Tetraamminezinc(2+) carbonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test: No mutagenic potential of the test item was reported in the bacterial reverse mutation test (Ames test) in the absence and the presence of metabolic activation.

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:

27 January 2017 - 10 February 2017

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:

21 Jul 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

30 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

Aug 1998,

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Kampagne 02/2014
- Expiration date of the lot/batch: February 18, 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9 mix from phenobarbital (i.p.) and β-naphthoflavone (p.o) induced rat liver

Test concentrations with justification for top dose:

0; 100; 333; 1000; 3333; 10000 and 30000 μg/plate (Standard Plate Assay)
0; 100; 333; 1000; 3333; 10000 and 30000 μg/plate (Preincubation Test)
In this study, due to the purity of the test substance 30 mg/plate was used as top dose in all experiments.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used:
ultrapure water
- Justification for choice of solvent/vehicle:
Due to the good solubility of the test substance in water, water was used as vehicle.

Untreated negative controls:

yes

Remarks:

ultrapure water

Negative solvent / vehicle controls:

yes

Remarks:

ultrapure water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (2-AA)

Untreated negative controls:

yes

Remarks:

ultrapure water

Negative solvent / vehicle controls:

yes

Remarks:

ultrapure water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)

Untreated negative controls:

yes

Remarks:

ultrapure water

Negative solvent / vehicle controls:

yes

Remarks:

ultrapure water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylenediamine (NOPD)

Untreated negative controls:

yes

Remarks:

ultrapure water

Negative solvent / vehicle controls:

yes

Remarks:

ultrapure water

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Untreated negative controls:

yes

Remarks:

ultrapure water

Negative solvent / vehicle controls:

yes

Remarks:

ultrapure water

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation

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

NUMBER OF REPLICATIONS: 3 test plates per dose or per control

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.

Evaluation 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.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

A bacteriotoxic effect (reduced his- background growth, decrease in the number of his+ or trp+ revertants) was occasionally observed depending on the strain and test conditions from about 10000 μg/plate onward

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

A bacteriotoxic effect (reduced his- background growth, decrease in the number of his+ or trp+ revertants) was occasionally observed depending on the strain and test conditions from about 10000 μg/plate onward

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

A bacteriotoxic effect (reduced his- background growth, decrease in the number of his+ or trp+ revertants) was occasionally observed depending on the strain and test conditions from about 10000 μg/plate onward

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

A bacteriotoxic effect (reduced his- background growth, decrease in the number of his+ or trp+ revertants) was occasionally observed depending on the strain and test conditions from about 10000 μg/plate onward

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

A bacteriotoxic effect (reduced his- background growth, decrease in the number of his+ or trp+ revertants) was occasionally observed depending on the strain and test conditions from about 10000 μg/plate onward

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No test substance precipitation was found with and without S9 mix.

HISTORICAL CONTROL DATA: Please refer to tables 5 and 6.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
A bacteriotoxic effect (reduced his- background growth, decrease in the number of his+ or trp+revertants) was occasionally observed depending on the strain and test conditions from about
10000 μg/plate onward. Please refer to table 7.

Table 1: Without metabolic activation (Standard Plate Test)

Strain	Test group	Dose [µg/mL]	Mean revertants per plate	Standard deviation	Factor	Individual revertant colony counts
TA 1535	Water	-	12.3	2.3	-	11,11,15
	Test item	100	11.3	3.5	0.9	15, 8, 11
		333	13.3	4.2	1.1	18, 12, 10
		1000	12.0	1.0	1.0	13, 11, 12
		3333	11.3	3.5	0.9	8,11,15
		10000	11.7	0.6	0.9	11, 12, 12
		30000	10.7	1.2	0.9	10, 10,12
	MNNG	5.0	3644.0	519.9	295.5	4241, 3400, 3291
TA 100	Water	-	110.0	10.6	-	122, 102, 106
	Test item	100	124.7	8.1	1.1	134, 119, 121
		333	120.3	13.6	1.1	125, 105, 131
		1000	109.0	6.6	1.0	110, 115, 102
		3333	100.7	13.1	0.9	105, 111, 86
		10000	106.0	9.0	1.0	97, 115, 106
		30000	120.3	10.5	1.1	131, 110, 120
	MNNG	5.0	4493.0	23.9	40.8	4488, 4472, 4519
TA 1537	Water	_	6.7	2.5	-	9, 7, 4
	Test item	100	9.3	3.2	1.4	13, 7, 8
		333	10.7	2.1	1.6	10, 9, 13
		1000	6.3	3.2	1.0	4, 5,10
		3333	6.7	2.5	1.0	9, 7, 4
		10000	7.3	2.5	1.1	10, 7, 5
		30000	9.0	0.0	1.4	9, 9, 9
	AAC	100	763.7	72.6	114.6	680, 801, 810
TA 98	Water	-	19.3	4.6	-	22, 14, 22
	Test item	100	18.0	1.7	0.9	17, 20, 17
		333	18.7	6.7	1.0	17, 26, 13
		1000	22.3	3.5	1.2	22, 26, 19
		3333	18.0	6.2	0.9	20, 11,23
		10000	24.0	2.6	1.2	26, 21,25
		30000	21.0	4.0	1.1	21, 25, 17
	NOPD	10	575.7	10.0	29.8	575, 566, 586
E. coli	Water	-	28.7	3.5	-	25, 32, 29
	Test item	100	35.7	1.5	1.2	37, 34, 36
		333	25.7	5.9	0.9	19, 30, 28
		1000	29.3	15.0	1.0	15, 28, 45
		3333	23.7	3.1	0.8	23, 27, 21
		10000	19.0	1.0	0.7	20, 18, 19
		30000	21.3	6.7	0.7	29, 17, 18
	4-NQO	5	1079.0	47.0	37.6	1077, 1127, 1033

Table 2: With metabolic activation (Standard Plate Test)

Strain	Test group	Dose	Mean	Standard	Factor	Individual revertant colony
		(µg/plate)	revertants per plate	deviation		counts
TA 1535	Water	-	13.3	1.5	-	15, 13, 12
	Test item	100	7.3	2.5	0.6	10, 7, 5
		333	10.3	4.2	0.8	7, 15, 9
		1000	7.7	1.2	0.6	7, 7, 9
		3333	9.0	1.7	0.7	11, 8, 8
		10000	7.7	2.1	0.6	7, 10 ,6
		30000	10.3	2.9	0.8	12, 12, 7
	2-AA	2.5	207.7	9.0	15.6	207, 199, 217
TA 100	Water	-	125.3	10.5	-	115, 125, 136
	Test item	100	112.3	14.7	0.9	107, 129, 101
		333	130.7	11.9	1.0	127, 144, 121
		1000	122.7	9.6	1.0	133, 121, 114
		3333	112.3	15.0	0.9	121, 95, 121
		10000	123.3	9.7	1.0	121, 115,134
		30000	126.7	16.1	1.0	115, 145,120
	2-AA	2.5	2560.3	279.1	20.4	2499, 2865, 2317
TA 1537	Water	-	9.7	4.2	-	5, 11,13
	Test item	100	7.0	1.7	0.7	8, 8, 5
		333	8.7	1.5	0.9	9, 7, 10
		1000	7.0	1.7	0.7	6, 9, 6
		3333	10.0	2.6	1.0	11,7,12
		10000	10.3	3.1	1.1	11,13, 7
		30000	9.0	1.0	0.9	8, 10, 9
	2-AA	2.5	206.7	31.7	21.4	243, 192,185
TA98	Water	_	23.3	3.8	-	19, 25, 26
	Test item	100	31.0	1.0	1.3	32, 30, 31
		333	25.3	5.0	1.1	30, 26, 20
		1000	26.7	3.5	1.1	27, 23, 30
		3333	26.7	4.2	1.1	30, 22, 28
		10000	27.0	8.7	1.2	23, 21, 37
		30000	21.7	4.7	0.9	27, 18, 20
	2-AA	2.5	1579.7	106.4	67.7	1596,1466, 1677
E. Coli	Water	-	23.7	5.5	-	30, 21, 20
	Test item	100	30.3	6.7	1.3	36, 23, 32
		333	22.7	5.9	1.0	27, 25, 16
		1000	22.0	2.6	0.9	21, 25, 20
		3333	19.7	4.7	0.8	25, 16, 18
		10000	26.0	8.5	1.1	18, 25, 35
		30000	24.7	4.9	1.0	27, 19, 28
	2-AA	60	131.7	2.5	5.6	134, 132,129

Table 3: Without metabolic activation (Preincubation Test)

Strain	Test group	Dose (µg/mL)	Mean revertants per plate	Standard deviation	Factor	Individual revertant colony counts
TA 1535	Water	-	9.3	2.3	-	8, 8, 12
	Test item	100	9.7	2.1	1.0	9, 12, 8
		333	10.0	5.3	1.1	8, 6, 16
		1000	9.7	1.5	1.0	8, 10, 11
		3333	11.0	5.2	1.2	14, 5, 14
		10000	13.3	3.2	1.4	12,11,17
		30000	0.0	0.0	0.0	0B, 0B, 0B
	MNNG	5.0	2935.7	92.8	314.5	2830, 3004, 2973
TA 100	Water	-	104.7	6.7	-	99, 103, 112
	Test item	100	110.7	9.9	1.1	104, 122, 106
		333	127.0	8.7	1.2	122. 137, 122
		1000	121.7	8.1	1.2	123, 113, 129
		3333	112.7	2.1	1.1	112, 115, 111
		10000	103.3	6.7	1.0	99, 111, 100
		30000	108.7	22.2	1.0	129 B, 112 B, 85 B
	MNNG	5.0	2313.0	134.0	22.1	2271, 2205, 2463
TA 1537	Water	_	8.0	3.6	-	12, 5, 7
	Test item	100	9.3	0.6	1.2	9, 10, 9
		333	10.7	1.5	1.3	12, 11,9
		1000	6.3	4.0	0.8	4, 4,11
		3333	9.7	0.6	1.2	9, 10, 10
		10000	8.0	1.0	1.0	8, 7, 9
		30000	4.7	3.1	0.6	4B, 2B, 8B
	AAC	100	500.0	151.7	62.5	675, 405, 420
TA 98	Water	-	15.3	1.5	-	14, 17, 15
	Test item	100	21.7	3.8	1.4	20, 19, 26
		333	13.3	3.1	0.9	10, 14, 16
		1000	14.7	1.5	1.0	13, 15, 16
		3333	14.3	2.1	0.9	16, 15, 12
		10000	19.0	4.4	1.2	22, 21, 14
		30000	13.3	3.1	0.9	16 B, 10 B, 14 B
	NOPD	10	604.3	41.1	39.4	649, 596, 568
E. coli	Water	-	34.0	7.0	-	27, 34, 41
	Test item	100	26.7	9.7	0.8	29, 16, 35
		333	28.7	14.8	0.8	25, 45, 16
		1000	30.7	10.0	0.9	27, 23, 42
		3333	29.3	10.2	0.9	25, 41,22
		10000	19.7	5.0	0.6	25, 15, 19
		30000	18.7	7.8	0.5	21 B, 10 B, 25 B
	4-NQO	5	187.0	41.1	5.5	230, 148, 183

B = Reduced background growth

Table 4: Without metabolic activation (Preincubation Test)

Strain	Test group	Dose	Mean	Standard	Factor	Individual revertant colony
		(µg/plate)	revertants per plate	deviation		counts
TA 1535	Water	-	10.0	1.0	-	9,11,10
	Test item	100	10.3	4.5	1.0	10, 6,15
		333	6.7	1.2	0.7	6, 8,6
		1000	7.7	3.2	0.8	10,4,9
		3333	10.7	0.6	1.1	11,11,10
		10000	11.3	2.1	1.1	13, 9,12
		30000	9.7	1.5	1.0	10B, 11 B, 8B
	2-AA	2.5	216.7	31.1	21.7	242,182, 226
TA 100	Water	-	114.0	13.5	-	129, 103, 110
	Test item	100	116.7	25.4	1.0	122, 139, 89
		333	107.7	22.0	0.9	129, 109, 85
		1000	112.0	11.4	1.0	107, 104, 125
		3333	112.7	9.3	1.0	117, 102, 119
		10000	104.3	9.5	0.9	95, 114, 104
		30000	159.7	8.1	1.4	169 B, 155 B, 155 B
	2-AA	2.5	2553.3	116.4	22.4	2647, 2423, 2590
TA 1537	Water	-	10.0	2.0	-	8, 10, 12
	Test item	100	11.7	4.2	1.2	15, 7,13
		333	16.0	3.5	1.6	18, 12, 18
		1000	15.3	3.8	1.5	18, 11, 17
		3333	15.0	3.0	1.5	15, 18, 12
		10000	14.0	2.6	1.4	17, 13, 12
		30000	3.7	4.0	0.4	8 B, 0B, 3B
	2-AA	2.5	168.3	22.3	16.8	185, 143, 177
TA98	Water	_	25.3	8.1	-	30, 30, 16
	Test item	100	32.7	5.0	1.3	32, 38, 28
		333	31.0	0.0	1.2	31,31, 31
		1000	29.3	6.4	1.2	34, 32, 22
		3333	36.7	5.8	1.4	40, 30,40
		10000	25.3	3.5	1.0	22, 25, 29
		30000	21.7	7.6	0.9	20, 30, 15
	2-AA	2.5	1884.7	148.4	74.4	1797,1801,2056
E. coli	Water	-	26.7	8.0	-	26, 35. 19
	Test item	100	24.3	7.5	0.9	20, 20, 33
		333	19.7	2.1	0.7	22, 18, 19
		1000	23.3	7.6	0.9	30, 15, 25
		3333	29.3	2.1	1.1	30, 31, 27
		10000	15.7	1.2	0.6	15, 17, 15
		30000	33.0	3.0	1.2	33, 30, 36
	2-AA	60	125.7	51.1	4.7	109, 85, 183

B = Reduced background growth

Table 5: Historical Negative Controls (based on Sorcerer Counter / Ames Study Manager System (Apr 15 - Apr 16))

Strain	S9 Mix	Vehicle	No. of Plates	No. of Values	Min	Max	Mean	SD
TA 1535	Without	(All)	495	178	6	17	10	2.2
	With	(All)	483	177	6	18	10	2.1
TA 100	Without	(All)	501	179	69	140	95	9.9
	With	(All)	492	179	76	150	100	12.3
TA 1537	Without	(All)	501	178	5	12	7	1.5
	With	(All)	504	178	5	16	8	2.2
TA98	Without	(All)	501	178	12	30	18	3.1
	With	(All)	495	178	13	38	24	4.7
E. coli	Without	(All)	468	177	12	33	22	4.0
	With	(All)	468	178	13	35	23	4.0

Table 6: Historical Positive Controls (based on Sorcerer Counter / Ames Study Manager System (Apr 15 - Apr 16))

Strain	S9 Mix	Positive control	No. of Plates	No. of Values	Min	Max	Mean	SD
TA 1535	Without With	MNNG 2-AA	342 336	116 115	1174 108	6612 367	4063 217	1308.8 50.0
TA 100	Without With	MNNG 2-AA	351 348	118 118	1125 361	5557 2819	3331 1792	1021.5 448.9
TA 1537	Without With	AAC 2-AA	345 348	117 117	253 56	1858 241	975 130	339.9 33.6
TA98	Without With	NOPD 2-AA	348 348	117 117	271 431	1119 2427	486 1427	183.3 361.9
E. coli	Without With	4-NQO 2-AA	333 330	115 115	133 54	1552 169	884 98	442.4 26.6

Table 7: Toxicity: Decreased revertant numbers were observed at following concentrations (μg/plate)

Experiment	S9	TA1535	TA100	TA 1537	TA 98	E.coli
1st-SPT	Without	-
	With	10000	-	-	-	-
2nd-PIT	Without	30000	-	30000	-	10000-30000
	With	-	-	30000	-	10000
Reduced background growth was observed at following concentrations (μg/plate):
Experiment	S9	TA1535	TA100	TA 1537	TA 98	E.coli
1st-SPT	Without	-
	With	-
2nd-PIT	Without	30000
	With	30000	30000	30000	-	-

- = no adverse effect observed

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Ames test

A study according OECD TG 471 was performed to detect the 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. The following strains were used: TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA. Standard plate test (SPT) and preincubation test (PIT) both with and without metabolic activation (liver S9 mix from induced rats) were performed using concentrations from 100 μg up to 30000 μg/plate. No precipitation of the test substance was found with and without S9 mix. A bacteriotoxic effect was observed depending on the strain and test conditions from about 10000 μg/plate onward. A biologically relevant increase in the number of his+ or trp+ revertants (factor ≥ 2: TA 100, TA98 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.

Besides, the results of the negative as well as the positive controls performed in parallel corroborated the validity of this study, since the values fulfilled the acceptance criteria. In this study with and without S9 mix, the number of revertant colonies in the negative controls was within or marginally above the range of the historical negative control data for each tester strain. In addition, the positive control substances with and without S9 mix induced a significant increase in the number of revertant colonies within the range of the historical positive control data.

Under the experimental conditions of this study, the test substance is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation (BASF, 2017).

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data is reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. As a result the substance is not considered to be classified for genetic toxicity under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.