Oxidation products of D-Glucose with nitric acid, sodium salts

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21/02/2017 - 08/05/2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Oxidation products of D-Glucose with nitric acid, sodium salts

Molecular formula:

Reaction products consisting mainly of sodium salts of D-glucarate, mono, di and tri carboxylic acids and keto gluconates. See remarks

IUPAC Name:

Oxidation products of D-Glucose with nitric acid, sodium salts

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source of test material: The test material was supplied by Rivertop Renewables.
- Lot no: BL04R1D1EA
- Expiration date of the lot/batch: June 30, 2017
- Purity : 97.7% purity of solids, 52.9% solids in water

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient conditions (15-30 °C)

Method

Target gene:

Not applicable

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9 liver homogenate from male Sprague-Dawley rats treated with Aroclor 1254

Test concentrations with justification for top dose:

0.31, 0.63,1.3, 2.5, and 5.0 μL/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: None
Justification for choice of solvent/vehicle: In preliminary solubility testing it was determined that D-Glucose, reaction products with nitric acid and sodium nitrite (1:1), sodium salts was completely soluble in water at 50 μL/mL

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

sodium azide

benzo(a)pyrene

methylmethanesulfonate

other: Cyclophosphamide monohydrate, 9-Aminoacridine hydrochloride monohydrate, 2-Aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: In agar (plate incorporation) and preincubation

DURATION
- Preincubation period: at 37 ± 2°C for 67 to 69 hours

NUMBER OF REPLICATIONS: Duplicate and triplicate plates were used for each treatment
or control condition

DETERMINATION OF CYTOTOXICITY
The cytotoxicity of the test material was assessed in a preliminary assay.

Evaluation criteria:

A negative result (i.e. no evidence of genotoxicity) is concluded if there is no significant increase (p > 0.01) in the mean number of revertant colonies per plate in comparison with the concurrent and historical negative control data. A negative result indicates that the test item is non-mutagenic in S. typhimurium or E. coli
There are several criteria for determining a positive result, such as a concentration-related increase over the range tested and/or a reproducible statistically significant increase (p ≤ 0.01) at one or more concentrations in the number of revertant colonies per plate in comparison with the concurrent negative control in at least one strain with or without metabolic activation system. For cases where historical data is available, a positive result should present an increase in the number of revertant colonies per plate in comparison with the historical data for at least one experimental condition. Biological relevance of the results will be considered first. A statistical method may be used as an aid in evaluating the test results but it will not be the only determining factor.
A positive result indicates that the test item induces point mutations in S. typhimurium and/or E. coli. There is no requirement for verification of a clear positive response . In such an event only one experiment, plate incorporation or preincubation, may be performed and reported.

An equivocal result is concluded if no definitive judgment can be made to fit the above criteria, even after repeated experiments. An equivocal result indicates that a definitive result cannot be made by performing the bacterial reverse mutation assay under the conditions described in this study plan.
In the event that the controls fall slightly outside the normal (historical) range, the Study Director will be allowed discretion in accepting the results of the experiment as valid based on the biological significance.

Statistics:

Statistical analysis was applied to test item treatment conditions for which the range of colony counts (mean ± SD) exceeded the upper limit of the concurrent negative control range. The colony counts were square root-transformed to normalize the data prior to performing statistical analysis (5). Data that passed normality testing was evaluated using ANOVA with Dunnett’s method. Results were considered significant if p ≤ 0.01 compared to the concurrent negative control.
Statistical analysis was applied to the transformed colony counts of all positive controls using a t-test. Results were considered significant if p ≤ 0.01 compared to the concurrent negative control

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium, other: TA98, TA100, TA1535 and TA1537

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:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

E. coli WP2 uvr A

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:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: In the preliminary study, test item precipitation was not observed at any concentration in the presence and absence of S9 metabolic activation. Test item toxicity, evidenced by a reduction in the background lawn or a reduction of colony counts when compared to the concurrent negative control, was not observed for any condition or strain.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%): Please see appendix 3 of report attached

Remarks on result:

other: No indicaton of mutagenicity

Any other information on results incl. tables

Mean revertant counts: main study (plate incorporation assay)

Concentration (µL/plate)	TA98		TA100		TA1535		TA1537		WP2 uvrA
	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
-Control	23	31	111	125	11	17	17	24	28	38
Untreated control	24		114		16		13		22
0.31	22	32	110	129	15	17	17	15	30	36
0.63	21	30	111	121	13	17	16	13	25	29
1.3	22	31	115	128	12	10	20	18	26	35
2.5	21	27	114	112	15	18	15	22	32	36
5.0	24	28	103	115	15	16	19	17	29	38
+Control 2-NF, 5 μg	6851
B[α]P, 5 μg		461		1832				229
NaAz, 5 μg			1922		1760
CP, 200 μg						170
9-AA, 100 μg							2894
MMS, 1 μL									440
2-AMA, 100 μg										158

 

Mean revertant counts: main study (pre-incubation assay)

Concentration (µL/plate)	TA98		TA100		TA1535		TA1537		WP2 uvrA
	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
-Control	27	26	106	116	18	16	16	20	26	35
Untreated control	26		108		14		13		23
0.31	21	31	113	114	15	14	17	18	30	32
0.63	19	29	110	94	17	17	16	19	27	31
1.3	26	30	109	113	14	15	16	19	30	33
2.5	19	29	120	119	14	13	14	16	24	33
5.0	222	26	121	102	14	13	14	13	23	28
+Control 2-NF, 5 μg	4922
B[α]P, 5 μg		628		1703				217
NaAz, 5 μg			2049		1734
CP, 200 μg						304
9-AA, 100 μg							1414
MMS, 1 μL									1258
2-AMA, 100 μg										189

 

2-NF: 2-Nitrofluorene

NaAz: Sodium azide B[α]P: Benzo[α]pyrene

9-AA: 9-Aminoacridine CP: Cyclophosphamide

2-AMA: 2-Aminoanthracene MMS: Methyl methanesulfonate

Applicant's summary and conclusion

Conclusions:

D-Glucose, reaction products with nitric acid and sodium nitrite (1:1), sodium salts was not mutagenic to S. typhimurium strains TA98, TA100, TA1535, TA1537 and E. coli strain WP2 uvrA, up to the maximum recommended exposure concentration of 5 μL/plate, under the conditions of the test.

Executive summary:

D-Glucose, reaction products with nitric acid and sodium nitrite (1:1), sodium salts and its potential metabolites were evaluated for their potential to induce point mutations in Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and Escherichia coli strain WP2 uvrA. The experimental design followed the OECD Guideline for Testing of Chemicals - 471, Bacterial Reverse Mutation Test. Based on preliminary testing, the concentration levels of the test item in the main study plate incorporation and pre-incubation tests were designed to reach the maximum recommended exposure concentration of 5 μL/plate. In this study, test item precipitation was not observed at any concentration in the presence and absence of S9 metabolic activation. Test item toxicity, evidenced by a reduction in the background lawn or a reduction of colony counts when compared to the concurrent negative control, was not observed for any condition or strain.

For the main study (plate incorporation test), there were five concentrations available for evaluation of mutagenicity for all conditions: 5.0, 2.5, 1.3, 0.63 and 0.31 μL/plate. The colony counts per plate of D-Glucose, reaction products with nitric acid and sodium nitrite (1:1), sodium salts were all within or below the concurrent negative control range for all tester strains and conditions. The more sensitive pre-incubation test was performed to confirm the results of the plate incorporation test. There were five concentrations available for evaluation for mutagenicity for all conditions: 5.0, 2.5, 1.3, 0.63 and 0.31 μL/plate. The colony counts per plate of D-Glucose, reaction products with nitric acid and sodium nitrite (1:1), sodium salts were all within or below the concurrent negative control range for all tester strains and conditions with one exception (WP2 uvrA without S9). However, this slight increase in colony counts was not statistically significant (p > 0.01). In addition, the mean transformed colony counts for WP2 uvrA without S9 in the pre-incubation test were all within the historical negative control range. Therefore, the test item was considered to be negative for mutagenicity. The genotyping, concurrent negative/positive/spontaneous reversion controls and results for the test item were all accepted as valid.

D-Glucose, reaction products with nitric acid and sodium nitrite (1:1), sodium salts was not mutagenic to S. typhimurium strains TA98, TA100, TA1535, TA1537 and E. coli strain WP2 uvrA, up to the maximum recommended exposure concentration of 5 μL/plate, under the conditions of the test.