(2-hydroxypropyl)ammonium citrate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 Apr to 10 May 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

July 21, 1997

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

31 May 2008

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

(2-hydroxypropyl)ammonium citrate

EC Number:

250-342-1

EC Name:

(2-hydroxypropyl)ammonium citrate

Cas Number:

30798-32-4

Molecular formula:

C6H8O7.xC3H9NO

IUPAC Name:

tris(2-hydroxypropan-1-aminium) citrate

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source of test material: Zschimmer and Schwarz GmbH + Co. KG.
- Identification: 1739 MIPA-CITRAT
- Batch: 207459-01
- Purity/Composition: UVCB

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Test item storage: At room temperature
- Stable under storage conditions until: 13 January 2018 (expiry date)
- Solubility in vehicle: Water: Not indicated; Dimethyl sulfoxide: Not indicated
- Stability in vehicle: Water: Stable; Dimethyl sulfoxide: Not indicated

OTHER SPECIFICS
- Appearance: Clear yellow liquid
- Purity/Composition correction factor: Yes, according to the purity: 1.49

Method

Target gene:

- S. typhimurium: his
- E. coli: trp

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9 mix from Aroclor 1254 (i.p.) induced rats livers

Test concentrations with justification for top dose:

DOSE RANGE FINDING TEST
Strains TA100 and the WP2uvrA, with and without S9-mix: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate

EXPERIMENT 1 (DIRECT PLATE ASSAY)
- Strains TA98, TA1535 and TA1537, with and without S9-mix: 52, 164, 512, 1600, 5000 μg/plate

EXPERIMENT 2 (PRE-INCUBATION ASSAY)
- Strains TA98, TA100, TA1535, TA1537 and WP2 uvrA, with and without S9-mix: 52, 164, 512, 1600, 5000 μg/plate

Vehicle / solvent:

DOSE RANGE FINDING TEST and EXPERIMENT 1 and 2
- Vehicle used: Milli-Q water

REPEAT EXPERIMENT 2
- Vehicle used: dimethyl sulfoxide

JUSTIFICATION
The use of two solvents has no effect on the study integrity.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Milli-Q water or dimethyl sulfoxide

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: see section "Any other information on materials and methods incl. tables"

Details on test system and experimental conditions:

METHOD OF APPLICATION
- DOSE-RANGE FINDING TEST and EXPERIMENT 1: in agar (plate incorporation)
- EXPERIMENT 2: preincubation

DURATION
- Preincubation period (EXPERIMENT 2 only): 30 minutes by 70 rpm at 37°C
- Exposure duration: 48 ± 4 h in the dark at 37.0 ± 1.0°C

NUMBER OF REPLICATIONS
3 plates/dose

COLONY COUNTING
Revertant colonies (histidine independent (His+) for Salmonella typhimurium bacteria and tryptophan independent (Trp+) for Escherichia coli) were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test item precipitate to interfere with automated colony counting were counted manually. Evidence of test item precipitate on the plates and the condition of the bacterial background lawn were evaluated when considered necessary, macroscopically and/or microscopically by using a dissecting microscope.

Evaluation criteria:

see section "Any other information on materials and methods incl. tables"

Results and discussion

Test results

Test results 1

Species / strain:

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

Remarks:

Dose range Experiment and Experiment 1: Direct Plate Assay

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

Remarks:

Dose range Experiment: Direct Plate Assay

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

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

Remarks:

Experiment 2: Pre-Incubation Assay

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

E. coli WP2 uvr A

Remarks:

Experiment 2: Pre-Incubation Assay

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 100

Remarks:

Experiment 2: Pre-Incubation Assay

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 6

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity, as evidenced by a reduction in the bacterial background lawn, was only observed in tester strain TA100 in the presence of S9-mix at the highest tested concentration.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation, Direct Plate Assay: Precipitation of the test item on the plates was not observed at the start or at the end of the incubation period.
- Precipitation, Pre-Incubation Assay: Precipitation of the test item on the plates was not observed at the start or at the end of the incubation period.