Magnesium glycerophosphate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

To evaluate the genotoxic properties of the test item following in vitro studies were performed:

1) In Vitro Bacterial Reverse Mutation Test (according to OECD 471)

2) In Vitro Mammalian Chromosomal Aberration Test (according to OECD 473)

3) In Vitro Mammalian Cell Gene Mutation Tests using the Hprt and xprt genes (according to OECD 476)

Based on the negative results of all three in vitro assays the test item is considered to be non mutagenic and non clastogenic.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-08-16 till 2017-09-12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Identification: Magnesium glycerophosphate
Batch: INVG003917
CAS No.: 972-20-8
EINECS No. / EC No.: Not available
Purity: 96.5% (w/w) Magnesium 2,3 hydroxypropyl phosphate
3.5% (w/w) water
The product can be regarded as a pure substance.
Physical State / Appearance: Solid white
Expiry Date: 10 June 2021
Storage Conditions: At room temperature, moisture protected, light protected*
Purpose of Use: Industrial chemical
Stability in solvent: Not indicated by the Sponsor

* only valid for storage condition, not for test performance

Species / strain / cell type:

other: TA 1535, TA 1537, TA 98, TA 100, and WP2 uvrA

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/Beta-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment II: 33; 100; 333; 1000; 2500; and 5000 µg/plate

Vehicle / solvent:

Solvent used: deionized water
Justification for choice of solvent: best suitable solvent, because of its solubility properties and its relative nontoxicity to the bacteria

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine, 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar plate incorporation; pre-incubation

DURATION:
Preincubation period: 60 Minutes
exposure duration: 72 hours

NUMBER OF REPLICATIONS: 3 plates for each concentration including the controls

DETERMINATION OF CYTOTOXICITY: Evident as a reduction in the number of spontaneous revertants (below the induction factor of 0.5) or a clearing of the bacterial background lawn.

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Key result

Species / strain:

other: TA 1535, TA 1537, TA 98, TA 100, and WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST SPECIFIC CONFOUNDING FACTORS
Effects of pH: none
Water solubility: soluble
Precipitation: The test item precipitated in the overlay agar in the test tubes in experiment I at 5000 µg/plate. In experiment II no precipitation of the test item was observed in the overlay agar in the test tubes. No precipitation of the test item was observed in the overlay agar on the incubated agar plates in both experiments.
Other confounding effects: none

COMPARISON WIT HISTORICAL CONTROL DATA: performed, no deviation
ADDITIONAL INFORMATION ON CYTOTOXICITY: No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation.

Summary of Experiment I

Study Name: 1855514	Study Code: Envigo 1855514
Experiment: 1855514 VV Plate	Date Plated: 16.08.2017
Assay Conditions:	Date Counted: 23.08.2017

Metabolic Activation	Test Group	Dose Level (per plate)		TA 1535 Revertant Colony Counts (Mean ±SD)	TA 1537 Revertant Colony Counts (Mean ±SD)	TA 98 Revertant Colony Counts (Mean ±SD)	TA 100 Revertant Colony Counts (Mean ±SD)	WP2 uvrA Revertant Colony Counts (Mean ±SD)
Without	Deionised water			10 ± 3	10 ± 3	29 ± 10	173 ± 24	40 ± 8
Activation	Untreated			13 ± 4	11 ± 4	33 ± 7	177 ± 10	38 ± 4
	Magnesium	3 µg		10 ± 2	9 ± 2	30 ± 3	193 ± 31	34 ± 10
	glycerophosphate	10 µg		12 ± 3	13 ± 2	33 ± 6	177 ± 6	36 ± 6
		33 µg		9 ± 2	9 ± 3	28 ± 7	169 ± 2	39 ± 4
		100 µg		13 ± 1	14 ± 4	22 ± 1	170 ± 8	48 ± 6
		333 µg		13 ± 4	8 ± 3	25 ± 6	179 ± 8	41 ± 10
		1000 µg		12 ± 5	12 ± 3	30 ± 6	182 ± 6	41 ± 6
		2500 µg		10 ± 2	10 ± 4	31 ± 4	175 ± 6	49 ± 2
		5000 µg		11 ± 1	10 ± 1	28 ± 8	188 ± 15	44 ± 3
	NaN3	10 µg		1205 ± 48			1873 ± 87
	4-NOPD	10 µg				345 ± 22
	4-NOPD	50 µg			91 ± 6
	MMS	2.0 µL						891 ± 15
With	Deionised water			11 ± 3	13 ± 3	38 ± 7	170 ± 20	46 ± 6
Activation	Untreated			17 ± 2	13 ± 5	40 ± 1	182 ± 7	58 ± 5
	Magnesium	3 µg		13 ± 6	17 ± 7	45 ± 2	186 ± 30	47 ± 0
	glycerophosphate	10 µg		13 ± 3	13 ± 3	42 ± 5	185 ± 19	51 ± 7
		33 µg		13 ± 6	17 ± 7	42 ± 5	175 ± 17	49 ± 4
		100 µg		15 ± 6	17 ± 6	39 ± 3	175 ± 9	53 ± 9
		333 µg		16 ± 5	17 ± 6	41 ± 4	163 ± 13	56 ± 8
		1000 µg		15 ± 4	13 ± 5	36 ± 6	159 ± 13	48 ± 9
		2500 µg		13 ± 3	17 ± 6	43 ± 6	158 ± 6	45 ± 5
		5000 µg		9 ± 2	15 ± 4	42 ± 6	165 ± 2	54 ± 11
	2-AA	2.5 µg		329 ± 36	141 ± 23	2964 ± 270	2908 ± 579
	2-AA	10.0 µg						416 ± 8

Summary of Experiment II

Study Name: 1855514	Study Code: Envigo 1855514
Experiment: 1855514 HV2 Pre	Date Plated: 06.09.2017
Assay Conditions:	Date Counted: 12.09.2017

Metabolic Activation	Test Group	Dose Level (per plate)		TA 1535 Revertant Colony Counts (Mean ±SD)	TA 1537 Revertant Colony Counts (Mean ±SD)	TA 98 Revertant Colony Counts (Mean ±SD)	TA 100 Revertant Colony Counts (Mean ±SD)	WP2 uvrA Revertant Colony Counts (Mean ±SD)
Without	Deionised water			11 ± 3	9 ± 3	27 ± 4	158 ± 14	45 ± 2
Activation	Untreated			9 ± 2	10 ± 2	23 ± 3	178 ± 13	42 ± 9
	Magnesium	33 µg		10 ± 1	9 ± 3	27 ± 2	178 ± 10	48 ± 2
	glycerophosphate	100 µg		13 ± 6	10 ± 1	29 ± 3	172 ± 20	50 ± 8
		333 µg		10 ± 3	8 ± 2	24 ± 3	174 ± 3	44 ± 6
		1000 µg		11 ± 2	9 ± 2	26 ± 2	190 ± 24	50 ± 7
		2500 µg		8 ± 2	10 ± 4	29 ± 10	181 ± 8	47 ± 9
		5000 µg		10 ± 4	9 ± 3	31 ± 3	160 ± 2	52 ± 4
	NaN3	10 µg		1358 ± 30			2003 ± 28
	4-NOPD	10 µg				483 ± 13
	4-NOPD	50 µg			94 ± 1
	MMS	2.0 µL						612 ± 77
With	Deionised water			13 ± 2	13 ± 3	40 ± 6	161 ± 14	56 ± 6
Activation	Untreated			10 ± 1	12 ± 4	43 ± 7	182 ± 8	53 ± 8
	Magnesium	33 µg		12 ± 2^U M	13 ± 2	37 ± 6	167 ± 13	54 ± 4
	glycerophosphate	100 µg		11 ± 4^U M	13 ± 2	40 ± 6	155 ± 18	65 ± 12
		333 µg		11 ± 3^U M	12 ± 3	40 ± 9	180 ± 11	59 ± 14
		1000 µg		12 ± 3^U M	14 ± 2	41 ± 1	172 ± 33	62 ± 4
		2500 µg		11 ± 3^U M	11 ± 1	41 ± 5	174 ± 4	61 ± 7
		5000 µg		8 ± 2^U M	11 ± 1	39 ± 8	181 ± 5	63 ± 5
	2-AA	2.5 µg		387 ± 12	113 ± 9	4119 ± 240	2455 ± 100
	2-AA	10.0 µg						426 ± 27

Key to Plate Postfix Codes:              

U: Air bubbles

M: Manual count

Key to positive controls:

NaN3: sodium azide

4 -NOPD: 4 -nitro-o-phenylene-diamine

MMS: methyl methane sulfonate

2-AA: 2 -aminoanthracene

Conclusions:

During the described mutagenicity test (Ames Test) and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
Therefore, Magnesium glycerophosphate is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

Magnesium glycerophosphate was assessed for its potential to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) usingSalmonella typhimuriumstrains TA 1535, TA 1537, TA 98, TA 100, and theEscherichia colistrain WP2 uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration and the controls were tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment/Experiment I:  3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment II:                           33; 100; 333; 1000; 2500; and 5000 µg/plate

The test item precipitated in the overlay agar in the test tubes in experiment I at 5000 µg/plate. In experiment II no precipitation of the test item was observed in the overlay agar in the test tubes. No precipitation of the test item was observed in the overlay agar on the incubated agar plates in both experiments.

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used.

 

No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation.

 

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Magnesium glycerophosphate at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

 

Appropriate reference mutagens were used as positive controls. They showed a distinct in­crease in induced revertant colonies.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

September 18, 2018 to February 13, 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

Adopted 29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: in vitro cytogenicity / chromosome aberration study

Specific details on test material used for the study:

Identification: Magnesium glycerophosphate
CAS No.: 972-20-8
The product can be regarded as a pure substance.
Physical State / Appearance: Solid white
Expiry Date: 10 June 2021
Storage Conditions: At room temperature, moisture protected, light protected*


* only valid for storage condition, not for test performance

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: National Institute of Cell Science, Pune, India
- Suitability of cells: Suitable
- Cell cycle length, doubling time or proliferation index: 16-18 hours
- Sex, age and number of blood donors if applicable:
- Whether whole blood or separated lymphocytes were used if applicable:
- Number of passages if applicable:
- Methods for maintenance in cell culture if applicable:
- Modal number of chromosomes: 21±2
- Normal (negative control) cell cycle time:

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:Minimal Essential Medium
- Properly maintained: [yes/no] YES
- Periodically checked for Mycoplasma contamination: [yes/no] YES
- Periodically checked for karyotype stability: [yes/no) YES
- Periodically 'cleansed' against high spontaneous background: [yes/no] YES

Cytokinesis block (if used):

colcemid (10 µg/mL)

Metabolic activation:

with and without

Metabolic activation system:

aroclor 1254 induced rat liver homogenate

Test concentrations with justification for top dose:

1 mg/mL culture media both in the absence (-S9) as well as in the presence of metabolic activation (+S9)
Justification: In the cytotoxicity experiment, the test concentration 2.0 (T3) mg/mL of culture media showed more than 55% reduction in RICC when compared to the respective negative control both in the presence or absence of metabolic activation.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Mimimal essential media (Culture Media)
- Justification for choice of solvent/vehicle:as it is the medium where cells were grown and test item soluble in the same medium

Untreated negative controls:

yes

Remarks:

Minimal Essential Medium (MEM0)

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk
- Cell density at seeding (if applicable):

DURATION
- Preincubation period:
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 21 hours
- Selection time (if incubation with a selection agent):
- Fixation time (start of exposure up to fixation or harvest of cells): 3 hours

SELECTION AGENT (mutation assays):

SPINDLE INHIBITOR (cytogenetic assays): colcemid

STAIN (for cytogenetic assays): Giemsa stain

NUMBER OF REPLICATIONS:02

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Droping Cell suspension

NUMBER OF CELLS EVALUATED: 1000

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 300

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:

DETERMINATION OF CYTOTOXICITY : mitotic index/ relative total growth
- Method: mitotic index, relative total growth
- Any supplementary information relevant to cytotoxicity: No

OTHER EXAMINATIONS:
- Determination of polyploidy:
- Determination of endoreplication:
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable):

- OTHER:

Evaluation criteria:

A test item can be classified as non - clastogenic if:
 the number of induced structural chromosome aberrations in all evaluated dose groups is within the range of the negative control.
A test item can be classified as clastogenic if:
 the number of induced structural chromosome aberrations will be increased at least 2-3 fold when compare to negative data and either a concentration-related or a significant increase of the number of structural chromosome aberrations is observed

Statistics:

Statistical significance at the p < 0.05 was evaluated by means of the two tailed – t test.
The result of treatment groups and positive controls in Phase I and II were compared with Negative control.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

As cytotoxicity observed at 2 mg/ ml concetration, the main study was conducted upto 1 mg/ ml concnetration

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

  

Statistical significance at the p < 0.05 was evaluated by means of the two tailed – t test.

The result of treatment groups and positive controls in Phase I and II were compared with Negative control.

Negative control Versus test group	Phase I - Number of aberrant cells
	-S9		+S9
	p value	Significance	p value	Significance
0.25 mg of Test item/mL Culture media (T1)	1.0000	-	0.6985	-
0.5 mg of Test item/mL Culture media (T2)	1.0000	-	0.2929	-
1 mg of Test item/mL Culture media (T3)	0.2929	-	0.4226	-
Positive Control	0.0025*	-	0.0018*	-

 

Negative control Versus test group	Phase II - Number of aberrant cells
	-S9		+S9
	p value	Significance	p value	Significance
0.25 mg of Test item/mL Culture media (T1)	1.0000	-	1.0000	-
0.5 mg of Test item/mL Culture media (T2)	1.0000	-	1.0000	-
1 mg of Test item/mL Culture media (T3)	0.2929	-	0.4226	-
Positive Control	0.0047*	-	0.0040*	-

*Significant at p ≤ 0.05 level with Negative control;

1.1                 

Treatment	Percent Aberrant Cells
	Phase I
	In the Absence of Metabolic Activation (-S9)		In the Presence of Metabolic Activation (1% S9)
	Mean	SD	Mean	SD
NC	0.333	0.471	0.333	0.471
T1 (0.25 mg/mL)	0.333	0.471	0.333	0.471
T2 (0.50 mg/mL)	0.333	0.471	0.667	0.000
T3 (1 mg/mL)	0.667	0.000	0.667	0.000
PC	10.000	0.943	11.333	0.943

 

Treatment	Percent Aberrant Cells
	Phase II
	In the Absence of Metabolic Activation (-S9)		In the Presence of Metabolic Activation (2% S9)
	Mean	SD	Mean	SD
NC	0.333	0.471	0.333	0.471
T1 (0.25 mg/mL)	0.333	0.471	0.333	0.471
T2 (0.50 mg/mL)	0.333	0.471	0.333	0.471
T3 (1 mg/mL)	0.667	0.000	0.667	0.000
PC	11.000	1.414	10.000	0.943

Key: NC = Negative Control, SD = Standard Deviation, PC = Positive Control

Conclusions:

under the experimental conditions and results observed, it is concluded that Magnesium glycerophosphate is “non-clastogenic” both in the presence (1% and 2%) and in the absence of metabolic activation.

Executive summary:

This study was conducted to determine the chromosomal aberration induction potential of Magnesium glycerophosphate in Chinese hamster Ovary cell lines. The methods followed were as per OECD guideline No. 473, adopted on 29thJuly 2016. The experiment was conducted using Chinese Hamster Ovary cell lines both in the presence and absence of metabolic activation system with 72 h grown cell cultures. The cells were treated with metaphase arresting substance (colcemid) 3 h prior to harvesting and stained. The metaphase cells were analysed microscopically and a minimum number of 1000 cells per slide were counted and number of metaphases were recorded in different fields to determine the mitotic index. The number of cells with aberrations were recorded (150 cells per slide) to calculate percent aberrant cells.

On the basis of solubility and precipitation test, Minimum Essential Eagle Media (MEM0) was selected as the vehicle for treatment. The pH of test item in culture medium was assessed at 0 h and 4 h after incubation at 37°C. No significant change in pH was observed at 0 h and 4 h when compared with negative controls. Hence, 2.0 mg/mL was selected as the highest concentration for cytotoxicity test.

Before conducting the chromosomal aberration study,Magnesium glycerophosphate was evaluated for cytotoxicity both in the absence and presence of metabolic activation system (1%). Cytotoxicity was assessed at the concentrations of 0.0 (NC), 0.5 (T1), 1.0 (T2) and 2.0 (T3) mg/mL of culture media. Cytotoxicity was observed in both absence and presence of metabolic activation (1%) at treated concentration T3 (2 mg/ml).

In the absence of S9 mix, the Relative Increase in Cell Count (RICC) in percentage observed was 100.00 (NC), 73.58 (T1), 53.46 (T2), 37.74 (T3) and 44.03 (PC). In the presence of S9 mix, the Relative Increase in Cell Count (RICC)in percentage observed was 100.0 (NC), 70.63 (T1), 54.38 (T2), 39.38 (T3) and 43.75 (PC). In the absence of S9 mix, the mean mitotic index observed was 10.06 (NC), 7.96 (T1), 6.08 (T2), 3.48 (T3) and 8.13 (PC). In the presence of S9 mix, the mean mitotic index observed was 10.29 (NC), 8.26 (T1), 6.34 (T2), 3.88 (T3) and 8.53 (PC). In the cytotoxicity experiment, the test concentration 2.0(T3) mg/mL of culture media showed more than 55% reduction in RICC when compared to the respective negative control both in the presence or absence of metabolic activation. Hence, the concentrations [1, 0.50 and 0.25 mg/mL] were selected for the main study. Hence, 1 mg/mL of test item was selected as the highest concentration for main study both in the presence and in the absence of metabolic activation. The main study was performed in two independent phases

 

Phase I

In the experiment, the cultures were exposed to Magnesium glycerophosphate for a short period of time (4 h) both in the absence and in the presence of metabolic activation system (1%). In the absence of S9 mix, theRelative Increase in Cell Count (RICC)in percentage observed was 100.0 (NC), 87.34 (T1), 74.68 (T2), 51.27 (T3) and 46.84 (PC). In the presence of S9 mix, theRelative Increase in Cell Count (RICC)in percentage observed was 100.0 (NC), 88.13 (T1), 74.38 (T2), 45.63 (T3) and 44.38 (PC).

During the treatment with test item in the absence and presence of S9 mix, there was no reduction in mitotic index observed at the tested concentrations.The observed mean mitotic indexin the absence of metabolic activation were 10.09, 9.01, 7.94, 6.39 and 8.27 and in the presence of metabolic activation were 10.12, 9.30, 8.33, 6.48 and 8.55 for0.0 (NC), 0.25 (T1), 0.50 (T2) and 1 (T3) mg/mLconcentrations, respectively.

There was no significant increase in the percentage of aberrant cells both in the absence and presence of metabolic activation when compared with negative control. However significant increase in the percentage of aberrant cells was observed in the positive control when compared with negative control.

The mean percentage of aberrant cells was 0.333 (NC), 0.333 (T1), 0.333 (T2), 0.667 (T3) and 10.000 (PC) in the absence of metabolic activation and 0.333 (NC), 0.333 (T1), 0.667 (T2), 0.667 (T3) and 11.333 (PC)in the presence of metabolic activation at the concentration of 0.0 (NC), 0.25 (T1), 0.5 (T2) and 1 (T3) mg/mL and positive controls, respectively.

Treatment with Mitomycin C at the concentration of 1.0 µg/mL in the absence of metabolic activation and Cyclophosphamidemonohydrate at the concentration of 5 µg/mL in the presence of metabolic activation (1%) causedsignificant increase in percent aberrant cells. Even though the analysis did not reveal any statistical significance, the increase was biologically significant.

 

Phase II

The phase II experiment was performed to confirm the negative results obtained in the absence and in the presence of metabolic activation in Phase I. In the Phase II, test item concentrations used were 0.0 (NC), 0.25 (T1), 0.50 (T2) and 1 (T3) mg/mLculture both in absence and presence of metabolic activation (2%). The duration of exposure to the test item in presence of metabolic activation system was 4 hours and in absence of metabolic activation the duration of exposure was 17 hours. In the absence of S9 mix, theRICCin percentage observed was 100.0 (NC), 86.79 (T1), 76.73 (T2), 61.64 (T3) and 44.65 (PC). In the presence of S9 mix, theRICCin percentage observed was 100.0 (NC), 87.04 (T1), 75.31 (T2), 67.90 (T3) and 45.68 (PC).

Treatment with test item in the absence and presence of S9 mix, did not show anyreduction in mitotic index at the tested concentrations. The observed mean mitotic indexin the absence of metabolic activation were 10.06, 8.75, 7.92, 6.02 and 8.24 andin the presence ofmetabolic activation were 10.28, 9.22, 8.55, 6.20 and 8.44 for0.0 (NC), 0.25 (T1), 0.5 (T2) and 1 (T3) mg/mLconcentrations, respectively.

The mean percent aberrant cells were 0.333 (NC), 0.333 (T1), 0.333 (T2), 0.667 (T3) and 11.000 (PC) in the absence of metabolic activation and 0.333 (NC), 0.333 (T1), 0.333 (T2), 0.667 (T3) and 10.000 (PC) in the presence of metabolic activation at the concentration of 0.0 (NC), 0.25 (T1), 0.50 (T2) and 1 (T3) mg/mL of culture and positive control, respectively.

Treatment with Mitomycin C at the concentration of 1.0 µg/mL in the absence of metabolic activation and Cyclophosphamidemonohydrate at the concentration of 5µg/mL in the presence of metabolic activation (2%) causedsignificant increase in percent aberrant cells.Though the analysis did not reveal any statistical significance, the increase was biologically significant.

The number of aberrations observed in the concurrent positive control groups (Phase I and II) demonstrated the sensitivity of the test system, suitability of the methods and conditions employed in the experiment.

Note: NC: Negative control; T1: Test concentration1; T2: Test concentration 2; T3: Test concentration 3; PC: Positive Control.

 

Conclusion

In conclusion, under the experimental conditions and results observed, it is concluded that Magnesium glycerophosphate is “non-clastogenic” both in the presence (1% and 2%) and in the absence of metabolic activation.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

December 26, 2017 to January 28, 2019

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:

adopted 29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: In vitro Mammalian Cell Gene Mutation Assay at the hypoxanthine-guanine phosphoribosyl transferase Locus (HPRT) In Chinese Hamster V79 Cells with Magnesium glycerophosphate

Specific details on test material used for the study:

Identification: Magnesium glycerophosphate
CAS No.: 972-20-8
The product can be regarded as a pure substance.
Physical State / Appearance: Solid white
Expiry Date: 10 June 2021
Storage Conditions: At room temperature, moisture protected, light protected*

* only valid for storage condition, not for test performance

Target gene:

HPRT

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: National Centre for Cell Science, Pune, INDIA
- Suitability of cells:The V79 cell line has been used successfully in the in vitro experiments for many years. Especially the high proliferation rate and good cloning efficiency of untreated cells (as a rule more than 50%) both necessary for appropriate performance of the study, recommended the use of this cell line. The Cells have a stable karyotype with a modal chromosome number of 22 (±3)
- Cell cycle length, doubling time or proliferation index:doubling time 13-16 hours
- Sex, age and number of blood donors if applicable:
- Whether whole blood or separated lymphocytes were used if applicable:
- Number of passages if applicable:
- Methods for maintenance in cell culture if applicable:
- Modal number of chromosomes:22±3
- Normal (negative control) cell cycle time:

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: DMEM medium
- 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:

AroclorTM 1254 induced liver homogenate (s9 fraction).

Test concentrations with justification for top dose:

The maximum recommended test concentration selected was 2.0 mg/mL for Magnesium glycerophosphate.
To evaluate the cytotoxicity of test item, six concentrations (2.0 mg/mL, 1.0 mg/mL, 0.5 mg/mL, 0.25 mg/mL, 0.125 mg/mL, and 0.062 mg/mL) were tested as there was no cytotoxicity observed at te highest tested dose i.e., 2.0 mg/mL, the same was selected for the highest test concentration

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMEM medium
- Justification for choice of solvent/vehicle: as test item disolved in the culture media hence the same was used as vehicle

Untreated negative controls:

yes

Remarks:

DFMEM Medium

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: In DMEM medium
- Cell density at seeding (if applicable):2x10^6

DURATION
- Preincubation period:
- Exposure duration:4 hours /24 hours
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 7days
- Fixation time (start of exposure up to fixation or harvest of cells): NA

SELECTION AGENT (mutation assays):6-thioguanine

SPINDLE INHIBITOR (cytogenetic assays):

STAIN (for cytogenetic assays):

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:

NUMBER OF CELLS EVALUATED:

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

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth; :

OTHER EXAMINATIONS:
- Determination of polyploidy:
- Determination of endoreplication:
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable):

- OTHER:

Evaluation criteria:

A test item is classified as positive if
• At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent vehicle control.
• The increase is concentration-related
Any of the results are outside the distribution of the historical negative control data.

Statistics:

The number of mutant colonies obtained for the groups treated with the test item was compared to the solvent control groups using Mann-Whitney test.
A trend is judged as significant whenever the p-value (probability value) is below 0.05.
However, both, biological relevance 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

Untreated negative controls validity:

valid

Positive controls validity:

valid

Calculations used for the study                      

PRE-TEST

Adjusted CE	=	CE x Number of cells at the end of treatment
		Number of cells at the beginning of treatment

 

CE	=	Number of colonies
		Number of cells seeded

 

RS	=	Adjusted CE in treated culture	x100
		Adjusted CE in the solvent control

MAIN EXPERIMENT

Cloning efficiency (CE) (survival, absolute)	=	Mean number of colonies per flask
		Number of cells seeded

 

Cloning efficiency (CE) (survival, relative)	=	Mean number of colonies per flask of treatment	x100
		Mean number of colonies per flask of vehicle control

 

Cell density (% of control)	=	Cell density at first sub cultivation	x100
		Cell density first sub cultivation of vehicle control

 

Mutant colonies/ 10^6cells	=	Cloning efficiency of mutant colonies in selective medium x10^6
		Cloning efficiency in non-selective medium

 

Induction Factor	=	Mutant colonies per 10^6cells
		Mutant colonies per 10^6cells of corresponding solvent control

SUMMARY OF RESULTS - TABLE

	Conc., mg/mL	Without S9 mix		Culture I				Culture II
				CE Relative Survival	CE Relative viability	Mutant colonies /10^6Cells	Induction factor	CE Relative Survival	CE Relative viability	Mutant colonies /10^6Cells	Induction factor
Phase I
NC	0	-	100		100	13.9	1.0	100	100	12.7	1.0
T1	0.25	-	93		94	11.8	0.85	96	94	12.1	0.95
T2	0.5	-	91		91	11.4	0.82	95	95	11.1	0.87
T3	1.0	-	90		93	12.9	0.93	93	94	12.4	0.98
T4	2.0	-	94		90	12.3	0.88	94	94	11.3	0.89
PC	150	-	77		72	122.4	8.81	80	82	104.6	8.24

	Conc., mg/mL	With S9 mix	Culture I				Culture II
			CE Relative Survival	CE Relative viability	Mutant colonies /10^6Cells	Induction factor	CE Relative Survival	CE Relative viability	Mutant colonies /10^6Cells	Induction factor
Phase I
NC	0	+	100	100	13.4	1.0	100	100	14.4	1.0
T1	0.25	+	95	96	11.7	0.87	95	97	13.1	0.91
T2	0.5	+	93	94	11.3	0.84	92	95	12.0	0.83
T3	1.0	+	96	91	13.0	0.97	92	93	14.1	0.98
T4	2.0	+	94	92	12.0	0.90	93	94	13.1	0.91
PC	1.3	+	81	74	642.3	47.93	82	79	594.1	41.28

Key:NC = Negative control, CE = Cloning Efficiency, PC= Positive control, T=Test concentrations

	Conc., mg/mL	Without S9 mix	Culture I				Culture II
			CE Relative Survival	CE Relative viability	Mutant colonies /10^6Cells	Induction factor	CE Relative Survival	CE Relative viability	Mutant colonies /10^6Cells	Induction factor
Phase II
NC	0	-	100	100	13.6	1.0	100	100	12.5	1.0
T1	0.25	-	95	95	12.9	0.95	93	97	12.0	0.96
T2	0.5	-	90	91	13.4	0.99	94	96	10.8	0.86
T3	1.0	-	93	93	11.7	0.86	91	95	11.6	0.93
T4	2.0	-	89	90	13.1	0.96	88	94	12.2	0.98
PC	150	-	78	76	147.4	10.84	78	88	132.6	10.61

Key:NC = Negative control, CE = Cloning Efficiency, PC= Positive control, T=Test concentrations

 

	Conc., mg/mL	With S9 mix	Culture I				Culture II
			CE Relative Survival	CE Relative viability	Mutant colonies /10^6Cells	Induction factor	CE Relative Survival	CE Relative viability	Mutant colonies /10^6Cells	Induction factor
Phase II
NC	0	+	100	100	11.8	1.0	100	100	12.5	1.0
T1	0.25	+	94	94	11.1	0.94	95	98	11.5	0.92
T2	0.5	+	90	95	10.5	0.89	91	96	11.0	0.88
T3	1.0	+	92	92	11.6	0.98	92	95	11.9	0.95
T4	2.0	+	89	88	10.8	0.92	88	97	12.1	0.97
PC	1.3	+	84	77	495.7	42.01	77	91	423.2	33.86

Key:NC = Negative control, CE = Cloning Efficiency, PC= Positive control, T=Test concentrations

Conclusions:

In conclusion it is stated that during the mutagenicity test described and under the experimental conditions reported, the test item did not induce mutations at HPRT locus in V79 cells of the Chinese hamster both in absence and presence of metabolic activation system. Therefore Magnesium glycerophosphate was considered to be “non-mutagenic”in this HPRT assay.

Executive summary:

This study was conducted to investigate the potential of Magnesium glycerophosphate to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster according to OECD guideline No. 476, adopted on29thJuly 2016. The assay was performed in two independent experiments, using two parallel cultures each. The experiment I was performed with and without liver microsomal activation at a treatment period of 4 hours. Experiment II was performed for a treatment period of 24 hours without metabolic activation. 2.0 mg/mL concentration was chosen as the highest dose for the cytotoxicity experiment, based on the solubility and precipitation properties of the test item. The following concentrations were selected for the cytotoxicity experiment 2.0 mg/mL, 1.0 mg/mL, 0.5 mg/mL, 0.25 mg/mL, 0.125 mg/mL and 0.062 mg/mL both in the presence and absence of metabolic activation.

No relevant cytotoxic effect was observed as indicated by the Relative survival (RS)i.e.,cloning efficiency (CE) of cells plated immediately after treatment, adjusted by any loss of cells during treatment, based on cell count, as compared with adjusted cloning efficiency in negative controls (assigned a survival of 100%) with the RS for the test item. The RS of the other tested concentrations were found to be more than 50% and hence the doses were selected for the main experiment (Phase-I and Phase-II) are2.0 mg/mL, 1.0 mg/mL, 0.5 mg/mL, and 0.25 mg/mL. In both the cultures, there was no distinct increase in the mutant frequency of Magnesium glycerophosphate in the all tested concentrations, when compared to the respective negative control for phase I and phase II both in the presence and absence of metabolic activation system. No significant and reproducible dose dependent increase in mutant colony numbers was observed either in the Phase I or Phase II of the experiment. The positive controls used, EMS in the absence of metabolic activation and DMBA in the presence of metabolic activation, revealed a significant increase in mutant colonies and the induction factor is more than three times of negative control indicating that the sensitivity of the test system used and validity of the experiment.

Note: PC: Positive Control; EMS: (ethyl methanesulfonate); DMBA:(Dimethyl benz(a)anthracene).

Conclusion

In conclusion it is stated that during the mutagenicity test described and under the experimental conditions reported, the test item did not induce mutations at HPRT locus in V79 cells of the Chinese hamster both in absence and presence of metabolic activation system.

Therefore Magnesium glycerophosphate was considered to be “non-mutagenic”in this HPRT assay

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Experimental data of the in vitro bacterial mutagenicity test (Ames Test) and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore Magnesium glycerophosphate is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Furthermore an in vitro study in mamalian cells was performed to assess the potential of the test item to induce gene mutations using the Chinese hamster V79 cell lines in the presence and absence of metabolic ativation.This in vitro test is an assay for the detection of forward gene mutations in mammalian cells. During the mutagenicity test described and under the experimental conditions reported, the test item did not induce mutations at HPRT locus in V79 cells of the Chinese hamster both in absence and presence of metabolic activation system. Therefore, Magnesium glycerophosphatewas considered to be “non-mutagenic”in this HPRT assay.

An in vitro cytogenicity assay was performed to determine the potential of the test item to induce chromosomal abberration in chinese hamster ovary cell lines in the presence and absence of metabolic ativation. This in vitrotest is an assay for the detection of structural and numerical chromosomal aberrations. In conclusion, under the experimental conditions and results observed, it is concluded that Magnesium glycerophosphate is “non-clastogenic" both in the presence and absence of metabolic activation.

Justification for classification or non-classification

no classification

Taken together based on in vitro bacterial reverse mutation test, In Vitro Mammalian Chromosomal Aberration Test and In Vitro Mammalian Cell Gene Mutation Tests using the Hprt gene the test item is considered to be non mutagenic and non clastogenic.