Pyridoxine hydrochloride

Genetic toxicity: in vitro

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:

09 FEB 2015 - 23 FEB 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

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:

Pyridoxine hydrochloride

EC Number:

200-386-2

EC Name:

Pyridoxine hydrochloride

Cas Number:

58-56-0

Molecular formula:

C8H11NO3.ClH

IUPAC Name:

4,5-bis(hydroxymethyl)-2-methylpyridin-3-ol hydrochloride

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): Pyridoxine Hydrochloride (Vitamin B6)
- Substance type: organic
- Physical state: white powder
- Storage condition of test material: At room temperature, protected from light and humidity

Method

Target gene:

- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene

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:

Rat liver S9-mix induced by Aroclor 1254 (500 mg/kg)

Test concentrations with justification for top dose:

Experiment 1
Preliminary test (without and with 5% S9) TA100 and WP2uvrA: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate
Main study: TA1535, TA1537 and TA98:
Without and with 5% S9-mix: 52, 164, 512, 1600 and 5000 µg/plate
Experiment 2:
Without and with 10% S9-mix: 492, 878, 1568, 2800 and 5000 µg/plate

Vehicle / solvent:

- Solvent used: Milli-Q
- Preparation of test solutions started with solutions of 50 mg/ml resulting in a clear colourless solution. The lower test concentrations were prepared by subsequent dilutions in Milli-Q water.

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Remarks:

TA1535: sodium azide (5 µg/ plate); TA1537: ICR-191 (2.5 µg/ plate); TA98: 2-nitrofluorene (10 µg/ plate); TA100: methylmethanesulfonate (650 µg/ plate); WP2uvrA: 4-nitroquinoline N-oxide (10 µg/ plate)

Remarks:

without metabolic activation

Controls 2

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Remarks:

TA1535: 2.5µg/ plate (5 and 10% S9-mix); TA 1537: 2.5 and 5µg/ plate (5 and 10% S9-mix); TA98: 1µg/ plate (5 and 10% S9-mix); TA100: 1 and 2µg/ plate (5 and 10% S9-mix); WP2uvrA: 15µg/ plate (5 and 10% S9-mix)

Positive control substance:

other: 2-aminoanthracene

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hour

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted (with 5% and 10% S9-mix respectively).

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies.

OTHER EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was determined.

Evaluation criteria:

A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three (3) times the concurrent vehicle control.
b) The negative response should be reproducible in at least one follow-up experiment.
A test substance is considered positive (mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is greater than three (3) times the concurrent vehicle control.
b) In case a follow up experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

Statistics:

No formal hypothesis testing was done.

Results and discussion

Test results

Test results 1

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

but tested up to limit concentration

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

but tested up to limit concentration

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation was observed up to and including the top dose of 5000 µg/plate

RANGE-FINDING/SCREENING STUDIES:
- No toxicity or mutagenicity was observed up to and including the top dose of 5000 µg/plate

COMPARISON WITH HISTORICAL CONTROL DATA:
- The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.
- No toxicity or mutagenicity was observed up to and including the top dose of 5000 µg/plate

Remarks on result:

other: all strains/cell types tested

Applicant's summary and conclusion

Conclusions:

Interpretation of results:
negative

In an AMES test, performed according to OECD guideline and GLP principles, Pyridoxine Hydrochloride (Vitamin B6) was found not to be mutagenic with or without metabolic activation.

Executive summary:

An AMES test was performed with Pyridoxine Hydrochloride (Vitamin B6) according to OECD guideline and GLP principles. All bacterial strains showed negative responses up to 5000ug/plate, i.e. no significant dose-related increase in the number of revertants with or without metabolic activation was seen. No cytotoxicity and/or precipitation of the test substance was observed. The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Based on the results of this study it is concluded that Pyridoxine Hydrochloride (Vitamin B6) is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay with or without metabolic activation.