Bis(glycinato-N,O)zinc

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

-study according to OECD guideline 471, Zinc bisglycinate was tested for genotoxicity in the Tester strains TA98, TA100, TA102, TA1535 and TA1537 up to the limit concentration of 5000 µg/plate. No cytotoxicity of the test item and no increase of the number of revertants was observed, negative

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-12-03 to 2020-02-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted July 21st, 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

his locus

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system: S9 Mix
- source of S9 :S9 was obtained by Trinova Biochem GmbH, Gießen. Batch nos. 4115
- method of preparation of S9 mix :produced from the livers of male Sprague-Dawley rats which were treated with 500 mg Aroclor 1254/kg body weight intraperitoneally.
- concentration or volume of S9 mix and S9 in the final culture medium:
S9-Mix
Phosphate buffer 22.5 mL
0.1M NADP-solution 1.0 mL
1M G6P-solution 0.125 mL
Salt solution 0.5 mL
Rat liver S9 1.0 mL
500 µL per 2000 µL top-agar (preincubation method) or 500 µL per 2700µL (plate incorporation method)

Test concentrations with justification for top dose:

Nominal concentrations: 50, 150, 500, 1500, 5000 µg/plate for the first experiment (plate incorporation)
Nominal concentrations: 78, 156, 313, 625, 1250, 2500, 5000 µg/plate for the second experiment (preincubation method)
Concentrations were based on preliminary tests

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: aqueous solvents (water)

- Justification for choice of solvent/vehicle: Based on the non-GLP pre-test, a test item suspension in demin. water was used, be-
cause this solvent shows the most stable suspension with the test item and does not have any effects on the viability of the bacteria or the number of spontaneous revertants in the tested concentrations.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

benzo(a)pyrene

other: 4-Nitro-1,2-phenylene diamine; without metabolic activation; 20 µg in DMSO for TA 98 and 30 µg for TA102 and TA1537; 2-Amino-anthracene; with metabolic activation; 1 µg in DMSO for TA100, TA1535 and 2.4 µg in DMSO for TA 102, TA1537

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate) : Treatment with test item: triplicates; Spontaneous revertants: triplicates; Determination of titre: duplicates; Toxicity control: duplicates; Sterility control: four replicates; Positive controls: triplicates
- Number of independent experiments : two

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): at least E+09 cells/mL
- Test substance added in medium; in agar (plate incorporation, experiment one) and preincubation (experiment two)

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 20 min at 37 ± 1°C

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition

Rationale for test conditions:

As recommended by OECD guideline 471

Evaluation criteria:

Five different analysable and non-toxic concentrations should be used for the evaluation of the mutagenic potential of the test item.
A substance is considered to be mutagenic, if a reproducible increase with or without metabolic activation of revertant colonies per plate exceeding an increase factor of 2 for the bacteria strains TA98, TA100, TA102, TA1535 and TA1537 compared to vehicle controls
in at least one strain can be observed.
A concentration-related increase over the range tested is also taken as a sign of mutagenic activity.
A substance is not mutagenic if it does not meet these criteria. If the criteria listed above are not clearly met, the results will be assessed as equivocal and will be discussed.

Key result

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 examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

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 examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

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 examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

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 examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

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 examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: sufficient for using demin. water as vehicle
- Precipitation and time of the determination: Not observed

RANGE-FINDING/SCREENING STUDIES (if applicable): Preliminary non-GLP test to determine the solubility of the test item

STUDY RESULTS
- Concurrent vehicle negative and positive control data
Please refer to the 'Any other information on results incl. tables' section

For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship where possible : Due to lack of toxicity and mutagenicity not possible.

Ames test:
- Signs of toxicity : No signs of toxicity

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: Please refer to the 'Any other information on results incl. tables' section
- Negative (solvent/vehicle) historical control data: Please refer to the 'Any other information on results incl. tables' section

Spontaneous Revertants demin. water (colonies per plate) Demin. water; experiment 1

Strain	TA98		TA100		TA102		TA1535		TA1537
Induction	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Repl. 1	15	18	62	70	280	280	9	17	8	9
Repl. 2	13	10	78	72	248	272	9	17	8	10
Repl. 3	18	17	72	76	264	272	12	19	6	8
Mean	15	15	71	73	264	275	10	18	7	9
sd	2.5	4.4	8.1	3.1	16.0	4.6	1.7	1.2	1.2	1.0

Spontaneous Revertants demin. water (colonies per plate) Demin. water; experiment 2

Strain	TA98		TA100		TA102		TA1535		TA1537
Induction	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Repl. 1	19	20	96	96	352	368	9	10	6	9
Repl. 2	20	19	92	92	384	384	12	10	5	7
Repl. 3	20	19	84	80	376	376	10	11	4	7
Mean	20	19	91	89	371	376	10	10	5	8
sd	0.6	0.6	6.1	8.3	16.7	8.0	1.5	0.6	1.0	1.2

Spontaneous Revertants demin. water (colonies per plate) DMSO, experiment 1

Strain	TA98		TA100		TA102		TA1535		TA1537
Induction	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Repl. 1	11	15	60	62	256	288	15	15	11	8
Repl. 2	14	13	62	80	272	264	17	10	8	9
Repl. 3	14	19	58	66	248	280	18	14	8	8
Mean	13	16	60	69	259	277	17	13	9	8
sd	1.7	3.1	2.0	9.5	12.2	12.2	1.5	2.6	1.7	0.6

Spontaneous Revertants demin. water (colonies per plate) DMSO, experiment 2

Strain	TA98		TA100		TA102		TA1535		TA1537
Induction	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Repl. 1	18	19	76	80	352	360	9	11	6	9
Repl. 2	18	20	80	84	360	368	8	15	6	6
Repl. 3	18	21	88	84	352	368	10	10	6	8
Mean	18	20	81	83	355	365	9	12	6	8
sd	0.0	1.0	6.1	2.3	4.6	4.6	1.0	2.6	0.0	1.5

Historical Data of Spontaneous Revertants

Strain		TA98		TA100		TA102		TA1535		TA1537
Induction		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Demin. Water	Mean	28	30	91	96	303	318	15	15	6	8
	Min	6	8	46	53	85	67	6	6	3	5
	Max	93	109	147	141	509	587	36	40	15	22
	SD	16	15	16	16	70	77	6	6	2	2
	Exp1	15	15	71	73	264	275	10	18	7	9
	Exp2	20	19	91	89	371	376	10	10	5	8
DMSO	Mean	28	29	88	91	302	311	15	15	6	7
	Min	7	8	37	42	79	80	6	6	4	4
	Max	104	108	143	199	531	499	35	37	15	20
	SD	16	15	16	17	70	69	6	6	4	4
	Exp1	13	16	60	69	259	277	17	13	9	8
	Exp2	18	20	81	83	355	365	9	12	6	8
Positive controls	Mean	433	188	529	829	1138	1209	289	151	124	111
	Min	77	39	218	273	491	408	55	45	79	100
	Max	s.g.	s.g.	1256	1912	2331	6083	s.g.	s.g.	165	123
	SD	219	159	214	275	364	473	119	106	17	7
	Exp1	s.g.	98	s.g.	s.g.	605	s.g.	221	175	232	168
	Exp2	s.g.	111	s.g.	s.g.	765	872	248	167	152	160

Mean Revertants First Experiment (1)

Strain		TA98		TA100		TA102		TA1535		TA1537
Induction		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Demin. Water	Mean	15	15	71	73	264	275	10	18	7	9
	SD	2.5	4.4	8.1	3.1	16.0	4.6	1.7	1.2	1.2	1.0
DMSO	Mean	13	16	60	69	259	277	17	13	9	8
	SD	1.7	3.1	2.0	9.5	12.2	12.2	1.5	2.6	1.7	0.6
Positive controls	Mean	s.g.	98	s.g.	s.g.	605	s.g.	221	175	232	168
	SD	--	17.1	--	--	28.1	--	6.1	4.6	8.0	21.2
	f(l)	> 2	6.13	> 2	> 2	2.34	> 2	22.10	13.46	25.78	21.00
5000µg/plate	Mean	15	19	125	120	256	256	13	13	9	12
	SD	0.6	2.9	5.0	8.0	16.0	13.9	1.2	2.5	1.2	1.5
	f(l)	1.00	1.27	1.76	1.64	0.97	0.93	1.30	0.72	1.29	1.33
1500 µg/plate	Mean	17	17	94	107	253	269	11	14	12	9
	SD	3.8	2.5	7.2	4.6	18.5	20.1	1.5	2.3	2.5	0.6
	f(l)	1.13	1.13	1.32	1.47	0.96	0.98	1.10	0.78	1.71	1.00
500 µg/plate	Mean	12	15	67	89	272	264	11	12	9	10
	SD	2.5	3.1	1.2	2.3	16.0	8.0	2.1	0.6	0.6	1.5
	f(l)	0.80	1.00	0.94	1.22	1.03	0.96	1.10	0.67	1.29	1.11
150 µg/plate	Mean	13	13	62	65	267	261	12	13	9	8
	SD	3.1	4.0	2.0	5.0	12.2	18.5	1.7	0.6	1.2	1.0
	f(l)	0.87	0.87	0.87	0.89	1.01	0.95	1.20	0.72	1.29	0.89
50 g/plate	Mean	10	14	62	63	264	264	12	12	10	10
	SD	1.7	3.0	2.0	3.1	8.0	0.0	0.6	2.1	2.1	2.1
	f(l)	0.67	0.93	0.87	0.86	1.00	096	1.20	0.67	1.43	1.11

Mean Revertants Second Experiment (2)

Strain		TA98		TA100		TA102		TA1535		TA1537
Induction		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Demin. Water	Mean	20	19	91	89	371	376	10	10	5	8
	SD	0.6	0.6	6.1	8.3	16.7	8.0	1.5	0.6	1.0	1.2
DMSO	Mean	18	20	81	83	355	365	9	12	6	8
	SD	0.0	1.0	6.1	2.3	4.6	4.6	1.0	2.6	0.0	1.5
Positive controls	Mean	s.g.	111	s.g.	s.g.	765	872	248	167	152	160
	SD	--	2.3	--	--	12.2	21.2	13.9	2.3	18.3	13.9
	f(l)	> 2	5.55	> 2	> 2	2.15	2.39	24.80	13.92	25.33	20.00
5000µg/plate	Mean	19	24	108	108	368	349	10	12	7	8
	SD	1.5	1.0	4.0	4.0	8.0	4.6	0.6	2.1	1.5	1.0
	f(l)	0.95	1.26	1.19	1.21	0.99	0.93	1.00	1.20	1.40	1.00
2500 g/plate	Mean	19	21	92	88	363	349	13	12	7	8
	SD	1.0	1.0	4.0	6.9	23.1	12.2	1.5	1.7	2.1	0.6
	f(l)	0.95	1.11	1.01	0.99	0.98	0.93	1.30	1.20	1.40	1.00
1250µg/plate	Mean	18	20	89	97	373	355	10	11	6	6
	SD	1.0	1.2	8.3	6.1	12.2	18.5	0.6	1.0	0.6	1.0
	f(l)	0.90	1.05	0.98	1.09	1.01	0.94	1.00	1.10	1.20	0.75
625 µg/plate	Mean	19	22	87	95	360	368	10	8	7	6
	SD	0.6	1.0	2.3	2.3	8.0	16.0	1.0	1.0	1.5	1.7
	f(l)	0.95	1.16	0.96	1.07	0.97	0.98	1.00	0.80	1.40	0.75
313 µg/plate	Mean	19	18	87	92	355	347	9	10	5	6
	SD	1.2	0.6	2.3	4.0	16.7	16.7	1.0	2.0	0.6	0.6
	f(l)	0.95	0.95	0.96	1.03	0.96	0.92	0.90	1.00	1.00	0.75
156 g/plate	Mean	19	22	87	95	360	368	10	8	7	6
	SD	0.6	1.0	2.3	2.3	8.0	16.0	1.0	1.0	1.5	1.7
	f(l)	0.95	1.16	0.96	1.07	0.97	0.98	1.00	0.80	1.40	0.75
78 g/plate	Mean	19	18	87	92	355	347	8	10	5	6
	SD	1.2	0.6	2.3	4.0	16.7	16.7	0.6	2.0	0.6	0.6
	f(l)	0.95	0.95	0.96	1.03	0.96	0.92	0.80	1.00	1.00	0.75

Conclusions:

Based on the results of this studyconducted according to OECD guideline 471 it is concluded that the test item Zinc bisglycinate is not mutagenic in the Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 in the presence and absence of metabolic activation under the experimental conditions in this study.

Executive summary:

In a reverse gene mutation assay in bacteria according to OECD guideline 471 (1997), Salmonella typhimurium strains TA98, TA100, TA102,TA1535 and TA1537 in the presence and absence of metabolic activation were exposed to Zinc bisglycinate in demin. water and DMSO, respectively, at concentrations of 50, 150, 500, 1500 and 5000 µg/plate (pre-incubation method) and to 78, 156, 313, 625, 1250, 2500 and 5000 µg/plate (plate incorporation method).

 

Zinc bisglycinate was tested up to limit concentration of 5000 µg/plate, there was no evidence of induced mutant colonies over background.

The positive controls induced the appropriate responses in the corresponding strains.

 

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data. Thus, the test item is not classified as mutagen according to Regulation (EC) No. 1272/2008 (CLP) or the Globally Harmonized System of Classification and Labelling of Chemicals (GHS).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

In a reverse gene mutation assay in bacteria according to OECD guideline 471 (1997), Salmonella typhimurium strains TA98, TA100, TA102,TA1535 and TA1537 in the presence and absence of metabolic activation were exposed to Zinc bisglycinate in demin. water and DMSO, respectively, at concentrations of 50, 150, 500, 1500 and 5000 µg/plate (pre-incubation method) and to 78, 156, 313, 625, 1250, 2500 and 5000 µg/plate (plate incorporation method).

 

Zinc bisglycinate was tested up to limit concentration of 5000 µg/plate, there was no evidence of induced mutant colonies over background.

The positive controls induced the appropriate responses in the corresponding strains.

 

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data. Thus, the test item is not classified as mutagen according to Regulation (EC) No. 1272/2008 (CLP) or the Globally Harmonized System of Classification and Labelling of Chemicals (GHS).