Calcium glycerophosphate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item Calcium 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 at 5000 µg/plate. Precipitation of the test item in the overlay agar on the incubated agar plates was observed at 5000 µg/plate. The undissolved particles had no influence on the data recording.

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.

 

One single minor toxic effect, evident as a reduction in the number of revertants (below the indication factor of 0.5), was observed in experiment I in strain TA98 without S9 mix at 5000 µg/plate. No other toxic effects occurred in in the remaining test groups neither with nor and without metabolic activation.

 

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Calcium 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.

Link to relevant study records

Reference

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 2017-08-30

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: guideline-conform study under GLP without deviations

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:

IIdentification: Calcium glycerophosphate
Batch: CGP0416003
CAS No.: 27214-00-2
EINECS No. / EC No.: Not available
Purity: 85.9% (w/w) Calcium 2,3-dihydroxypropyl phosphate
5.8% (w/w)Calcium 1,3-dihydroxypropyl phosphate
8.3% Water
The product can be regarded as a pure substance
Physical State / Appearance: Solid white
Stability in solvent: Not indicated by the Sponsor
Expiry Date: 29 June 2021
Storage Conditions: At room temperature, moisture protected, light protected*
Purpose of Use: Industrial chemical

* only valid for storage condition, not for test performance
No correction for purity was made.

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: DMSO
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: 600 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:

cytotoxicity

Remarks:

Strain TA 98 without S9 mix

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: not soluble
Precipitation: The test item precipitated in the overlay agar in the test tubes at 5000 µg/plate. Precipitation of the test item in the overlay agar on the incubated agar plates was observed at 5000 µg/plate. The undissolved particles had no influence on the data recording.
Other confounding effects: none

COMPARISON WIT HISTORICAL CONTROL DATA: performed; no deviations
ADDITIONAL INFORMATION ON CYTOTOXICITY: experiment I: strain TA98 without S9 mix at 5000 µg/plate.

Remarks on result:

other: reverse mutation assay migrated from the field Test System

Summary of Experiment I

Study Name: 1855513	Study Code: Envigo 1855513
Experiment: 1855513 VV Plate	Date Plated: 16.08.2017
Assay Conditions:	Date Counted: 21.08.2017

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	DMSO		11 ± 4	10 ± 1	25 ± 2	200 ± 7	34 ± 5
Activation	Untreated		10 ± 3	10 ± 5	24 ± 4	195 ± 23	32 ± 6
	Calcium	3 µg	11 ± 3	11 ± 1	21 ± 1	170 ± 8	35 ± 6
	glycerophosphate	10 µg	10 ± 4	11 ± 3	19 ± 3	156 ± 13	32 ± 1
		33 µg	11 ± 4	11 ± 5	21 ± 8	173 ± 25	32 ± 6
		100 µg	9 ± 2	10 ± 1	25 ± 2	173 ± 11	33 ± 3
		333 µg	9 ± 2	11 ± 5	25 ± 2	163 ± 27	33 ± 2
		1000 µg	8 ± 4	11 ± 4	24 ± 7	181 ± 6	33 ± 7
		2500 µg	11 ± 4	8 ± 5	23 ± 5	181 ± 7	29 ± 2
		5000 µg	12 ± 3^P M	7 ± 3^P M	11 ± 4^P M	170 ± 8^P	32 ± 3^P
	NaN3	10 µg	1104 ± 35			1736 ± 257
	4-NOPD	10 µg			417 ± 21
	4-NOPD	50 µg		95 ± 7
	MMS	2.0 µL					762 ± 25
With	DMSO		12 ± 2	14 ± 3	35 ± 5	165 ± 15	51 ± 5
Activation	Untreated		16 ± 4	15 ± 5	43 ± 3	175 ± 6	45 ± 2
	Calcium	3 µg	12 ± 4	12 ± 3	42 ± 2	171 ± 12	50 ± 6
	glycerophosphate	10 µg	16 ± 5	18 ± 3	38 ± 2	162 ± 3	44 ± 6
		33 µg	14 ± 6	12 ± 2	43 ± 14	156 ± 8	41 ± 15
		100 µg	9 ± 2	19 ± 4	44 ± 7	153 ± 6	42 ± 12
		333 µg	14 ± 4	24 ± 3	44 ± 4	154 ± 9	41 ± 10
		1000 µg	16 ± 2	16 ± 3	40 ± 3	156 ± 8	56 ± 11
		2500 µg	15 ± 6	19 ± 3	40 ± 10	129 ± 26	49 ± 1
		5000 µg	8 ± 2^P M	9 ± 3^P M	21 ± 5^P M	140 ± 7^P	55 ± 11^P
	2-AA	2.5 µg	384 ± 5	169 ± 14	3756 ± 752	2808 ± 53
	2-AA	10.0 µg					430 ± 21

Summary of Experiment II

Study Name: 1855513	Study Code: Envigo 1855513
Experiment: 1855513 HV2 Pre	Date Plated: 23.08.2017
Assay Conditions:	Date Counted: 30.08.2017

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	DMSO		12 ± 2	10 ± 4	26 ± 3	112 ± 16	37 ± 1
Activation	Untreated		10 ± 2	12 ± 2	23 ± 3	156 ± 20	40 ± 7
	Calcium	33 µg	15 ± 1	11 ± 4	32 ± 6	98 ± 7	38 ± 4
	glycerophosphate	100 µg	14 ± 3	7 ± 2	22 ± 6	105 ± 4	42 ± 5
		333 µg	11 ± 4	10 ± 1	32 ± 2	114 ± 8	37 ± 3
		1000 µg	14 ± 2	11 ± 2	25 ± 7	94 ± 19	42 ± 6
		2500 µg	11 ± 3	10 ± 2	27 ± 1	110 ± 1	43 ± 4
		5000 µg	11 ± 1^P	6 ± 4^P	29 ± 6^P	106 ± 3^P	42 ± 9^P
	NaN3	10 µg	1035 ± 64			1932 ± 93
	4-NOPD	10 µg			422 ± 39
	4-NOPD	50 µg		101 ± 3
	MMS	2.0 µL					667 ± 57
With	DMSO		10 ± 6	13 ± 3	34 ± 7	100 ± 24	48 ± 5
Activation	Untreated		9 ± 2	13 ± 3	50 ± 2	129 ± 7	52 ± 6
	Calcium	33 µg	10 ± 4	12 ± 3	37 ± 11	93 ± 9	47 ± 9
	glycerophosphate	100 µg	10 ± 1	14 ± 2	31 ± 6	103 ± 19	54 ± 6
		333 µg	11 ± 4	16 ± 2	42 ± 13	91 ± 18	50 ± 11
		1000 µg	7 ± 2	18 ± 2	28 ± 6	101 ± 12	45 ± 10
		2500 µg	10 ± 1	14 ± 5	37 ± 9	93 ± 11	55 ± 3
		5000 µg	12 ± 2^P	15 ± 3^P	36 ± 5^P	115 ± 19^P	57 ± 1^P
	2-AA	2.5 µg	310 ± 37	105 ± 6	3738 ± 713	2456 ± 474
	2-AA	10.0 µg					393 ± 15

Key to Plate Postfix Codes:              

P: Precipitate

M: Manuel 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, Calcium glycerophosphate is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

The test item Calcium 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 at 5000 µg/plate. Precipitation of the test item in the overlay agar on the incubated agar plates was observed at 5000 µg/plate. The undissolved particles had no influence on the data recording.

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.

 

One single minor toxic effect, evident as a reduction in the number of revertants (below the indication factor of 0.5), was observed in experiment I in strain TA98 without S9 mix at 5000 µg/plate. No other toxic effects occurred in in the remaining test groups neither with nor and without metabolic activation.

 

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Calcium 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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available (further information necessary)

Justification for classification or non-classification

no classification

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Calcium glycerophosphate at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix).