Reaction products of sodium glucoheptonate with manganese sulfate and sodium hydroxide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted by the Council on July 21, 1997, and corrected on June 26, 2020

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reaction products of sodium glucoheptonate with manganese sulfate and sodium hydroxide

EC Number:

946-400-7

Molecular formula:

Not specified (UVCB substance).Molecular formula of the main substance:C14H30O22SMn2

IUPAC Name:

Reaction products of sodium glucoheptonate with manganese sulfate and sodium hydroxide

Test material form:

solid: granular

Method

Target gene:

his

Species / strain

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:
- source of S9: Meshram genotox services (Nagpur, Maharashtra)
- method of preparation of S9 mix: The S9 fraction is buffered and supplemented with the essential co-factors β-NADP and glucose-6-phosphate to form the “S9 mix”.

Co-factor Mix
D-Glucose–6-phosphate: 0.80 g
beta Nicotinamide adenine dinucleotide Phosphate (beta NADP): 1.75 g
Magnesium chloride: 0.90 g
Potassium chloride: 1.35 g
Sodium phosphate, dibasic: 6.40 g
Sodium phosphate, monobasic: 1.40 g
Distilled water: 450 mL
The prepared co-factor mix was dispensed to suitable volumes and stored below 0 °C.

S9 Mix (10 mL)
Constituent 5% v/v S9 mix (10 mL) 10% v/v S9 mix (10 mL)
Co-factor mix 9.5 mL 9.0 mL
S9 fraction 0.5 mL 1.0 mL

The S9 mix was prepared fresh by adding the required quantity of S9 fraction to the thawed co-factors and maintained in an ice bath. The remaining portions of the S9 mix were discarded.

- concentration or volume of S9 mix and S9 in the final culture medium: A volume of 0.1 mL of S9 mix (5% v/v S9 mix for initial toxicity-mutation test and 10% v/v S9 mix for confirmatory mutation test) prepared for treatment was added aseptically to 2 mL of the top agar, mixed thoroughly, and this mixture was poured onto the MGA plate.
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): An acceptable positive control in the presence of S9 for a specific strain was evaluated as having demonstrated integrity of the S9 mix and ability of the tester strain to detect a mutagen.

Test concentrations with justification for top dose:

Initial Toxicity-Mutation Test: 0, 1.5, 5, 15, 50, 150, 500, 1500, 5000 µg/plate
Confirmatory Mutation Test: 0, 156.25, 312.5, 625, 1250, 2500, 5000 µg/plate
Precipitation was not observed at the tested concentration of 5000 µg/plate in a solubility and precipitation test. Hence, 5000 µg/plate was selected as the highest concentration to be tested for initial toxicity-mutation test both in the absence and presence (5% v/v S9 mix) of metabolic activation.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: distilled water

- Justification for choice of solvent/vehicle: The test item was found to be soluble in distilled water (stock A, 50000 µg/mL). Hence, distilled water was selected as vehicle for treatment. A volume of 100 µL from stock A (50000 µg/mL) was added to 2 mL of top agar and poured on minimal glucose agar plate, to assess the precipitation. Precipitation was not observed at the tested concentration of 5000 µg/plate.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

mitomycin C

other: 2-Aminoanthracene

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): duplicate (initial toxicity-mutation test), triplicate (confirmatory mutation test)
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 1 - 3 x 10E9 bacterial cells/mL
- Test substance added in agar (plate incorporation)

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 48 h
- Harvest time after the end of treatment (sampling/recovery times):

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition

METHODS FOR MEASUREMENTS OF GENOTOXICIY
increase in the number of revertants

Evaluation criteria:

A result is considered positive if concentration-related increase over the range tested and/or a reproducible increase at one or more concentrations in the number of revertant colonies per plate in at least one strain with or without metabolic activation system.

Strains TA1535, TA1537
Data sets were judged positive, if the increase in mean revertants at the peak of the dose response was equal to or greater than 3.0 times the mean negative control value.

Strains TA98, TA100, and TA102
Data sets were judged positive, if the increase in mean revertants at the peak of the dose response was equal to or greater than 2.0 times the mean negative control value.

Statistical analysis was used as an aid in the evaluation of dose response.

A response that did not meet all three of the above criteria (magnitude, concentration-responsiveness, reproducibility) was not evaluated as positive.

Negative results obtained in the initial toxicity-mutation test were confirmed by a second trial, using the same method as specified above, with an alteration in concentration spacing.

Statistics:

Simple linear regression analysis was performed for tester strains TA1537, TA1535, TA98, TA100, and TA102, separately, to assess the dose dependent nature of any increase in revertant colonies.

Results and discussion

Test results

Test results 1

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

Test results 2

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

Test results 3

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

Test results 4

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

Test results 5

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

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Possibility of evaporation from medium: no
- Water solubility: high
- Precipitation and time of the determination: no

RANGE-FINDING/SCREENING STUDIES (if applicable): Before commencing the confirmatory mutation test, the test item was tested for the initial toxicity-mutation test using all five tester strains of Salmonella typhimurium (TA1537, TA1535, TA98, TA100, and TA102). The experiment was conducted using the plate incorporation method and tested at concentrations 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate. The experiment was conducted both in the absence and presence of the metabolic activation system (5% v/v S9 mix).

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

For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship where possible
- Statistical analysis; p-value if any
- Any other criteria: e.g. GEF for MLA

Ames test:
- Signs of toxicity: No, normal bacterial background lawn was observed up to the concentration of 5000 µg/plate, in all tester strains (TA1537, TA1535, TA98, TA100, and TA102) in the absence and presence of the metabolic activation system (5% v/v S9 mix).
- Individual plate counts: Please refer to 'Any other information on results incl. tables'
- Mean number of revertant colonies per plate and standard deviation: Please refer to 'Any other information on results incl. tables'

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 'Attached illustration'
- Negative (solvent/vehicle) historical control data: Please refer to 'Attached illustration'

Any other information on results incl. tables

TABLE 1: Mean Count of His⁺ Revertant Colonies in Negative Control, Positive Controls and Treatment Plates in the Absence of Metabolic Activation (Initial Toxicity-Mutation Test)

Concentration of DABQUEL COMPLEX MnP (µg/plate)	His^+ Revertant Colonies/Plate (Absence of Metabolic Activation)
	TA1537		TA1535		TA98		TA100		TA102
	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD
NC (DW)	9.50	0.71	15.00	1.41	27.50	3.54	131.00	1.41	227.50	12.02
1.5	8.00	1.41	13.00	1.41	27.00	2.83	118.50	7.78	228.00	9.90
5	10.50	2.12	14.00	0.00	27.00	1.41	132.50	0.71	216.50	9.19
15	8.50	3.54	16.50	6.36	26.00	2.83	123.00	2.83	225.50	10.61
50	9.00	0.00	14.00	1.41	24.50	0.71	137.50	0.71	227.00	8.49
150	8.50	2.12	14.00	1.41	23.50	0.71	129.00	9.90	215.50	6.36
500	8.50	0.71	14.50	3.54	26.00	0.00	122.50	12.02	224.00	7.07
1500	8.50	3.54	14.00	1.41	25.00	4.24	124.50	4.95	230.50	3.54
5000	8.00	2.83	16.00	0.00	25.50	2.12	126.00	1.41	234.50	9.19
PC	185.00	11.31	270.00	9.90	386.50	16.26	830.00	59.40	1025.50	6.36
2AA	NA	NA	NA	NA	NA	NA	139.50	2.12	NA	NA

Key:   SD = Standard Deviation, NC = Negative Control, DW = Distilled Water, PC = Positive Control {TA1537 = 9-Aminoacridine Hydrochloride Monohydrate (75 µg/plate), TA1535 = Sodium Azide (0.5 µg/plate), TA98 = 2-Nitrofluorene (7.5 µg/plate), TA100 = Sodium Azide (5 µg/plate), TA102 = Mitomycin-C (0.5 µg/plate)}, 2AA = 2-Aminoanthracene (5 µg/plate for TA100), NA = Not Applicable.

 

TABLE 2: Mean Count of His⁺ Revertant Colonies in Negative Control, Positive Control and Treatment Plates in the Presence of Metabolic Activation (Initial Toxicity-Mutation Test)

Concentration of DABQUEL COMPLEX MnP (µg/plate)	His^+ Revertant Colonies/Plate [Presence of Metabolic Activation (5% v/v S9 mix)]
	TA1537		TA1535		TA98		TA100		TA102
	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD
NC (DW)	9.00	2.83	15.50	4.95	26.00	5.66	126.00	16.97	224.00	1.41
1.5	8.00	1.41	14.50	3.54	25.00	7.07	132.50	3.54	231.00	7.07
5	7.00	0.00	16.00	1.41	25.00	1.41	124.50	2.12	216.00	5.66
15	8.50	0.71	19.50	0.71	26.50	2.12	121.00	18.38	221.00	15.56
50	9.00	1.41	13.50	2.12	24.00	5.66	125.50	7.78	238.00	14.14
150	7.50	0.71	15.00	1.41	25.00	2.83	129.50	12.02	219.50	27.58
500	10.00	1.41	13.50	0.71	27.00	1.41	132.50	13.44	217.00	12.73
1500	8.50	0.71	16.50	0.71	25.00	4.24	129.50	10.61	231.00	18.38
5000	10.00	2.83	15.50	4.95	26.50	3.54	137.50	4.95	219.00	22.63
PC	208.00	42.43	281.00	12.73	385.50	64.35	806.50	36.06	1,006.00	12.73

Key:   SD = Standard Deviation, NC = Negative Control, DW = Distilled Water, PC = Positive control {2AA = 2-Aminoanthracene (10 µg/plate for TA1537, TA1535, TA102 and 5 µg/plate for TA98 and TA100)}.

 

 

TABLE 3: Mean Count of His⁺ Revertant Colonies in Negative Control, Positive Controls and Treatment Plates in the Absence of Metabolic Activation (Confirmatory Mutation Test)

Concentration of DABQUEL COMPLEX MnP (µg/plate)	His^+ Revertant Colonies/Plate (Absence of Metabolic Activation)
	TA1537		TA1535		TA98		TA100		TA102
	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD
NC (DW)	10.00	2.00	15.67	3.79	25.67	2.52	124.00	16.64	194.67	35.95
156.25	10.00	2.65	17.67	1.15	25.67	7.02	128.33	12.50	227.00	4.58
312.5	9.00	1.73	15.00	1.73	25.67	2.52	132.33	8.14	231.00	11.53
625	9.00	1.73	14.33	3.21	26.00	3.61	132.33	8.96	229.67	1.53
1250	9.33	1.15	17.33	4.16	25.00	7.55	136.67	5.86	210.33	13.01
2500	9.33	1.53	16.33	5.77	27.33	3.21	131.33	12.10	216.67	11.06
5000	10.33	2.52	16.33	3.06	25.00	2.00	138.33	3.21	224.67	4.04
PC	189.67	24.58	274.67	61.98	373.67	45.45	728.00	36.66	1032.33	13.01
2AA	NA	NA	NA	NA	NA	NA	135.00	9.17	NA	NA

Key:   SD = Standard Deviation, NC = Negative Control, DW = Distilled Water, PC = Positive Control {TA1537 = 9-Aminoacridine Hydrochloride Monohydrate (75 µg/plate), TA1535 = Sodium Azide (0.5 µg/plate), TA98 = 2-Nitrofluorene (7.5 µg/plate), TA100 = Sodium Azide (5 µg/plate), TA102 = Mitomycin-C (0.5 µg/plate)}, 2AA = 2-Aminoanthracene (5 µg/plate for TA100), NA = Not Applicable.

  

TABLE 4: Mean Count of His⁺ Revertant Colonies in Negative Control, Positive Control and Treatment Plates in the Presence of Metabolic Activation (Confirmatory Mutation Test)

Concentration of DABQUEL COMPLEX MnP (µg/plate)	His^+ Revertant Colonies/Plate [Presence of Metabolic Activation (10% v/v S9 mix)]
	TA1537		TA1535		TA98		TA100		TA102
	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD
NC (DW)	9.00	2.65	16.33	2.08	26.67	4.04	123.00	18.08	212.67	8.08
156.25	9.00	1.00	15.33	2.52	26.33	6.11	135.00	9.54	214.33	14.98
312.5	9.67	1.15	18.33	3.21	27.00	6.00	123.33	15.31	219.33	8.14
625	8.33	3.21	17.67	2.52	26.67	1.53	130.00	14.73	214.67	15.82
1250	9.00	2.00	17.67	3.51	29.67	3.51	133.33	6.81	227.67	5.03
2500	9.67	2.52	18.33	3.06	27.00	3.00	135.00	6.08	217.00	14.00
5000	9.67	2.52	17.33	3.06	30.67	4.73	129.67	14.98	217.67	13.65
PC	194.67	22.50	325.67	66.94	377.33	32.58	760.33	88.79	1048.67	34.53

Key:   SD = Standard Deviation, NC = Negative Control, DW = Distilled Water, PC = Positive Control {2AA = 2-Aminoanthracene (10 µg/plate for TA1537, TA1535, TA102 and 5 µg/plate for TA98 and TA100)}.

 

Table 5: Individual Plate Count (Initial Toxicity-Mutation Test) 

Absence of Metabolic Activation

Concentration of DABQUEL COMPLEX MnP (µg/plate)	Number of Revertant Colonies
	TA1537		TA1535		TA98		TA100		TA102
	R1	R2	R1	R2	R1	R2	R1	R2	R1	R2
NC (DW)	9	10	14	16	25	30	132	130	236	219
1.5	7	9	14	12	29	25	113	124	235	221
5	9	12	14	14	26	28	132	133	210	223
15	6	11	21	12	28	24	125	121	218	233
50	9	9	13	15	25	24	137	138	233	221
150	10	7	15	13	23	24	122	136	211	220
500	8	9	12	17	26	26	131	114	229	219
1500	11	6	13	15	22	28	121	128	233	228
5000	6	10	16	16	27	24	125	127	228	241
PC	177	193	263	277	375	398	872	788	1021	1030
2AA	NA	NA	NA	NA	NA	NA	141	138	NA	NA

Presence of Metabolic Activation (5% v/v S9 mix)

Concentration of DABQUEL COMPLEX MnP (µg/plate)	Number of Revertant Colonies
	TA1537		TA1535		TA98		TA100		TA102
	R1	R2	R1	R2	R1	R2	R1	R2	R1	R2
NC (DW)	7	11	12	19	30	22	114	138	225	223
1.5	9	7	17	12	20	30	135	130	236	226
5	7	7	15	17	24	26	123	126	212	220
15	9	8	19	20	28	25	108	134	232	210
50	10	8	15	12	28	20	131	120	228	248
150	8	7	14	16	23	27	121	138	200	239
500	9	11	13	14	28	26	142	123	226	208
1500	8	9	16	17	28	22	122	137	218	244
5000	8	12	19	12	24	29	141	134	235	203
PC	178	238	272	290	340	431	832	781	1,015	997

Key: R = Replicate, NC = Negative control, DW = Distilled Water, PC = Positive control {Absence of S9: TA1537 = 9-Aminoacridine hydrochloride monohydrate (75 µg/plate), TA1535 = Sodium azide (0.5 µg/plate), TA98 = 2-Nitrofluorene (7.5 µg/plate), TA100 = Sodium azide (5 µg/plate), TA102 = Mitomycin-C (0.5 µg/plate), 2AA = 2-Aminoanthracene (5 µg/plate for TA100); Presence of S9: 2AA = 2-Aminoanthracene (10 µg/plate for TA1537, TA1535, TA102 and 5 µg/plate for TA98 and TA100)}, NA = Not applicable.

 

 

Table 6: Individual Plate Count (Confirmatory Mutation Test)

Absence of Metabolic Activation

Concentration of DABQUEL COMPLEX MnP (µg/plate)	Number of Revertant Colonies
	TA1537			TA1535			TA98			TA100			TA102
	R1	R2	R3	R1	R2	R3	R1	R2	R3	R1	R2	R3	R1	R2	R3
NC (DW)	10	8	12	13	20	14	26	23	28	131	136	105	166	183	235
156.25	13	8	9	17	19	17	25	33	19	114	134	137	223	232	226
312.5	8	8	11	13	16	16	26	28	23	123	136	138	220	243	230
625	11	8	8	13	18	12	27	22	29	138	122	137	228	230	231
1250	10	8	10	22	14	16	32	26	17	139	130	141	223	197	211
2500	8	11	9	13	23	13	26	31	25	145	122	127	227	205	218
5000	8	10	13	13	19	17	23	25	27	136	142	137	224	221	229
PC	164	213	192	342	220	262	381	415	325	696	720	768	1045	1033	1019
2AA	NA	NA	NA	NA	NA	NA	NA	NA	NA	145	133	127	NA	NA	NA

Presence of Metabolic Activation (10% v/v S9 mix)

Concentration of DABQUEL COMPLEX MnP (µg/plate)	Number of Revertant Colonies
	TA1537			TA1535			TA98			TA100			TA102
	R1	R2	R3	R1	R2	R3	R1	R2	R3	R1	R2	R3	R1	R2	R3
NC (DW)	12	8	7	17	14	18	22	29	29	104	140	125	214	204	220
156.25	8	10	9	13	18	15	21	25	33	140	124	141	202	210	231
312.5	9	11	9	17	16	22	33	27	21	141	115	114	225	223	210
625	6	12	7	20	15	18	25	28	27	113	138	139	211	232	201
1250	7	11	9	21	14	18	30	26	33	141	131	128	223	233	227
2500	12	10	7	21	15	19	30	24	27	139	128	138	211	207	233
5000	7	10	12	20	14	18	27	29	36	113	134	142	220	203	230
PC	169	211	204	402	277	298	400	340	392	788	661	832	1084	1015	1047

Key:   R = Replicate, NC = Negative control, DW = Distilled Water, PC = Positive control {Absence of S9: TA1537 = 9-Aminoacridine hydrochloride monohydrate (75 µg/plate), TA1535 = Sodium azide (0.5 µg/plate), TA98 = 2-Nitrofluorene (7.5 µg/plate), TA100 = Sodium azide (5 µg/plate), TA102 = Mitomycin-C (0.5 µg/plate), 2AA = 2-Aminoanthracene (5 µg/plate for TA100); Presence of S9: 2AA = 2-Aminoanthracene (10 µg/plate for TA1537, TA1535, TA102 and 5 µg/plate for TA98 and TA100)}, NA = Not Applicable.

Applicant's summary and conclusion

Conclusions:

DABQUEL COMPLEX MnP did not induce any significant increase in the number of revertants, in trials with and without S9 mix, in any histidine deficient mutant tester strains of Salmonella typhimurium (i.e., TA1537, TA1535, TA98, TA100, and TA102). Values for the negative control were within the historical control ranges of the laboratory. Positive controls showed an increase in the number of revertant colonies, demonstrating the efficiency of the test system.

Executive summary:

This study was performed to evaluate the mutagenic activity of the test item using the bacterial reverse mutation test on five histidine deficient mutant tester strains of Salmonella typhimurium (i.e., TA1537, TA1535, TA98, TA100, and TA102). The study was done according to OECD Test Guideline 471 under GLP-compliance. The test item was tested in two independent experiments in the absence and presence of metabolic activation.

Bacterial cultures were exposed to the test item at 8 concentration levels (two plates/concentration) ranging from 1.5 to 5000 µg/plate in the initial toxicity-mutation test, using the plate incorporation method. Normal bacterial background lawn was observed up to the concentration of 5000 µg/plate, in all tester strains (TA1537, TA1535, TA98, TA100, and TA102) in the absence and presence of the metabolic activation system (5% v/v S9 mix). No increase in the number of revertant colonies (no mutagenic effect) was observed in the absence and presence of the metabolic activation system (5% v/v S9 mix) in any tester strain.

To confirm the negative results obtained in the initial toxicity mutation test, the confirmatory mutation test was conducted, using the plate incorporation method with an increased S9 concentration, i.e., 10% v/v S9 mix and concentration spacing modification. Bacterial cultures were exposed to the test item at 6 concentration levels (three plates/concentration) ranging from 156.25 to 5000 µg/plate for tester strains TA1537, TA1535, TA98, TA100, and TA102 in the absence and presence (10 % v/v S9 mix) of the metabolic activation. The revertant colonies were scored after 48 hours of incubation at 37 ± 1 °C. The test item did not induce any significant increase in the number of revertants, in trials with and without S9 mix, in any tester strain. Values for the negative control were within the historical control ranges of the laboratory. Positive controls showed an increase in the number of revertant colonies, demonstrating the efficiency of the test system.

All criteria for a valid study were met. Based on the results of this study, under specified experimental conditions, the test item is concluded to be non-mutagenic in the Bacterial Reverse Mutation Test using Salmonella typhimurium.