Neutralisation product of iron oxide hydroxide, phosphoric acid, phosphonic acid, lithium hydroxide or lithium carbonate, D-Glucose, 4-O-β-D-galactopyranosyl-, hydrate (1:1) and amylopectin

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:

Mar 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

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 Jul 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Rheinland-Pfalz

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Neutralisation product of iron oxide hydroxide, phosphoric acid, phosphonic acid, lithium hydroxide or lithium carbonate, D-Glucose, 4-O-β-D-galactopyranosyl-, hydrate (1:1) and amylopectin

EC Number:

942-575-9

Molecular formula:

not available

IUPAC Name:

Neutralisation product of iron oxide hydroxide, phosphoric acid, phosphonic acid, lithium hydroxide or lithium carbonate, D-Glucose, 4-O-β-D-galactopyranosyl-, hydrate (1:1) and amylopectin

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: 21747297V09
- Purity: 100% UVCB (UVCB = Substance of Unknown or Variable composition)
- Content water: 0.3 g/100 g

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature (protected against humidity)
- Stability under test conditions: The stability of the test substance under storage conditions was guaranteed until 22 Jan 2018 as indicated by the sponsor, and the sponsor holds this responsibility
- Stability of the test substance in the solvent/vehicle: Due to the use of water as vehicle the verification of the stability of the test substance in the vehicle was not required.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test substance was weighed and topped up with the chosen vehicle to achieve the required concentration of the stock solution. The test substance was suspended in ultrapure water. To achieve homogeneity of the test substance in the vehicle, the test substance preparation was shaken thoroughly. The further concentrations were diluted according to the planned doses. To keep the test substance homogeneously in the vehicle, the test substance preparation was carefully pipetted before removal. All test substance formulations were prepared immediately before administration.

FORM AS APPLIED IN THE TEST (if different from that of starting material): suspended in ultrapure water

Method

Target gene:

his, trp

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

S9 mix (supplemented with cofactors) derived from phenobarbital (i.p.) and β-naphthoflavone (orally) induced rat liver

Test concentrations with justification for top dose:

0; 33; 100; 333; 1000; 2500 and 5000 μg/plate (SPT)
0; 33; 100; 333; 1000; 2500 and 5000 μg/plate (PIT)

The maximum dose was tested even in the case of relatively insoluble test compounds to detect possible mutagenic impurities.

3 test plates per dose or per control

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ultrapure water
- Justification for choice of solvent/vehicle: In comparison to other commonly used vehicles (e.g. DMSO, acetone) water is the most suitable.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

other: 2-aminoanthracene (60 μg/plate (E.coli) or 2.5 µg/plate (Salmonella strains), dissolved in DMSO; with S9 mix)

Remarks:

Sterility control: Additional plates were treated with soft agar, S9 mix, buffer, vehicle and the test substance but without the addition of tester strains.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation
- Cell density at testing: approx. 10^9 cells per mL

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 – 72 hours

DETERMINATION OF CYTOTOXICITY
- Toxicity detected by a
• decrease in the number of revertants (factor ≤ 0.6)
• clearing or diminution of the background lawn (= reduced his- or trp- background growth)
was recorded for all test groups both with and without S9 mix in all experiments. Single values with a factor ≤ 0.6 were not detected as toxicity in low dose groups.

Evaluation criteria:

Acceptance criteria
The experiment was considered valid if the following criteria were met:
• The number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain.
• The sterility controls revealed no indication of bacterial contamination.
• The positive control substances both with and without S9 mix induced a distinct increase in the number of revertant colonies within the range of the historical positive control data or above.
• Fresh bacterial culture containing approximately 10^9 cells per mL were used.

Assessment criteria
The test substance was considered positive in this assay if the following criteria were met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. at least doubling (bacteria strains with high spontaneous mutation rate, like TA 98, TA 100 and E.coli WP2 uvrA) or tripling (bacteria strains with low spontaneous mutation rate, like TA 1535 and TA 1537) of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.
A test substance was generally considered non-mutagenic in this test if:
• The number of revertants for all tester strains were within the range of the historical negative control data under all experimental conditions in at least two experiments carried out independently of each other.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Homogeneity: The homogeneity of the test substance was ensured by mixing before preparation of the test substance solutions.
- Precipitation: Test substance precipitation was found from about 2500 μg/plate onward with and without S9 mix.

Applicant's summary and conclusion

Conclusions:

Under the experimental conditions chosen here, it is concluded that the test substance is not mutagenic in the bacterial reverse mutation test in the absence and the presence of metabolic activation.

Executive summary:

The test substance was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay.

STRAINS: TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA

DOSE RANGE: 33 μg - 5000 μg/plate (SPT)

33 μg - 5000 μg/plate (PIT)

TEST CONDITIONS: Standard plate test (SPT) and preincubation test (PIT) both with and without metabolic activation (liver S9 mix from induced rats).

SOLUBILITY: Precipitation of the test substance was found from about 2500 μg/plate onward with and without S9 mix.

TOXICITY: No bacteriotoxic effect was observed under all test conditions.

MUTAGENICITY: A relevant increase in the number of his+ or trp+ revertants (factor ≥ 2: TA 100, TA 98 and E.coli WP2 uvrA or factor ≥ 3: TA 1535 and TA 1537) was not observed in the standard plate test or in the preincubation test without S9 mix or after the addition of a metabolizing system.