- Solvent used: water
- Justification for choice of solvent: A solubility test was conducted to select the vehicle. The test was conducted using water. The test article was tested to determine the vehicle that permitted preparation of the highest soluble or workable stock concentration, up to 50 mg/mL for aqueous solvents.

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

Remarks:

Without S9

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 1.0 μg/plate 2-aminoanthracene for the strains TA 100, TA 98, TA 1537 and TA 1535, and 10 μg/plate 2-aminoanthracene for the strain WP2uvrA

Remarks:

With S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: approximately 48 to 72 hours

NUMBER OF REPLICATIONS:
Initial Toxicity-Mutation Assay in duplicate
Confirmatory Mutagenicity Assay in triplicate

DETERMINATION OF CYTOTOXICITY
The condition of the bacterial background lawn was evaluated for evidence of test substance toxicity by using a dissecting microscope.

Evaluation criteria:

For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated and were reported.
For the test article to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test article. Data sets for tester strains TA1535 and TA1537 were judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than three times the mean vehicle control value. Data sets for tester strains TA98, TA100 and WP2 uvrA were judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than two times the mean vehicle control value.

Statistics:

Not performed.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

but tested up to and including 5000 μg/plate.

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

but tested up to and including 5000 μg/plate.

Vehicle controls validity:

valid

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

Remarks:

but tested up to and including 5000 μg/plate.

Vehicle controls validity:

valid

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

Remarks:

but tested up to and including 5000 μg/plate.

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

but tested up to and including 5000 μg/plate.

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation:
Initial Toxicity Mutation Assay at concentrations of 1800 and 5000 μg/plate.
Confirmatory Mutagenicity Assay at the concentration of 5000 μg/plate.

HISTORICAL CONTROL DATA

Historical Negative and Positive Control Values 1998-2000

revertants per plate

Strain Control Activation
None Rat Liver
Mean SD Min Max Mean SD Min Max

TA98 Neg 16 7 4 59 21 7 7 58
Pos 425 206 21 1536 592 322 56 2454

TA100 Neg 128 31 53 288 138 34 74 258
Pos 568 159 129 1371 736 301 198 2871

TA1535 Neg 12 5 1 45 12 4 1 42
Pos 378 164 6 978 104 84 18 1640

TA1537 Neg 6 3 0 30 7 3 1 29
Pos 708 409 13 2786 88 106 12 2060

WP2uvrA Neg 14 5 4 48 16 6 4 115
Pos 190 138 34 961 317 299 22 2632

SD=standard deviation; Min=minimum value; Max=maximum value; Neg=negative control (including but not limited to deionized water, dimethyl sulfoxide, ethanol and acetone); Pos=positive control

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: No toxicity was observed up to and including 5000 μg/plate.

Conclusions:

The substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and not mutagenic in the Escherichia coli reverse mutation assay performed according to OECD 471 guideline and GLP principles.

Executive summary:

The mutagenic activity of Diammonium phosphate was evaluated in accordance with OECD 471 guideline and GLP principles.

The test was performed in two independent direct plate experiments, using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100, and Escherichia coli strain WP2uvrA. Dose levels up to and including 5000 μg/plate were tested in the absence and presence of S9-mix. Adequate positive controls and solvent controls were included.

In the Initial Toxicity Mutation Assay precipitation was obseved at concentrations of 1800 and 5000 μg/plate and in the Confirmatory Mutagenicity Assay precipitation was observed at the concentration of 5000 μg/plate. No toxicity was observed up to and including the concentration of 5000 μg/plate.

No increase in revertant colony numbers of any of the five tester strains was observed following treatment with the substance at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). Based on the results, Diammonium phosphate is considered to be non-mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Reference 5

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

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

Adequacy of study:

key study

Study period:

2018

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Read-across information.

Justification for type of information:

See the attached read-across document in section 13.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

but tested up to and including 5000 μg/plate.

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

but tested up to and including 5000 μg/plate.

Vehicle controls validity:

valid

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

Remarks:

but tested up to and including 5000 μg/plate.

Vehicle controls validity:

valid

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

Remarks:

but tested up to and including 5000 μg/plate.

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

other: TA92, TA1535, TA100, TA1537, TA94 and TA98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not specified

Positive controls validity:

not specified

Key result

Species / strain:

other: TA97, TA98, TA100 and TA104

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

up to and including 2000 µg/plate

Vehicle controls validity:

valid

Positive controls validity:

other: with S9: yes, without S9: no

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

but tested up to and including 5000 μg/plate.

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Read-across result from Ammonium sulphate.

Conclusions:

The substance Ammonium dihydrogen citrate in water is not mutagenic in the Salmonella typhimurium reverse mutation assay and not mutagenic in the Escherichia coli reverse mutation assay performed according to OECD 471 guideline, based on the results of the source substances.

Reference 6

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

No data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: In this publication a chromosome aberration test was conducted using a similar OECDTG 473 protocol without GLP.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

no evaluation on precipitate, no determination of cytotoxicity, no positive controls included, exposure with S9-mix not investigated.

GLP compliance:

not specified

Type of assay:

other: in vitro cytogenicity / chromosome aberration study in mammalian cells

Species / strain / cell type:

lymphocytes: human peripheral blood

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: whole blood in a Ficoll gradient

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: McCoy's 5A medium supplemented with 20% (v/v) fetal calf serum, 0.06 mL phytohaemagglutimin M, 100 units per mL penicillin and 0.1 mg per mL dihydrostreptomycin sulphate.

Metabolic activation:

without

Test concentrations with justification for top dose:

3.2 M (ca. 423 mg/mL)

Vehicle / solvent:

Vehicle: water, medium or Alu I shipping buffer (KPO4 = 20 mM ; KCl= 50 mM; EDTA = 0 .1 mM ; dithioerythritol = 10 mM; glycerin = 50% (v/v); pH = 7.5)

Negative solvent / vehicle controls:

yes

Positive controls:

no

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 20 h and 46 h after start of cultures
- Fixation time (start of exposure up to fixation or harvest of cells): 50 h

SPINDLE INHIBITOR (cytogenetic assays): Colcemid (0.08 µg/mL)

STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: Up to 7 independent experiments were performed with blood from 5 different donors

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Preparations were made following a routine protocol

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 100 per donor

DETERMINATION OF CYTOTOXICITY
Not performed.

OTHER EXAMINATIONS:
- Determination of polyploidy: yes

Evaluation criteria:

No data.

Key result

Species / strain:

lymphocytes: human peripheral blood

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Positive controls validity:

not examined

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
No data.

RANGE-FINDING/SCREENING STUDIES: Not performed.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
No data.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: not determined.

Conclusions:

In a chromosome aberration test performed with Ammonium sulphate equivalent to OECD 473 guideline without GLP, Ammonium sulphate was found to be not clastogenic without S9-mix.

Executive summary:

A chromosome aberration test was performed with Ammonium sulphate equivalent to OECD 473 guideline without GLP.

Two experiments were performed in human peripheral blood cells with an Ammonium sulphate concentration of 3.2 M (ca. 423 mg/mL) for 20 and 46 hr after start of the cultures without S9 -mix.

Ammonium sulphate did not induce a dose dependent significant increase in the frequency of chromosome aberrations in both treatment times compared with the untreated cultures. Also no increase in the incidence of polyploid cells was observed at 20 or 46 hr after the start of the cultures.

Based on the results of this study it is concluded that Ammonium sulphate is not clastogenic in the chromosome aberration test with human peripheral blood cells without S9 -mix.

Reference 7

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

No data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: In this publication a chromosome aberration test was conducted using a similar OECDTG 473 protocol without GLP.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

no details on positive controls, no evaluation on precipitate, exposure with S9-mix not investigated.

GLP compliance:

no

Type of assay:

other: in vitro cytogenicity / chromosome aberration study in mammalian cells

Species / strain / cell type:

other: Chinese hamster fibroblast cell line, CHL

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Originally established from the lung of a newborn female at the Cancer Research Institute, Tokyo (Koyama, Utakoji & Ono, 1970).
- Suitability of cells: no data
- Cell cycle length, doubling time or proliferation index: doubling time approximately 15 hr
- Number of passages if applicable: 4-day passages
- Modal number of chromosomes: 25

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Minimum Essential Medium (MEM; GIBCO) supplemented by 10% calf serum.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: no data
- Periodically checked for karyotype stability: no data

Metabolic activation:

without

Test concentrations with justification for top dose:

Experiment 1: three different doses max dose 1000 mg/mL
The maximum dose was selected by a preliminary test in which the dose needed for 50% cell-growth inhibition was estimated.

Vehicle / solvent:

- Solvent used: physiol. saline
- Justification for choice of solvent: no data

Negative solvent / vehicle controls:

yes

Remarks:

physiol. saline

True negative controls:

yes

Positive controls:

not specified

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 24 and 48 hr without S9
- Fixation time (start of exposure up to fixation or harvest of cells): 24 and 48 hr

SPINDLE INHIBITOR (cytogenetic assays): Colcemid (0.2 μg/mL)

STAIN (for cytogenetic assays): Giemsa (1.5% solution)

NUMBER OF REPLICATIONS: no data

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
The cell were trypsinized and suspended in a hypotonic KCl solution (0.075M) for 13 min. at room temperature. After centrifugation the cells were fixed with acetic acid-methanol (1:3, v/v) and spread on clean glass slides.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 100

DETERMINATION OF CYTOTOXICITY
- Method: The maximum dose was selected by a preliminary test in which the dose needed for 50% cell-growth inhibition was estimated using a cell densitometer.

OTHER EXAMINATIONS:
- Determination of polyploidy: yes

Evaluation criteria:

The results were considered to be negative if the incidence was less than 4.9%, equivocal if it was between 5.0 and 9.9%, and positive if it was more than 10.0%.
When no reasonable dose-response relationships were found, additional experiments were carried out at similar dose levels.

Key result

Species / strain:

other: Chinese hamster fibroblast cell line, CHL

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

50% cell-growth inhibition

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH:
- Effects of osmolality: Previous studies indicated that the osmotic pressure of the medium generally rose with sample concentrations of more than 10 mM, so that the maximum dose for some samples was limited to around this level.
- Precipitation: no data

RANGE-FINDING/SCREENING STUDIES: The maximum dose was selected by a preliminary test in which the dose needed for 50% cell-growth inhibition was estimated using a cell densitometer.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
Untreated cells and solvent-treated cells served as negative controls, in which the incidence of aberrations was usually less than 3.0%.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The maximum dose was selected by a preliminary test in which the dose needed for 50% cell-growth inhibition was estimated using a cell densitometer.

Conclusions:

In a chromosome aberration test performed with Citric acid equivalent to OECD 473 guideline without GLP, Citric acid was found to be not clastogenic without S9-mix.

Executive summary:

A chromosome aberration test was performed with Citric acid equivalent to OECD 473 guideline without GLP.

One single experiment was performed in a Chinese hamster fibroblast cell line with Citric acid concentrations up to and including 1000 mg/mL for 24 and 48 hr without S9 -mix. The maximum dose was selected by a preliminary test in which the dose needed for 50% cell-growth inhibition was estimated. No increase in the incidence of polyploid cells or in the incidence of cells with structural aberrations was observed at 24 or 48 hr after treatment.

Based on the results of this study it is concluded that Citric acid is not clastogenic in the chromosome aberration test with CHL cells without S9 -mix.

Reference 8

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

See the attached read-across document in section 13.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

other: Chinese hamster fibroblast cell line, CHL

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

50% cell-growth inhibition

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Key result

Species / strain:

lymphocytes: human peripheral blood

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Positive controls validity:

not examined

Additional information on results:

In a chromosome aberration test performed with Citric acid equivalent to OECD 473 guideline without GLP, Citric acid was found to be not clastogenic without S9-mix.
In a chromosome aberration test performed with Ammonium sulphate equivalent to OECD 473 guideline without GLP, Ammonium sulphate was found to be not clastogenic without S9-mix.

Remarks on result:

other: Results of Citric acid

Conclusions:

Based on the results of the source substances Citric acid and Ammonium sulphate, it is concluded that the target substance Ammonium dihydrogen citrate in water is not clastogenic in the chromosome aberration test without S9 -mix.

Reference 9

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 March 2010 - 02 June 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

21 July 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

30 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Version / remarks:

August 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Target gene:

HPRT locus in V79 cells.

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Laboratory for Mutagenicity Testing, Technical University, 64287 Darmstadt, Germany
- Suitability of cells: Especially the high proliferation rate (doubling time 12 - 16 h in stock cultures) and a good cloning efficiency of untreated cells (as a rule more than 50 %) both necessary for the appropriate performance of the study, recommend the use of this cell line.
- Modal number of chromosomes: The cells have a stable karyotype with a modal chromosome number of 22

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: MEM (minimal essential medium; SEROMED, 12247 Berlin, Germany) supplemented with 10 % fetal calf serum (FCS; PAA Laboratories GmbH, 35091 Cölbe, Germany) and 1 % neomycin. The cell cultures were incubated at 37 °C in a 4.5 % carbon dioxide atmosphere (95.5 % air). For the selection of mutant cells the medium was supplemented with 11 Pg/mL 6-thioguanine (6TG, SIGMA GmbH, 82041 Deisenhofen, Germany).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically 'cleansed' against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/β-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Dose range finding test:
Without and with S9-mix, 4 hours treatment: 10.3, 20.6, 41.3, 82.5, 165, 330, 660 and 1320 μg/mL
Without S9-mix, 24 hours treatment: 10.3, 20.6, 41.3, 82.5, 165, 330, 660 and 1320 μg/mL
Experiment 1:
Without and with S9-mix, 4 hours treatment: 41.3, 82.5, 165, 330, 660 and 1320 μg/mL
The following dose levels were selected to measure mutation frequencies at the HPRT-locus:
Without and with S9-mix, 4 hours treatment: 82.5, 165, 330, 660 and 1320 μg/mL
Experiment 2
With S9-mix, 4 hours treatment: 41.3, 82.5, 165, 330, 660 and 1320 μg/mL
Without S9-mix, 24 hours treatment: 41.3, 82.5, 165, 330, 660 and 1320 μg/mL
The following dose levels were selected to measure mutation frequencies at the HPRT-locus:
With S9-mix, 4 hours treatment: 82.5, 165, 330, 660 and 1320 μg/mL
Without S9-mix, 24 hours treatment: 82.5, 165, 330, 660 and 1320 μg/mL

Vehicle / solvent:

- Solvent used: deionised water
- Justification for choice of solvent: The test item was dissolved in deionised water. The solvent was chosen according to its solubility properties and its relative nontoxicity to the cells.

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
Short-term treatment
With and without S9-mix: 4 hours
Prolonged treatment period
Without S9-mix: 24 hours
- Expression time (cells in growth medium): 3 or 4 days
- Selection time (if incubation with a selection agent): 7 days

SELECTION AGENT (mutation assays): 11 μg/mL 6-thioguanine (6TG)

NUMBER OF REPLICATIONS: Duplicate

STAINING TECHNIQUE USED: 10 % methylene blue in 0.01 % KOH solution

NUMBER OF CELLS EVALUATED: 500 cells/flask

DETERMINATION OF CYTOTOXICITY
- Method: relative cloning efficiency

Evaluation criteria:

Evaluation of Results:
A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points.
A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.
A positive response is described as follows:
A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concentrations in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
However, in a case by case evaluation this decision depends on the level of the corresponding solvent control data. If there is by chance a low spontaneous mutation rate in the range normally found (3.0 – 33.2 mutants per 106 cells) a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of solvent controls within all experiments of this study was also taken into consideration.

Acceptability of the Assay:
The gene mutation assay is considered acceptable if it meets the following criteria:
- the numbers of mutant colonies per 106 cells found in the solvent controls fall within the laboratory historical control data range.
- the positive control substances must produce a significant increase in mutant colony frequencies.
- the cloning efficiency II (absolute value) of the solvent controls must exceed 50 %.

Statistics:

A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT®11 (SYSTAT Software, Inc., 501, Canal Boulevard, Suite C, Richmond, CA 94804, USA) statistics software. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance were considered together.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

up to and including the concentration of 1320 μg/mL (~0.01 M).

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolarity: The pH and osmolarity at a concentration of 1320 μg/mL were 7.4 and 0.309 Osm/kg respectively (compared to 7.4 and 0.278 Osm/kg in the solvent control).
- Precipitation: No precipitation was observed up to and including of the maximum concentration of 1320 μg/mL with and without metabolic activation.

RANGE-FINDING/SCREENING STUDIES: The highest concentration in the pre-experiment was 1320 μg/mL equal to approximately 10 mM. No cytotoxic effects were noted in any of the experimental parts up to the maximum concentration.

HISTORICAL CONTROL DATA:
These values represent the historical control data from 2006 – 2009

Number of mutant colonies per 10^6 cells

without metabolic activation (4 hours treatment time)
Positive control Solvent control
EMS (medium, water, DMSO, ethanol, THF)
150 μg/mL
Range: 69.5 – 1386.4 3.4 – 31.7
Mean value: 176.5 14.0
Standard deviation: 172.5 6.6
Number of studies: 42 42

with metabolic activation (4 hours treatment time)
Positive control Solvent control
DMBA (medium, water, DMSO, ethanol, THF)
1.1 - 2.0 μg/mL
Range: 135.5 – 2574.2 3.0 – 33.2
Mean value: 965.5 15.2
Standard deviation: 457.5 6.5
Number of studies: 42 42

without metabolic activation (24 hours treatment time)
Positive control Solvent control
EMS (medium, water, DMSO, ethanol, THF)
75 – 300 μg/mL
Range: 54.3 – 710.5 3.9 – 31.5
Mean value: 254.2 15.3
Standard deviation: 124.8 6.7
Number of studies: 34 34

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No relevant cytotoxic effect as indicated by a relative cloning efficiency of less than 50% occurred up to and including of the maximum concentration of 1320 μg/mL with and without metabolic activation.

Conclusions:

A gene mutation assay in Chinese hamster V79 cells with the substance was conducted according to OECD 476 guideline and GLP principles. It is concluded that the substance is not mutagenic in the gene mutation assay under the experimental conditions of the study.

Executive summary:

A gene mutation assay in Chinese hamster V79 cells with the substance was conducted according to OECD 476 guideline and GLP principles.

Based on the results of the dose-range finding test, Ammonium sulphate was tested in two mutation assays. The first experiment was performed in the absence and presence of S9-mix with a 4 hour treatment

period. The second mutation experiment was performed in the absence of S9-mix with a 24 hour treatment period and in the presence of S9 -mix with a 4 hour treatment period. The following dose-range was selected for the mutagenicity tests in the absence and presence of S9-mix: 82.5, 165, 330, 660 and 1320 (~0.01 M) μg/mL exposure medium. Reliable solvent and positive controls were included.

No relevant cytotoxic effect as indicated by a relative cloning efficiency of less than 50% occurred up to and including of the maximum concentration of 1320 μg/mL with and without metabolic activation.

No relevant and reproducible increase in mutant colony numbers/10^6 cells was observed in the main experiments up to and including of the maximum concentration. It is concluded that the substance is not mutagenic in the gene mutation assay under the experimental conditions of the study.

Reference 10

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

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

Adequacy of study:

key study

Study period:

2018

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Read-across information.

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
See Read-across justification document attached in section 13.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

up to and including the concentration of 1320 μg/mL (~0.01 M).

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

A gene mutation assay in Chinese hamster V79 cells with the source substance was conducted according to OECD 476 guideline and GLP principles. It is concluded that Ammonium dihydrogen citrate in water is not mutagenic in the gene mutation assay based on the results of the source substance.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Micronucleus test: not mutagenic

Bone marrow chromosome aberration test: not mutagenic

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to a protocol that is similar to an appropriate OECD test guideline, with acceptable restrictions.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

Deviations:

yes

Remarks:

Limited details on test animals and environmental conditions, only 50 cells per animal were scored for aberrations.

GLP compliance:

no

Type of assay:

other: in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Flow Laboratories random-bred, closed colony
- Age at study initiation: 10 to 12 weeks
- Weight at study initiation: 280 to 350 g
- Assigned to test groups randomly: yes
- Fasting period before study: no data
- Housing: Rats were housed one to five to a cage
- Diet: Free access to commercial 4% fat diet
- Water: Free access to water
- Acclimation period: 4 - 11 days

ENVIRONMENTAL CONDITIONS
No data

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: physiol. saline
- Justification for choice of vehicle: no data

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The substance was suspended in 0.85% saline.

Duration of treatment / exposure:

6, 24 and 48 hours

Frequency of treatment:

Single dose

Dose / conc.:

1.2 mg/kg bw/day

Remarks:

test 1

Dose / conc.:

12 mg/kg bw/day

Remarks:

test 1

Dose / conc.:

120 mg/kg bw/day

Remarks:

test 1

Dose / conc.:

300 mg/kg bw/day

Remarks:

test 2

Dose / conc.:

500 mg/kg bw/day

Remarks:

test 2

Dose / conc.:

3 000 mg/kg bw/day

Remarks:

test 2

Dose / conc.:

3 500 mg/kg bw/day

Remarks:

test 2

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

Triethylene Melamine, 0.3 mg/kg bw.

Tissues and cell types examined:

Fifty metaphase spreads in bone marrow cells were scored per animal.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Based on toxicity test data.

DETAILS OF SLIDE PREPARATION: Four hours after the last substance administration, and two hours prior to killing, each animal was given 4 mg/kg bw colcemid intraperitoneally in order to arrest the bone marrow cells in c-mitosis. Animals were killed by using CO2, and the adhering muscle and epiphysis of one femur were removed. The cells were suspended in HBSS, centrifuged and resuspended in a hypotonic KCl solution. After incubation at 37°C, the cells were centrifuged and fixated in methanol/acetid acid and after overnight incubation resuspended and 2-3 drops of the suspension were allowed to drop onto a clean, dry slide and allowed to dry at room temperature overnight. The slides were stained using a 5% Giemsa solution, mounted and covered with a coverglass.

METHOD OF ANALYSIS: The specimens were scanned with 10x and 24x objectives and suitable metaphase spreads were examined using oil immersion flatfield apochromatic objectives. The chromosome of each cell were counted and only diploid cells were analyzed. Fifty metaphase spreads were scored per animal.

OTHER: Mitotic indices were obtained by counting at least 500 cells and the ratio of the number of cells in mitosis/the number of cells observed was expressed as the mitotic index.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
Dose range test 1: 100, 250, 500, 1000, 2000, 3000 mg/kg. One animal died at 500 mg/kg bw, 2 animals died at 1000 mg/kg bw and 3 animals died at 2000 and 3000 mg/kg bw. The LD50 was determined as 1700 mg/kg bw.
Dose range test 2: 5000 mg/kg, no deaths occurred. The LD50 was determined as >5000 mg/kg bw.

RESULTS OF DEFINITIVE STUDY
The substance produced no detectable significant aberration of the bone marrow metaphase chromosomes of rats.

Conclusions:

An in vivo bone marrow chromosome aberration study with Citric acid was performed equivalent to OECD 475 guideline, in male rats. It is concluded that the substance is not mutagenic in the in vivo bone marrow chromosome aberration assay.

Executive summary:

An in vivo bone marrow chromosome aberration test was performed with Citric acid equivalent to OECD 475 guideline without GLP.

Groups of 5 male rats were orally administered to the test substance up to and including 3500 mg/kg bw for 6, 24 and 48 hours in two independently performed experiments. Reliable vehicle controls and positive controls were included. No treatment-related increase in the number of cells with aberrations were observed. It is concluded that the substance is not mutagenic in the in vivo bone marrow chromosome aberration assay.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Type of information:

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

Adequacy of study:

key study

Study period:

2018

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Read-across information

Justification for type of information:

See the attached read-across document in section 13.

Reason / purpose for cross-reference:

read-across source

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

Results of Citric acid

Conclusions:

Based on the results of the source substances Citric acid, it is concluded that the target substance Ammonium dihydrogen citrate in water is not mutagenic in the in vivo bone marrow chromosome aberration test.

Reference 3

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

No data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: In this publication a chromosome aberration test was conducted using a similar OECDTG 474 protocol without GLP.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

Limited details on test animals and environmental conditions

GLP compliance:

not specified

Type of assay:

other: in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Species:

mouse

Strain:

other: ddY

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Shizuoka Agricultural Cooperative Association for Laboratory Animals, Shizuoka
- Age at study initiation: 8 weeks
- Weight at study initiation: no data
- Fasting period before study: no data
- Housing: no data
- Diet: Free access to food pellets CE2 (Japan Clea, Tokyo)
- Water: Free access to water
- Acclimation period: No data

ENVIRONMENTAL CONDITIONS
No data.

Route of administration:

intraperitoneal

Vehicle:

- Vehicle used: saline

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: No data.

Duration of treatment / exposure:

24 hour

Frequency of treatment:

single dose (pilot study) or 4 doses (main study), divided by 24 hour intervals

Post exposure period:

24 hours after dosing

Dose / conc.:

0 mg/kg bw (total dose)

Remarks:

Single dose

Dose / conc.:

62.5 mg/kg bw (total dose)

Remarks:

Single dose

Dose / conc.:

125 mg/kg bw (total dose)

Remarks:

Single dose

Dose / conc.:

250 mg/kg bw (total dose)

Remarks:

Single dose

Dose / conc.:

500 mg/kg bw (total dose)

Remarks:

Single dose

Dose / conc.:

0 mg/kg bw (total dose)

Remarks:

Four doses with 24 hour interval

Dose / conc.:

31.3 mg/kg bw (total dose)

Remarks:

Four doses with 24 hour interval

Dose / conc.:

62.5 mg/kg bw (total dose)

Remarks:

Four doses with 24 hour interval

Dose / conc.:

125 mg/kg bw (total dose)

Remarks:

Four doses with 24 hour interval

Dose / conc.:

250 mg/kg bw (total dose)

Remarks:

Four doses with 24 hour interval

No. of animals per sex per dose:

6

Control animals:

yes, concurrent vehicle

Positive control(s):

mitomycin C
- Route of administration: intraperitoneal
- Doses / concentrations: 2.0 mg/kg bw (single dose with 24 hour sampling time)

Tissues and cell types examined:

1000 polychromatic erythrocytes per mouse were scored and the number of micronucleated polychromatic erythrocytes (MNPCEs) was recorded.
The proportion of polychromatic erythrocytes (PCEs) among the total erythrocytes was also evaluated by observing 1000 erythrocytes.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
The maximum dose of the test substance were determined by a pilot experiment using the multi-sampling at multi-dose levels method (Hayashi et al. 1984).

DETAILS OF SLIDE PREPARATION:
Mice were killed by cervical dislocation at the appropriate time after an administration. Femoral marrow cells were flushed out with foetal bovine serum and smeared on clean glass slides. Cells were fixed with methanol for 5 minutes and stained with Acridine Orange for the pilot experiment and with Giemsa for the main test.

METHOD OF ANALYSIS: The MNPCE and PCE cells were scored using a light microscope with a high power objective (100x).

Evaluation criteria:

A positive result was recorded only when one or more treatment group(s) showed a statistically significant difference (p < 0.01) from the spontaneous level of MNPCEs and the Cochran-Armitage trend test indicated a positive dose response (p < 0.05).

Statistics:

The dose-response relationships were tested using the Cochran-Armitage trend test.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
No data.

RESULTS OF DEFINITIVE STUDY
The test substance showed no significant induction of micronuclei when compared the historical control data. See the attached illustration (picture/graph) for detailed results.

Conclusions:

An in vivo micronucleus study with Ammonium chloride was performed equivalent to OECD 474 guideline, in male mice. It is concluded that the substance is not mutagenic in the mouse micronucleus assay.

Executive summary:

An in vivo micronucleus test was performed with Ammonium chloride equivalent to OECD 474 guideline without GLP.

Groups of 6 male mice were administered to the test substance by intraperitoneal injection, either as single dose of up to and including 500 mg/kg bw or as 4 doses, divided by 24 -hour intervals at dose levels of up to and including 250 mg/kg bw. Reliable vehicle controls and positive controls (Mitomycin, 2 mg/kg bw) were included.

No deaths were observed. The test substance showed no significant induction of micronuclei when compared the historical control data.

It is concluded that the substance is not mutagenic in the mouse micronucleus assay.

Reference 4

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

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

Adequacy of study:

key study

Study period:

2018

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Read-across information

Justification for type of information:

See the attached read-across document in section 13.

Reason / purpose for cross-reference:

read-across source

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Results Ammonium chloride

Conclusions:

It is concluded that the substance is not mutagenic in the mouse micronucleus assay, based on the results of the source substance.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro data source substances:

Ames test:

An Ames test with Citric acid was performed equivalent to OECD 471 guideline without GLP.

One single direct plate experiment was performed with Salmonella typhimurium strains TA97, TA98, TA100 and TA104 up to and including 2000 µg/plate with and without S9 -mix. A reliable positive control with S9 -mix was included. Routine examination of the bacterial background lawn indicates the absence of toxicity. In the presence and absence of S9 -mix, the numbers of revertants in all tester strains for all concentrations tested were not significantly different from the respective negative controls.

Based on the results of this study it is concluded that Citric acid is not mutagenic in the Salmonella typhimurium assay with and without S9 -mix.

An Ames test with Citric acid was performed equivalent to OECD 471 guideline without GLP.

One single pre-incubation experiment was performed with Salmonella typhimurium strains TA92, TA94, TA1535, TA98, TA100 and TA1537 up to and including 5000 μg/plate with and without S9 -mix.

In the presence and absence of S9 -mix, no significant increases in the numbers of revertant colonies were detected in any Salmonella typhimurium strains at the maximum dose up to and including 5000 μg/plate.

Based on the results of this study it is concluded that Citric acid is not mutagenic in the Salmonella typhimurium assay with and without S9 -mix.

The mutagenic activity of Ammonium sulphate was evaluated in accordance with OECD 471 guideline. The test was performed in two independent experiments, experiment 1 a plate incorporation test and experiment 2 a pre-incubation test using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100. Dose levels up to and including 5000 μg/plate were tested in the absence and presence of S9-mix. Adequate positive controls and solvent controls were included.

No precipitation of the test item and no toxicity was observed up to and including the concentration of 5000 μg/plate.

No increase in revertant colony numbers of any of the four tester strains was observed following treatment with the substance at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). Based on the results, Ammonium sulphate is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

The mutagenic activity of Diammonium phosphate was evaluated in accordance with OECD 471 guideline and GLP principles.

The test was performed in two independent direct plate experiments, using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100, and Escherichia coli strain WP2uvrA. Dose levels up to and including 5000 μg/plate were tested in the absence and presence of S9-mix. Adequate positive controls and solvent controls were included.

In the Initial Toxicity Mutation Assay precipitation was obseved at concentrations of 1800 and 5000 μg/plate and in the Confirmatory Mutagenicity Assay precipitation was observed at the concentration of 5000 μg/plate. No toxicity was observed up to and including the concentration of 5000 μg/plate.

No increase in revertant colony numbers of any of the five tester strains was observed following treatment with the substance at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). Based on the results, Diammonium phosphate is considered to be non-mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Chromosome aberration test:

A chromosome aberration test was performed with Citric acid equivalent to OECD 473 guideline without GLP.

One single experiment was performed in a Chinese hamster fibroblast cell line with Citric acid concentrations up to and including 1000 mg/mL for 24 and 48 hr without S9 -mix. The maximum dose was selected by a preliminary test in which the dose needed for 50% cell-growth inhibition was estimated. No increase in the incidence of polyploid cells or in the incidence of cells with structural aberrations was observed at 24 or 48 hr after treatment.

Based on the results of this study it is concluded that Citric acid is not clastogenic in the chromosome aberration test with CHL cells without S9 -mix.

A chromosome aberration test was performed with Ammonium sulphate equivalent to OECD 473 guideline without GLP.

Two experiments were performed in human peripheral blood cells with an Ammonium sulphate concentration of 3.2 M (ca. 423 mg/mL) for 20 and 46 hr after start of the cultures without S9 -mix.

Ammonium sulphate did not induce a dose dependent significant increase in the frequency of chromosome aberrations in both treatment times compared with the untreated cultures. Also no increase in the incidence of polyploid cells was observed at 20 or 46 hr after the start of the cultures.

Based on the results of this study it is concluded that Ammonium sulphate is not clastogenic in the chromosome aberration test with human peripheral blood cells without S9 -mix.

Gene mutation study in mammalian cells:

A gene mutation assay in Chinese hamster V79 cells with the substance was conducted according to OECD 476 guideline and GLP principles.

Based on the results of the dose-range finding test, Ammonium sulphate was tested in two mutation assays. The first experiment was performed in the absence and presence of S9-mix with a 4 hour treatment

period. The second mutation experiment was performed in the absence of S9-mix with a 24 hour treatment period and in the presence of S9 -mix with a 4 hour treatment period. The following dose-range was selected for the mutagenicity tests in the absence and presence of S9-mix: 82.5, 165, 330, 660 and 1320 (~0.01 M) μg/mL exposure medium. Reliable solvent and positive controls were included.

No relevant cytotoxic effect as indicated by a relative cloning efficiency of less than 50% occurred up to and including of the maximum concentration of 1320 μg/mL with and without metabolic activation.

No relevant and reproducible increase in mutant colony numbers/10^6 cells was observed in the main experiments up to and including of the maximum concentration. It is concluded that the substance is not mutagenic in the gene mutation assay under the experimental conditions of the study.

In vivo data source substances:

Micronucleus test:

An in vivo micronucleus test was performed with Ammonium chloride equivalent to OECD 474 guideline without GLP.

Groups of 6 male mice were administered to the test substance by intraperitoneal injection, either as single dose of up to and including 500 mg/kg bw or as 4 doses, divided by 24 -hour intervals at dose levels of up to and including 250 mg/kg bw. Reliable vehicle controls and positive controls (Mitomycin, 2 mg/kg bw) were included.

No deaths were observed. The test substance showed no significant induction of micronuclei when compared the historical control data.

It is concluded that the substance is not mutagenic in the mouse micronucleus assay.

Bone marrow chromosome aberration test:

An in vivo bone marrow chromosome aberration test was performed with Citric acid equivalent to OECD 475 guideline without GLP.

Groups of 5 male rats were orally administered to the test substance up to and including 3500 mg/kg bw for 6, 24 and 48 hours in two independently performed experiments. Reliable vehicle controls and positive controls were included. No treatment-related increase in the number of cells with aberrations were observed. It is concluded that the substance is not mutagenic in the in vivo bone marrow chromosome aberration assay.

Justification for classification or non-classification

Based on the results of the in vitro and in vivo studies with the source substances, the substance does not need to be classified for genetic toxicity according to Regulation (EC) No. 1272/2008 and its amendments.