Magnesium carbonate hydroxide

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:

14/10/2020 - 18/11/2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Magnesium carbonate hydroxide

EC Number:

235-192-7

EC Name:

Magnesium carbonate hydroxide

Cas Number:

12125-28-9

Molecular formula:

Mg5(CO3)4(OH)2(H2O)4

IUPAC Name:

Magnesium carbonate hydroxide

Test material form:

solid: particulate/powder

Method

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 pKM 101

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:

- source of S9 : male Wistar rats induced with intraperitoneal injection of sodium phenobarbitone and β-naphthoflavone at 16 mg/mL and 20 mg/mL, respectively, for 3 days prior to sacrifice.

- method of preparation of S9 mix: Each batch of S9 homogenate was assessed for sterility by streaking the supernatant fluid on Nutrient Agar plates and incubated at 37±1ºC for 24 hours. It was found sterile and was further evaluated for its protein content (Modified Lowry Assay, Sword and Thomson, 1980) and for its ability to metabolize the promutagens 2-Aminoanthracene and Benzo(a)pyrene to mutagens using Salmonella typhimurium TA100 tester strain. The results were found to be acceptable for the tested parameters.

- concentration or volume of S9 mix and S9 in the final culture medium: A volume of 1 mL of S9 homogenate was thawed immediately before use and mixed with 9 mL of co-factor solution containing 4 mM Nicotinamide Adenine Dinucleotide Phosphate (NADP) disodium salt, 5 mM Glucose-6-phosphate, 8 mM MgCl2 and 33 mM KCl in Phosphate Buffer Saline (PBS) of pH 7.34 for initial cytotoxicity, plate incorporation and preincubation method to get the concentration of 10% (v/v).

- quality controls of S9: Control plates with S9 mix, PBS, 5 mg/plate of test item, soft agar, DMSO and MGA plate incubated at 37±1ºC for 48 hours and 3 minutes. The plates were maintained on the day of conduct of plate incorporation and preincubation method.

Test concentrations with justification for top dose:

Based on the results of precipitation test, an initial cytotoxicity test was conducted for the selection of test concentration for the mutation assay. Salmonella typhimurium TA100 tester strain was exposed to concentrations of 0.00625, 0.0125, 0.025, 0.05, 0.1, 0.2, 0.4, 0.8, 1.6, 3.2 and 5 mg/plate of test item in triplicate, both in the presence and absence of metabolic activation along with concurrent vehicle control (DMSO). Each concentration of test item was mixed with soft agar containing histidine and biotin, S9 mix (for presence of metabolic activation), PBS (for absence of metabolic activation), Salmonella typhimurium TA100 of cell density approximately 18×108 cells/mL overlaid on to pre-labeled minimal glucose agar plates. The plates were incubated at 37±1ºC for 64 hours and 25 minutes.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used:DMSO
- Justification for choice of solvent/vehicle: The test item found insoluble in distilled water, ethanol and acetone. The test item formed the suspension in DMSO at 50 mg/mL.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-Aminoanthracene (Concentration (ug/plate): 4)

Details on test system and experimental conditions:

- The strain battery that was approved under the OECD Guideline No. 471, “Bacterial Reverse Mutation Test”, adopted on 26th June 2020 allows the use of the following strains of bacteria:
Histidine auxotrophic strains of Salmonella typhimurium and tryptophan auxotrophic strains of Escherichia coli viz.,
a) TA98 and TA1537 : Frame shift mutation
b) TA100, TA1535 and E. coli WP2 uvrA (pKM101) : Base pair substitution

- Justification for Selection of Test System: Salmonella typhimurium TA98, TA100, TA1535, TA1537 and Escherichia coli WP2 uvrA (pKM101) are the recommended strains by regulatory agencies for conducting Bacterial Reverse Mutation Test.

- Source of the Test System: Salmonella typhimurium TA98, TA100, TA1535, TA1537 and Escherichia coli WP2 uvrA (pKM101) were procured from Molecular Toxicology, Inc. PO Box 1189 Boone, NC 28607 USA.

- Storage of the Test System: Stock cultures of tester strains in oxoid nutrient broth no. 2 were stored in the test facility as frozen permanents in -80±10ºC. Laboratory stocks of each strain were maintained on minimal glucose agar as master plates. These master plates were stored in a refrigerator between 2 to 8ºC for 3 months.

- Genetic Characterization of Tester Strains: After preparation of the master plates, the growth requirements and the genetic identity of Salmonella typhimurium strains like histidine requirement, sensitivity to UV radiation and resistance of strains TA98, TA100, TA1535 and TA1537 to ampicillin and rfa mutation of Salmonella typhimurium strains were checked along with the range of spontaneous revertants. Similarly the growth requirements and the genetic identity of strains like tryptophan requirement, sensitivity to UV radiation and resistance of Escherichia coli WP2 uvrA (pKM101) to ampicillin were checked along with the range of spontaneous revertants and results were acceptable.

Evaluation criteria:

The conditions necessary for determining a positive result are, there should be a dose related increase in the mean revertants per plate of at least that one tester strain over a minimum of two increasing doses of the test item either in the presence or absence of the metabolic activation system.
The test will be judged positive if the increase in mean revertants at the limit dose tested is equal to or greater than 2 times the mean vehicle control value in Salmonella typhimurium strains TA98, TA100 and Escherichia coli WP2 uvrA (pKM101) or equal to or greater than 3 times the mean vehicle control value in tester strains TA1535 and TA1537.
An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose responsive increase that does not achieve respective threshold cited above or a non-dose responsive increase that is equal to or greater than the respective threshold cited. A response will be evaluated as negative, if it is neither positive nor equivocal.
Based on the above interpretation, the test item can be evaluated as negative.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

not determined

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

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

not determined

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

Test results 3

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

not determined

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

Test results 4

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

not determined

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

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

not determined

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

Applicant's summary and conclusion

Conclusions:

Based on the results of the study it is concluded that the test item Magnesium Carbonate Hydroxide is “non-mutagenic” in the Bacterial Reverse Mutation Test up to the highest tested concentration of 5 mg/plate under the test conditions when tested on Salmonella typhimurium TA98, TA100, TA1535, TA1537 and E.coli WP2 uvrA (pKM101) tester strains.

Executive summary:

The test item Magnesium Carbonate Hydroxide was assayed for bacterial reverse mutation test at the concentrations of 0.05, 0.16, 0.5, 1.6 and 5 mg/plate for plate incorporation method and for preincubation method using Salmonella typhimurium TA98, TA100, TA1535, TA1537 and E.coli WP2 uvrA (pKM101) tester strains.

In the two trials conducted, the test item concentrations tested resulted in no appreciable increase in the number of revertant colonies over the vehicle control, while the positive controls tested simultaneously, resulted in 3.7 to 15.8 fold increase in the number of revertant colonies/plate under identical conditions.

This was observed for all the five tester strains.

Based on the results of the study it is concluded that the test item Magnesium Carbonate Hydroxide is “non-mutagenic” in the Bacterial Reverse Mutation Test up to the highest tested concentration of 5 mg/plate under the test conditions when tested on Salmonella typhimurium TA98, TA100, TA1535, TA1537 and E.coli WP2 uvrA (pKM101) tester strains.