Sulfates of potassium, sodium and calcium, byproduct from fermentation

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro gene mutation study in bacteria: negative with and without metabolic activation (Reliability 1 key study; GLP compliant; OECD 471 test guideline) (CiToxLAB Hungary Ltd, 2017).

In vitro gene mutation study in mammalian cells: read-across from three source analogues [Potassium sulfate (CAS 7778-80-5), Sodium sulfate (CAS 7757-82-6) and Calcium sulfate (CAS 7778-18-9)]. All three substances did not induce mutations in the mammalian cell systems used in the absence and presence of metabolic activation.

In vitro cytogenicity / chromosome aberration study in mammalian cells: read-across from three source analogues [Potassium sulfate (CAS 7778-80-5), Sodium sulfate (CAS 7757-82-6) and Calcium sulfate (CAS 7778-18-9)]. All three substances item did not induce structural chromosome aberrations in the mammalian cell systems used both with and without metabolic activation (in vivo micronucleus test for Calcium sulfate) and were therefore considered to be non-clastogenic.

Based on the analogy approach applied, Sulfates of potassium, sodium and calcium, by-product from fermentation is not considered to be genotoxic in vitro.

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:

From 3 April 2017 to 28 August 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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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: EXP LIberica Lote: AI16503047
- Expiration date of the lot/batch: 30 November 2019
- Purity test date: 1 February 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Controlled room temperature (15-25°C, below 70 RH%), protected from light and humidity
- Stability under test conditions: not specified
- Solubility and stability of the test substance in the solvent/vehicle:Test item was soluble at 50 mg/mL concentration using Distilled water.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
Distilled water was used as solvent to prepare the stock solution of the test material (50mg/ml). Test solutions were freshly prepared at the beginning of the experiments in the testing laboratory by diluting the stock solution using the selected solvent and were used within 4 hours after preparation.

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Additional strain / cell type characteristics:

other:

Remarks:

See attached-table 1

Metabolic activation:

with and without

Metabolic activation system:

Liver extract, S9 fraction from rats which were treated intraperitoneally with phenobarbital and beta- naphthoflavone

Test concentrations with justification for top dose:

Concentrations were selected on the basis of the Preliminary Solubility Test and Preliminary Range Finding Test (Informatory Toxicity Test). In the Initial Mutation Test and Confirmatory Mutation Test, the followingt concentrations were used :
- Examined concentrations in the Initial Mutation Test were 5000, 1581, 500, 158.1, 50 and 15.81 μg/plate.
- Examined concentrations in the Confirmatory Mutation Test were 5000, 1581, 500, 158.1, 50, 15.81 and 5 μg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: distilled water
- Justification for choice of solvent/vehicle: Test item was soluble at 50 mg/mL concentration using Distilled water. Partial dissolution was observed at the same concentration using DMSO and DMF. Distilled water was selected as vehicle (solvent) for the study.

Untreated negative controls:

yes

Remarks:

Untreated

Negative solvent / vehicle controls:

yes

Remarks:

Vehicle (Distilled Water)

True negative controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

methylmethanesulfonate

other: 4-nitro-1,2-phenylenediamine (TA98 without S9); 2-aminoanthracene (for all strains, with S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)(Inital Assay); preincubation (Confirmatory Assay)
- Cell density at seeding (if applicable): not applicable

DURATION
- Preincubation period: 20 minutes
- Exposure duration:48±1 hours at 37°C
- Fixation time (start of exposure up to fixation or harvest of cells): not applicable.

SELECTION AGENT (mutation assays): not applicable

NUMBER OF REPLICATIONS: triplicate were used

DETERMINATION OF CYTOTOXICITY
- Method: Viability of the bacterial cells was checked.
- Any supplementary information relevant to cytotoxicity: The viability of each testing culture was determined by plating 0.1 mL of the 10E5, 10E6, 10E7 and 10E8 dilutions prepared by sterile physiological saline on Nutrient Agar plates. The viable cell number of the cultures was determined by manual counting after approximately 24-hour incubation at 37°C.

Evaluation criteria:

The colony numbers on the untreated / negative (solvent) / positive control and test item treated plates were determined by manual counting. Visual examination of the plates was also performed; precipitation or signs of growth inhibition (if any) were recorded and reported. The mean number of revertants per plate, the standard deviation and the mutation factor values were calculated for each concentration level of the test item and for the controls using Microsoft ExcelTM software. The study was considered valid if:

- the number of revertant colonies of the negative (vehicle/solvent) and positive controls are in the relevant historical control range, generated at the test facility, in all tester strains of the main tests (with or without S9-mix);
- at least five analysable concentrations are presented in all strains of the main tests

Criteria for a Positive Response:
A test item was considered mutagenic if:
- a concentration-related increase in the number of revertants occurs
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.

An increase was considered biologically relevant if:
- the number of reversions is more than two times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA98, TA100 and Escherichia coli WP2 uvrA bacterial strains;
- the number of reversions is more than three times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA1535 and TA1537 bacterial strains.

Criteria for a Negative Response:
The test item was considered to have shown no mutagenic activity in this study if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.

Statistics:

Statistical method may be used as an aid in evaluating the test results. However, statistical significance should not be the only determining factor for a positive response.

Key result

Species / strain:

S. typhimurium TA 1535

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

Key result

Species / strain:

S. typhimurium TA 1537

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

Key result

Species / strain:

S. typhimurium TA 98

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

Key result

Species / strain:

S. typhimurium TA 100

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

Key result

Species / strain:

E. coli WP2 uvr A

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:

RANGE-FINDING/SCREENING STUDIES:
In the Preliminary Range Finding Test, the plate incorporation method was used. The preliminary test was performed using Salmonella typhimurium TA98 and Salmonella typhimurium TA100 tester strains in the presence and absence of metabolic activation system (±S9 Mix) with appropriate untreated, negative (solvent) and positive controls. Each sample (including the controls) was tested in triplicate.
The following concentrations were examined: 5000, 2500, 1000, 316, 100, 31.6 and 10 µg/plate.
In the preliminary experiment, the number of revertant colonies were mostly in the normal range (minor differences were detected in some sporadic cases, but they were without biological significance and considered as biological variability of the test system).
No precipitate was detected on the plates in the preliminary experiment.
Inhibitory or toxic effects of the test item were not detected in the preliminary experiment.
Based on the results of the Range Finding Test and the solubility findings, the maximum final concentration to be tested in the main experiments was 5000 µg/plate.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
See attached table 5

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: The viability of the bacterial cells was checked by a plating experiment in each test.

Table 2:Summary Table of the Range Finding Test

 

Concentrations (µg/plate)	Mean values ofrevertants/ Mutation factor (MF)	Salmonella typhimuriumtester strains
		TA98		TA100
		-S9	+S9	-S9	+S9
Untreated control	Mean	23.0	33.7	95.3	92.7
	MF	0.87	0.98	1.05	0.98
Distilled water control	Mean	-	-	91.3	-
	MF	-	-	1.00	-
DMSO control	Mean	25.0	25.3	-	96.3
	MF	0.95	0.74	-	1.01
Distilled water 100µL control	Mean	26.3	34.3	91.0	95.0
	MF	1.00	1.00	1.00	1.00
5000	Mean	23.0	21.0	68.3	69.3
	MF	0.87	0.61	0.75	0.73
2500	Mean	23.3	28.0	61.3	70.3
	MF	0.89	0.82	0.67	0.74
1000	Mean	28.7	25.0	65.0	76.3
	MF	1.09	0.73	0.71	0.80
316	Mean	22.3	24.3	78.7	83.3
	MF	0.85	0.71	0.86	0.88
100	Mean	21.7	26.0	75.0	94.3
	MF	0.82	0.76	0.82	0.99
31.6	Mean	23.3	24.3	80.0	92.7
	MF	0.89	0.71	0.88	0.98
10	Mean	22.0	27.0	81.3	96.3
	MF	0.84	0.79	0.89	1.01
NPD (4mg)	Mean	402.0	-	-	-
	MF	16.08	-	-	-
2AA (2mg)	Mean	-	2438.7	-	2397.3
	MF	-	96.26	-	24.89
SAZ (2mg)	Mean	-	-	1201.3	-
	MF	-	-	12.65	-

Table 3:Summary Table of the Initial Mutation Test

 

Concentrations (µg/plate)	Mean values ofrevertants/ Mutation factor (MF)	Salmonella typhimuriumtester strains								Escherichia coli
		TA98		TA100		TA1535		TA1537		WP2uvrA
		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Untreated control	Mean	20.7	30.7	95.3	107.3	8.7	12.0	9.3	8.7	32.0	34.7
	MF	0.83	1.08	1.03	1.11	0.59	1.29	0.85	0.81	1.00	0.99
Distilled water control	Mean	-	-	96.3	-	12.3	-	-	-	30.3	-
	MF	-	-	1.04	-	0.84	-	-	-	0.95	-
DMSO control	Mean	20.3	27.0	-	102.3	-	12.3	9.7	8.3	-	31.0
	MF	0.81	0.95	-	1.05	-	1.32	0.88	0.78	-	0.89
Distilled water 100µL control	Mean	25.0	28.3	92.3	97.0	14.7	9.3	11.0	10.7	32.0	35.0
	MF	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00
5000	Mean	20.7	24.3	79.3	103.7	13.0	9.7	10.0	10.0	31.3	36.3
	MF	0.83	0.86	0.86	1.07	0.89	1.04	0.91	0.94	0.98	1.04
1581	Mean	19.3	25.3	95.7	113.0	10.7	10.3	9.7	9.0	30.7	36.7
	MF	0.77	0.89	1.04	1.16	0.73	1.11	0.88	0.84	0.96	1.05
500	Mean	20.7	25.3	98.0	150.0	11.3	9.7	11.3	12.7	30.0	31.7
	MF	0.83	0.89	1.06	1.55	0.77	1.04	1.03	1.19	0.94	0.90
158.1	Mean	20.7	23.3	82.7	114.3	10.7	11.3	13.0	13.0	45.0	59.3
	MF	0.83	0.82	0.90	1.18	0.73	1.21	1.18	1.22	1.41	1.70
50	Mean	21.0	24.0	87.0	100.7	12.3	10.0	12.0	11.0	39.7	39.3
	MF	0.84	0.85	0.94	1.04	0.84	1.07	1.09	1.03	1.24	1.12
15.81	Mean	21.3	21.0	79.0	100.0	13.3	9.3	9.7	11.7	38.0	40.7
	MF	0.85	0.74	0.86	1.03	0.91	1.00	0.88	1.09	1.19	1.16
NPD (4mg)	Mean	414.0	-	-	-	-	-	-	-	-	-
	MF	20.36	-	-	-	-	-	-	-	-	-
2AA (2mg)	Mean	-	2431.3	-	2361.3	-	208.7	-	194.7	-	-
	MF	-	90.05	-	23.07	-	16.92	-	23.36	-	-
2AA (50mg)	Mean	-	-	-	-	-	-	-	-	-	193.7
	MF	-	-	-	-	-	-	-	-	-	6.25
SAZ (2mg)	Mean	-	-	1192.0	-	1213.3	-	-	-	-	-
	MF	-	-	12.37	-	98.38	-	-	-	-	-
9AA (50mg)	Mean	-	-	-	-	-	-	372.0	-	-	-
	MF	-	-	-	-	-	-	38.48	-	-	-
MMS (2mL)	Mean	-	-	-	-	-	-	-	-	1034.0	-
	MF	-	-	-	-	-	-	-	-	34.09	-

Table 4:Summary Table of the Confirmatory Mutation Test

 

Concentrations (µg/plate)	Mean values ofrevertants/ Mutation factor (MF)	Salmonella typhimuriumtester strains								Escherichia coli
		TA98		TA100		TA1535		TA1537		WP2uvrA
		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Untreated control	Mean	25.3	30.0	99.3	96.0	12.7	11.3	12.0	13.7	56.7	57.3
	MF	0.84	1.03	0.99	0.96	1.09	0.97	0.90	0.98	0.98	0.99
Distilled water control	Mean	--	--	98.3	--	11.7	--	--	--	58.7	--
	MF	--	--	0.98	--	1.00	--	--	--	1.02	--
DMSO control	Mean	25.3	28.3	--	95.7	--	11.7	12.3	11.7	--	55.3
	MF	0.84	0.98	--	0.96	--	1.00	0.93	0.83	--	0.95
Distilled water 100µL control	Mean	30.0	29.0	100.0	99.7	11.7	11.7	13.3	14.0	57.7	58.0
	MF	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00
5000	Mean	24.3	39.7	107.0	107.0	13.7	13.3	14.0	11.3	55.0	53.3
	MF	0.81	1.37	1.07	1.07	1.17	1.14	1.05	0.81	0.95	0.92
1581	Mean	26.3	40.0	98.0	99.3	10.3	13.0	12.7	13.3	53.3	56.7
	MF	0.88	1.38	0.98	1.00	0.89	1.11	0.95	0.95	0.92	0.98
500	Mean	33.3	30.3	96.0	106.0	10.7	15.3	12.7	12.7	58.0	53.3
	MF	1.11	1.05	0.96	1.06	0.91	1.31	0.95	0.90	1.01	0.92
158.1	Mean	27.7	31.0	101.3	106.3	12.3	12.7	10.7	11.7	55.3	62.3
	MF	0.92	1.07	1.01	1.07	1.06	1.09	0.80	0.83	0.96	1.07
50	Mean	28.7	29.7	99.7	102.3	14.7	12.0	12.7	12.0	54.7	54.0
	MF	0.96	1.02	1.00	1.03	1.26	1.03	0.95	0.86	0.95	0.93
15.81	Mean	30.7	29.3	95.7	114.3	12.3	11.0	10.3	13.3	56.0	58.7
	MF	1.02	1.01	0.96	1.15	1.06	0.94	0.78	0.95	0.97	1.01
5	Mean	31.7	30.3	92.0	109.3	10.0	9.7	9.7	12.7	55.0	55.0
	MF	1.06	1.05	0.92	1.10	0.86	0.83	0.73	0.90	0.95	0.95
NPD (4mg)	Mean	401.3	--	--	--	--	--	--	--	--	--
	MF	15.84	--	--	--	--	--	--	--	--	--
2AA (2mg)	Mean	--	2486.7	--	2416.0	--	228.0	--	203.3	--	--
	MF	--	87.76	--	25.25	--	19.54	--	17.43	--	--
2AA (50mg)	Mean	--	--	--	--	--	--	--	--	--	275.0
	MF	--	--	--	--	--	--	--	--	--	4.97
SAZ (2mg)	Mean	--	--	1192.0	--	1185.3	--	--	--	--	--
	MF	--	--	12.12	--	98.78	--	--	--	--	--
9AA (50mg)	Mean	--	--	--	--	--	--	400.7	--	--	--
	MF	--	--	--	--	--	--	32.49	--	--	--
MMS (2mL)	Mean	--	--	--	--	--	--	--	--	1018.7	--
	MF	--	--	--	--	--	--	--	--	17.36	--

Table 5 : Historical Control Data (Period of 2011-2015)

Untreated control data
	without metabolic activation (-S9 Mix)					with metabolic activation (+S9 Mix)
	TA98	TA100	TA1535	TA1537	E. coli	TA98	TA100	TA1535	TA1537	E. coli
Mean	22.7	103.4	11.4	6.9	33.7	30.0	111.3	11.3	8.7	39.8
St. dev.	5.8	22.9	5.0	3.2	10.3	6.8	20.6	3.7	3.6	10.2
Range	9-46	54-210	1-46	1-24	11-82	10-56	65-204	1-35	1-28	16-89
n	1108	1093	1101	1107	1104	1108	1095	1100	1110	1101
DMSO control data
	without metabolic activation (-S9 Mix)					with metabolic activation (+S9 Mix)
	TA98	TA100	TA1535	TA1537	E. coli	TA98	TA100	TA1535	TA1537	E. coli
Mean	21.5	98.6	11.6	7.1	32.6	29.1	109.4	11.3	8.7	39.0
St. dev.	5.8	22.3	4.9	3.3	10.1	7.1	21.8	3.6	3.6	10.1
Range	6-55	40-217	1-43	1-25	7-81	11-67	53-229	2-27	1-29	9-85
n	1183	1173	1179	1185	1182	1182	1170	1178	1185	1176
Distilled water control data
	without metabolic activation (-S9 Mix)					with metabolic activation (+S9 Mix)
	TA98	TA100	TA1535	TA1537	E. coli	TA98	TA100	TA1535	TA1537	E. coli
Mean	23.3	103.0	11.5	7.5	34.7	30.9	112.1	11.1	9.1	41.0
St. dev.	6.3	24.2	4.9	3.2	10.3	7.3	23.0	3.5	3.4	10.2
Range	11-45	45-215	2-47	2-24	12-84	10-53	64-222	3-39	1-20	17-91
n	201	1092	1101	201	1122	201	1101	1107	201	1116
DMF control data
	without metabolic activation (-S9 Mix)					with metabolic activation (+S9 Mix)
	TA98	TA100	TA1535	TA1537	E. coli	TA98	TA100	TA1535	TA1537	E. coli
Mean	20.3	89.2	11.1	6.8	35.3	28.4	99.6	10.9	7.9	38.6
St. dev.	5.6	18.0	4.8	3.0	12.5	7.0	19.2	3.5	3.0	10.2
Range	8-38	54-152	1-34	1-19	16-99	13-49	60-156	3-20	1-23	17-76
n	198	198	198	195	192	198	198	198	195	192
Acetone control data
	without metabolic activation (-S9 Mix)					with metabolic activation (+S9 Mix)
	TA98	TA100	TA1535	TA1537	E. coli	TA98	TA100	TA1535	TA1537	E. coli
Mean	22.6	97.6	11.9	7.2	35.9	29.3	108.9	11.2	8.4	41.7
St. dev.	5.2	16.1	6.0	2.9	9.8	6.8	14.5	3.4	3.1	9.1
Range	11-39	62-160	4-49	1-17	17-62	15-52	66-177	4-22	2-19	17-69
n	224	225	225	225	222	225	225	225	225	225
Positive reference control data
	without metabolic activation (-S9 Mix)					with metabolic activation (+S9 Mix)
	TA98	TA100	TA1535	TA1537	E. coli	TA98	TA100	TA1535	TA1537	E. coli
Mean	351.9	1263.9	1184.3	466.2	1054.1	2425.9	2445.4	238.0	223.1	267.3
St. dev.	124.9	211.5	217.0	186.0	144.8	346.2	316.9	150.5	61.0	123.4
Range	152-2336	880-2120	208-2440	149-2104	516-1708	312-4918	1192-5240	101-2216	117-838	125-2512
n	1108	1095	1101	1107	1107	1108	1095	1104	1110	1101

TA98:Salmonella typhimuriumTA98, TA100:Salmonella typhimuriumTA100, TA1535:Salmonella typhimuriumTA1535, TA1537:Salmonella typhimuriumTA1537, E. coli:Escherichia coliWP2uvrA;n: numberof cases

 

Conclusions:

Under the experimental conditions of the study, the test item Sulfates of potassium, sodium and calcium, by- product from fermentation (Batch Number: EXP LIberica Lote: AI16503047) has no mutagenic activity on the growth of the bacterial strains used in this study.

Executive summary:

The purpose of this GLP study was to evaluate the mutagenic potential of the test item by measuring its ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli WP2 uvrA strain in the presence and absence of activated rat liver S9 fraction.

The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537) and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9 fraction) prepared from the livers of phenobarbital/beta-naphthoflavone- induced rats.

The study included a Preliminary Compatibility Test, a Preliminary Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Method) and a Confirmatory Mutation Test (Pre-Incubation Method).

Based on the results of the Compatibility Test, the test  item  was  dissolved  in  Distilled water at a concentration of 50 mg/mL. Concentrations of 5000; 2500; 1000; 316; 100; 31.6 and 10 µg/plate were examined in the Range Finding Test in tester strains Salmonella typhimurium TA100 and TA98 in the absence and presence of metabolic activation. Based on the results of the Range Finding Test, the test item concentrations  in  the  Initial  Mutation  Test  were  5000,  1581,  500,  158.1,  50  and 15.81 μg/plate. In  the Confirmatory Mutation Test, they were 5000, 1581, 500, 158.1,  50, 15.81 and 5 μg/plate.

No precipitate was detected on the plates in the Preliminary Concentration Range Finding Tests and in the main tests in all examined bacterial strains with and without metabolic activation.

Inhibitory, cytotoxic effect of the test item was not detected in the Initial Mutation Test and Confirmatory Mutation Test.

In the Initial Mutation Test and Confirmatory Mutation Test, the number of revertant colonies did not show any biologically relevant increase compared to the solvent controls. There were no consistent dose-related trends and no indication of any treatment-related effect.

The mean values of revertant colonies of the negative (vehicle/solvent) control plates were within the historical control range, the reference mutagens showed the expected increase in the number of revertant colonies, the viability of the bacterial cells was checked by a plating experiment in each test. At least five analyzable concentrations were presented in all strains of the main tests, the examined concentration range was considered to be adequate. The study was considered to be valid.

Under the experimental conditions of the study, the test item Sulfates of potassium, sodium and calcium, by- product from fermentation (Batch Number: EXP LIberica Lote: AI16503047) has no mutagenic activity on the growth of the bacterial strains used in this study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Read-across from the source analogue Calcium sulfate (CAS 7778-18-9). The substance did not induce mutations in the in vivo system used (in vivo micronucleus test).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro Genotoxicity

A GLP Ames study (OECD TG 471, CiToxLAB Hungary Ltd, 2017) was performed on the following bacterial strains: TA98, 100, 1535, 1537 and E. Coli WP2. Bacteria were exposed with and without metabolic activation to different concentrations up to 5000 µg/plate and the number of revertant colonies was counted. No increase in the revertant colony number was observed in any of the experimental conditions. Based on the results of this study, Sulfates of potassium, sodium and calcium, by-product from fermentation was not considered to be mutagenic in a bacterial reverse mutation test.

Reliable experimental data for mutagenicity on bacteria are also available on the three analogue substances: Potassium sulfate (CAS 7778-80-5), Sodium sulfate (CAS 7757-82-6) and Calcium sulfate (CAS 7778-18-9).

Ames tests were conducted for calcium (CAS 7778-18-9), potassium sulfate (CAS 7778-80-5) (OECD 471; NIER, 2001 and Bioreliance, 2001 respectively) and Sodium sulfate (CAS 7757-82-6) (according to publication of Ames et al. 1975, Bayer AG, 1988). Bacterial strains were exposed to concentrations up to 5000 µg/plate with and without metabolic activation. No increase in the number of revertant colonies was observed for the three substances. Based on the results of the three studies, the three analogues were not considered to be mutagenic in a bacterial reverse mutation test.

The results of the in vitro gene mutation studies in bacteria on both the three source substances and the target substance are indicative of a similar behavior regarding mutagenicity in the bacterial system. The very similar behavior in the bacterial mutagenicity potential further reinforces the read across justification applied for the systemic toxicity endpoints.

No in vitro gene mutation in mammalian cells or in vitro cytogenicity /chromosomal aberrations tests are available for Sulfates of potassium, sodium and calcium, by-product from fermentation. However, data are available on the three sulfate salts: Sodium sulfate (CAS 7757-82-6), Potassium sulfate (CAS 7778-80-5) and Calcium Sulfate (CAS7778-18-9) and a read across approach was used to fulfill data requirement for this endpoint.

The read across approach is based on the results of the transformation/dissolution study performed on the registered substance (Envigo Research Limited, 2016). This study demonstrated that Sulfates of potassium sodium and calcium, by-product from fermentation is completely transformed/dissolved (98.1%) within 30 minutes at pH 2 and at 37 °C. Hence, upon systemic uptake into the body, the registered substance will be present in dissolved form as sulfate anions and potassium, sodium and calcium cations. Furthermore, the three salts Sodium sulfate, Potassium sulfate and Calcium Sulfate are also completely dissociated in aqueous media in Sulfate anions and the respective cations, then this justifies that toxicity data can be read across from the three salts Sulfates of potassium, Sulfates of sodium and Sulfates of calcium to the target substance for systemic toxicity endpoints.

An in vitro gene mutation study on mammalian cells was performed (OECD 476, Harlan laboratories, 2010) for Calcium sulfate (CAS 7778-18-9). L5187Y cells were exposed to the test item at concentrations up to 1361 µg/mL with and without metabolic activation. At the end of the study, the test item did not induce mutant frequency at the locus TK +/-. An OECD 476 was similarly conducted for Sodium sulfate (CAS 7757-82-6) at concentrations up to 1420 µg/mL. The results were negative with and without metabolic activation. A read across key study (OECD TG 476; NOTOX B.V., 2010) is available for Potassium Sulfate (CAS 7778-80-5). L5187Y cells were exposed Magnesium Sulphate Anhydrous at concentrations up to 17 µg/mL with and without metabolic activation. The substance did not induce mutant frequency at the locus TK +/- both with and without metabolic activation. An OECD 473 study (Harlan Cytotest Cell Research, 2010) is available for Sodium sulfate (CAS 7757-82-6). Chinese hamster lung fibroblast (V79) cells were exposed to the test item at concentrations up to 1420 µg/mL with and without metabolic activation system. At the end of the exposure period, no structural aberration related to the treatment was observed. A similar study was conducted with Potassium sulfate (CAS 7778-18-9) (OECD 473, BioReliance, 2001) at concentrations up to 1740 µg/mL using CHO cells. No structural aberration was observed with and without metabolic activation. No OECD 473 study was performed on Calcium sulfate (CAS 7778-18-9) as an in vivo micronucleus study is available for this substance (OECD 474).

In vivo Genotoxicity

One in vivo mutagenicity study (OECD 474, NIER, 2002) is available for Calcium sulfate (CAS 7778-18-9). Mice were exposed to Calcium sulfate at doses of 1250, 2500 and 5000 mg/kg bw. No increase in the micronucleus frequency in polychromatic erythrocytes was observed in bone marrow. Under the experimental conditions of this study, Calcium sulfate was not clastogenic or aneugenic in mice

The results of the above in vitro and in vivo genetic toxicity studies are indicative of a similar low toxicity for the three analogue substances. Hence, based on these data and on the analogy approach, Sulfates of potassium, sodium and calcium, by-product from fermentation is not considered to be genotoxic.

Justification for classification or non-classification

The target substance was negative in a bacterial reverse mutation test. While no in vitro or in vivo genotoxicity studies in mammalian cells or organisms are available for the target substance, all the three read-across substances showed no genotoxicity in the in vitro or in vivo tests. Based on the read-across rationale and the weight of evidence, the substance is not considered to meet the criteria for classification according to Regulation (EC) No 1272/2008.