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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
07/04/2015 to 24/04/2015
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report):Gluconic acid technical 50/5
- Molecular formula (if other than submission substance):
- Molecular weight (if other than submission substance):base form:196.2 g/mol
- Smiles notation (if other than submission substance):O[C@@H]([C@@H]([C@@H](CO)O)O)[C@H](C(O)=O)O
- InChl (if other than submission substance):InChI=1/C6H12O7/c7-1-2(8)3(9)4(10)5(11)6(12)13/h2-5,7-11H,1H2,(H,12,13)/t2-,3-,4+,5-/m1/s1
- Structural formula attached as image file (if other than submission substance): see Fig.
- Substance type:chemical
- Physical state:colorless to yellowish liquid
- Analytical purity:52% W/W
- Impurities (identity and concentrations):unknown
- Composition of test material, percentage of components:52% W/WGluconic acid
- Isomers composition:unknown
- Purity test date:52% W/W
- Lot/batch No.:SX4G6
- Expiration date of the lot/batch:23/07/15 (retest date)
- Radiochemical purity (if radiolabelling):-
- Specific activity (if radiolabelling):-
- Locations of the label (if radiolabelling):-
- Expiration date of radiochemical substance (if radiolabelling):-
- Stability under test conditions:6 months (up to 23/07/15 for batch SX4G6)
- Storage condition of test material:room temperature (+20°±5°C)
- Other:-

Method

Target gene:
His gene
Species / strain
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
rat S9-mix
Test concentrations with justification for top dose:
preliminary toxicity assay: 5000 - 1500 - 500 - 150 - 50 µg/plate with and without metabolic activation
Assay 1 and Assay 2: 5000 - 1500 - 500 - 150 - 50 µg/plate with and without metabolic activation
Vehicle:
- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: maximum solubility obtained in this solvent

Gluconic acid technical 50/50 was dissolved in water (sterile water for irrigation, qs a maximal initial concentration of 50 mg/mL of Gluconic acid in order to obtain the top dose of 5000 µg/plate when added at 100 µL/plate.
Successive dilutions were also prepared with water and used at 100 µL/plate.
Controls
Negative controls:
no
Solvent controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Remarks:
Without S9-mix: sodium azide (TA1535, TA100); 9-amino-acridine (TA1537); 2-nitro fluorene (TA98); mitomycin C (TA102)/ With S9-mix: 2-anthramine (TA1535, TA1537, TA98, TA100); benzo[a]pyrene (TA102)
Details on test system and conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation (assay 2 in presence of S9-mix)

DURATION
- Preincubation period: 60 min
- Exposure duration: 44 hours
- Expression time (cells in growth medium):-
- Selection time (if incubation with a selection agent):-
- Fixation time (start of exposure up to fixation or harvest of cells):-

SELECTION AGENT (mutation assays): medium without histidine
SPINDLE INHIBITOR (cytogenetic assays):-
STAIN (for cytogenetic assays):-

NUMBER OF REPLICATIONS: 2 assays both with and withoutS9-mix (the 2nd assay with S9-mix was perfromùed following the pre-incubtion method) / 3 plates per dose (6 for solvent control)

NUMBER OF CELLS EVALUATED:-

DETERMINATION OF CYTOTOXICITY
- Method: decrease in background growth / decrease in number of revertants

OTHER EXAMINATIONS:
- Determination of polyploidy:-
- Determination of endoreplication:-
- Other:

OTHER:-
Evaluation criteria:
Criteria based on biological significance:
Assessment of mutagenic response considering fold change:
Strains TA1535, TA1537 A test item causing a positive response proportional to the dose for at least 3 doses with, for the highest increase, a value greater than or equal to 3 times the value for the solvent control, is considered positive in the assay.
Strains TA98, TA100, TA102: A test item causing a positive response proportional to the dose for at least 3 doses with, for the highest increase, a value greater than or equal to 2 times the value for the solvent control, is considered positive in the assay.

Comparison to historical control data:
In some borderline cases, an additional criterion to be considered is the comparison between the number of revertants induced by the test item and the laboratory historical control data. Indeed, an increase in each individual value that is above the highest value of corresponding historical control data can help supporting a conclusion such as “equivocal” or “weak” mutagen.

Reproducibility:
If a test item causes a positive response during a single assay and that result cannot be reproduced in at least 1 independent assay, the initial positive result may be considered as not significant.

Providing that all acceptability criteria are fulfilled, a test item is considered to be clearly positive if, in any of the experimental conditions examined all the following criteria are fulfilled:
-at least one of the test doses exhibits a biologically significant increase (2 or 3 fold increase in the mean number of revertants depending on the strain) compared with the concurrent negative control and,
- the increase is dose-related,
- and the mean numbers of revertants of the test doses are outside the distribution of negative control data
The test item is then considered able to induce mutations in this test system.
Statistics:
Data are analysed by means of Dunnett's method (Mahon et al, 1989) allowing the comparison of the mean value for each dose to the mean value for the corresponding solvent control.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not tested
- Effects of osmolality: not tested
- Evaporation from medium: not tested
- Water solubility: soluble
- Precipitation: none
- Other confounding effects: none

RANGE-FINDING/SCREENING STUDIES: a preliminary toxicity study was performed to determine the dose to be tested in the main mutagenicity assays

COMPARISON WITH HISTORICAL CONTROL DATA: in both mutagenicity assay, the mean number of revertants in the solvent (and positive) control or for the doses tested of the test item) were comprised in the intervals of historical data of negative (or positive) controls of the laboratory

ADDITIONAL INFORMATION ON CYTOTOXICITY: In the preliminary toxicity assay or in both mutagenicity assays, no toxicity was noted, either with or without metabolic activation, when evaluating by the micrscopical evaluation of the bakgropund growth.

Any other information on results incl. tables

In two independent assays performed either with or without metabolic activation (the second assay with S9-mix was performed according to the pre-incubation protocol), no statistically or biologically significant increases in the mean number of revertants were noted in the five Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and TA102 tested, in the presence of Gluconic acid technical 50/50.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The mutagenic activity of the test item Gluconic acid technical 50/50 (Batch SX4G6) sponsored by Members of the Gluconic Acid and its Derivatives REACH Consortium was assessed by means of the Ames’ test in the five Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and TA102 tested either in presence or in absence of metabolic activation, in two independent assays according to OECD guideline (OECD 471, 1997).

The control of concentrations of Gluconic acid in treatment preparations was performed in a GLP-compliant laboratory following a validated method. The results are considered as reliable.
The treatment solutions used in the frame of the main assays were properly prepared (except a slight deviation from the expected value below the range -10% without any incidence in a mutagenicity point of view). This deviation was thus considered without any impact on the general conclusion of the current study.
In addition, Gluconic acid was not detected in the solvent.

The validity criteria for the assay were considered as fulfilled. The study was thus valid.

Under these experimental conditions, no mutagenic activity was revealed.
Executive summary:

Bacterial Reverse Mutation Test

On five strains of Salmonella typhimurium His-using B.N. AMES' Technique

 

SUMMARY

SPONSOR                                  : Members of the Gluconic Acid and its Derivatives REACH Consortium

TEST ITEM                               : Gluconic acid technical 50/50

BATCH NUMBER                    : SX4G6

STUDY LOCATION                  : INSTITUT PASTEUR DE LILLE

                                                                       Genetic Toxicology Laboratory

                                                                       1, rue du Professeur Calmette - B.P. 245

                                                                       59019CEDEX

 

THIS STUDY WAS CARRIED OUT IN COMPLIANCE WITH GOOD LABORATORY PRACTICE REGULATIONS

 

Study initiation date (date Study Director signed Study Plan):                                                07/04/2015

Experimental start date (weighing of the test item):                                                                07/04/2015

Experimental completion date:                                                                                              24/04/2015

Study completion:                                                                                                                22/02/2016

 

AIM

The search for any mutagenic activity of Gluconic acid technical 50/50 (BatchSX4G6) sponsored byMembers of the Gluconic Acid and its Derivatives REACH Consortiumwas studied by means of the Ames’ test (Salmonella his-/microsome system) in compliance with the Commission Regulation EC 440/2008 and the OECD Guideline 471.

 

METHODS

Strains used                                : TA1535, TA1537, TA98, TA100, TA102

Solvent                                       : water

Purity                                          : 52% w/w

Stability in the solvent                  : at 0.5 mg/mL: 30 days atca.< -18°C

                                                     at 200 mg/mL: 8 days atca.< -18°C

 

TOXICITY ASSAY

Preliminary test of toxic activity   : carried out on 5 strains both with and without metabolic activation using hepatic microsomes from rat livers induced by Aroclor 1254

                                                     Incubation period:ca.44 h

Sterility test                                 : absence of contamination

Limiting factor for maximum dose: maximum dose according to OECD Guideline,i.e.5000 µg/plate

Doses used in toxicity assay       :expressed asµg/plate of pureGluconic acid

The examination of the background growth demonstrated that Gluconic acid technical 50/50 induced only a slight toxicity at 1500 and 5000 µg/plate in strain TA1537 in presence of metabolic activation and at 5000 µg/plate in strain TA102 with metabolic activation. No other sign of toxicity was noted.

Otherwise, no precipitate was noted whatever the tested condition.

 

Therefore, the maximum dose retained for the first mutagenicity assay was 5000 µg/plate in all strains both with and without metabolic activation.

MUTAGENICITY ASSAYS

Mutagenicity test                         : carried out on 5 strains both with and without metabolic activation using hepatic microsomes from rat livers induced by Aroclor 1254

                                                     Incubation period:ca.44 h

Number of assays                       : 2 (the second assay with metabolic activation was performed according to pre-incubation method)

Limiting factor for maximum dose: maximum dose according to OECD Guideline,i.e.5000 µg/plate

Doses used in main test              :expressed asµg/plate of pureGluconic acid

Results: 

In two independent assays carried out in the 5 stains of Salmonella typhimurium TA1535, TA1537, TA98, TA100 and TA102, neither statistically nor biologically significant increases in the number of revertants were noted, both with and without metabolic activation.