Pyridoxine hydrochloride

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Reference 1

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.

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

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

Species / strain / cell type:

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

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.

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

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.

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

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

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.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: other: clastogenicity/aneugenicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-06-27 to 2016-11-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

Name: Pyridoxine Hydrochloride
Chemical Name: 2-Methyl-3-hydroxy-4,5-bis(hydroxymethyl)pyridine hydrochloride, Vitamin B6
CAS No.: 58-56-0
Batch No.: UQ60313097
Molecular Weight: 205.64 g/mol
Physical State: solid
Colour: white
Density: 1.44 g/cm3
pH Value: 2.4 – 3.0
Active Components: >99%
Purity: 100.1%
Expiry Date: 28 March 2019
Storage Conditions: room temperature, protected from light and humidity

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI
- Properly maintained: yes

Metabolic activation:

with and without

Metabolic activation system:

rat S9 liver microsomal fraction

Test concentrations with justification for top dose:

Pre-experiment:
7.8, 15.6, 31.3, 62.5, 125, 250, 500, 1000, 1500 and 2000 µg/mL

Experiment I with short exposure (4 h):
without and with metabolic activation:1000, 1500 and 2000 µg/mL

Experiment II with extended exposure (44 h):
without metabolic activation: 1000, 1500 and 2000 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used:
The test item was suspended in cell culture medium (RPMI medium) prior to treatment. The pH value detected with the test item was within the physiological range (7.0-7.4).
- Justification for choice of solvent/vehicle: Based on the results of the solubility test RPMI cell culture medium was used as solvent .

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

clastogenic control without metabolic activation (600 and 1200 µg/mL)

Positive control substance:

other: colchicine

Remarks:

aneugenic control without metabolic activation (0.02 and 0.4 µg/mL)

Positive control substance:

cyclophosphamide

Remarks:

clastogenic control with metabolic activation (12.5 µg/mL)

Details on test system and experimental conditions:

TREATMENT TIME:
4 hours (Experiment I with and without metabolic activation)
44 hours (Experiment II without metabolic activation)

FIXATION INTERVAL:
44 hours (Experiment I and II)

STAIN (for cytogenetic assays): acridin orange

NUMBER OF REPLICATIONS: duplicate cultures

NUMBER OF CELLS EVALUATED: 2000 cells per concentration (1000 cells per culture). In case of significant difference between both slides (generally factor ≥2) additional 1000 binucleated cells of the same concentration were screened to verify this analysis.

DETERMINATION OF CYTOTOXICITY
- Method: cytokinesis block proliferation index (CBPI)

Evaluation criteria:

There are several criteria for determining a positive result:
- at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control
- the increase is concentration-related in at least one experimental condition when evaluated with an appropriate trend test
- any of the results are outside the distribution of the historical negative/solvent control data (e.g. Poisson-based 95% control limits).

A test item is considered to be negative if there is no biologically relevant increase in the percentages of cells with micronuclei above concurrent control levels, at any dose group.

Statistics:

The nonparametric chi² Test was performed to verify the results in both experiments.
The chi² Test for trend was performed to test whether there is a concentration-related increase in the micronucleated cells frequency in the experimental conditions.

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

only at long-term treatment (44 h)

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

Interpretation of results:
negative

In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test item Pyridoxine Hydrochloride did not induce structural and/or numerical chromosomal damage in human lymphocytes.
Therefore, Pyridoxine Hydrochloride is considered to be non-genotoxic with respect to clastogenicity and/or aneugenicity in the in vitro Mammalian Cell Micronucleus Test.

Executive summary:

In order to investigate a possible potential of Pyridoxine Hydrochloride for its ability to induce micronuclei in human lymphocytes in vitro a Micronucleus assay was carried out.

The selection of the concentrations was based on data from the pre-experiment. In the main experiment without and with metabolic activation 2000 µg/mL test item was selected as the highest concentration.

The test item was dissolved in cell culture medium (RPMI).

The following concentrations were selected for microscopic analysis of micronucleus frequency:

Experiment I with short exposure (4 h):

without and with metabolic activation: 1000, 1500 and 2000 µg/mL

Experiment II with extended exposure (44 h):

withoutmetabolic activation: 1000, 1500 and 2000 µg/mL

No precipitate of the test item was noted in all dose groups evaluated in experiment I and II.

According to laboratory experience the limit for discrimination between not cytotoxic and a cytotoxic effect is a CBPI value of 70% compared to the negative/solvent control which corresponds to 30% of cytostasis.

In experiment I without and with metabolic activation no increase of the cytostasis above 30 % was noted.

In experiment II without metabolic activation no increase of the cytostasis above 30 % was noted up to a concentration of 1000 mg/mL. At a concentration of 1500 µg/mL a cytostasis of 36% and at a concentration of 2000 µg/mL a cytostasis of 49% was noted.

In experiment I without and with metabolic activation and in experiment II without metabolic activation no biologically relevant increase of the micronucleus frequency was noted after treatment with the test item.

The nonparametric chi²-Test was performed to verify the results in both experiments.

No statistically significant enhancement (p<0.05) of cells with micronuclei was noted in the dose groups of the test item evaluated in experiment I and II.

The chi²-Test for trend was performed to test whether there is a concentration-related increase in the micronucleated cells frequency in the experimental conditions. No statistically significant increase in the frequency of micronucleated cells under the experimental conditions of the study was observed in experiment I and II.

Ethylmethanesulfonate (EMS, 600 and 1200 µg/mL) and cyclophosphamide (CPA, 12.5 µg/mL) were used as clastogenic controls. Colchicine (Colc, 0.02 and 0.4 µg/mL) was used as aneugenic control. All induced distinct and statistically significant increases of the micronucleus frequency. This demonstrates the validity of the assay.

This study is classified as acceptable. This study satisfies the requirements for Test Guideline OECD 487 for in vitro mammalian cell mirconucleus test.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-10-05 to 2016-10-24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline Study

Qualifier:

according to guideline

Guideline:

other: OECD Guidline 490 (In vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: IWGT Recommendations

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Germany)

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine kinase

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Liver S9 of Wistar Phenobarbital and ß-Naphthoflavone-induced rat liver S9 mix

Vehicle / solvent:

RPMI cell culture medium was used as solvent (RPMI + 5% HS).

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

Positive controls:

yes

Positive control substance:

other: benzo(a)pyrene 2.5 µg/mL

Positive control substance:

other: ethylmethanesulfonate 300 µg/mL

Positive control substance:

methylmethanesulfonate

Remarks:

10 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: dissolved in medium
DURATION: 4 h (short-term exposure)
Expression time (cells in growth medium): 48 h
Selection time (if incubation with selection agent): approximately 12 days

SELECTION AGENT ( mutation assay) 5 µg/mL trifluorothymidine
NUMBER OF REPLICATIONS: one experiment with single exposure; cells were seeded in 4 plates and evaluated
NUMBER OF CELLS SEEDED: 2000 cells per well
DETERMINATION OF CYTOTOXICITY: relative total growth (RTG)

Evaluation criteria:

The test item is considered mutagenic if following criteria are met:
-The induced mutant frequency meets or exceeds the Global Evaluation factor (GEF) of 126 per 106 cells
- A dose-dependent increase in mutant frequency is detected.

Besides, combined with a positive effect in the mutant frequency, an increased occurrence of small colonies (≥40% of total colonies) is an indication for potential clastogenic effects and/or chromosomal aberrations.
According to the OECD guideline, the biological relevance is considered first for the interpretation of results. Statistical methods might be used as an aid in evaluation the test result.
A test item is considered to be negative if the induced mutant frequency is below the GEF and the trend test is negative.

Statistics:

The non-parametric Mann-Whitney test is applied to the mutation data to prove the dose groups for any significant difference in mutant frequency compared to the negative control.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

Interpretation of results:
negative

In conclusion, in the described mutagenicity test under the experimental conditions reported, the test item Pyridoxine Hydrochloride is considered to be non-mutagenic in the in vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L5178Y cells.

Executive summary:

The test item Pyridoxine Hydrochloride was assessed for its potential to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y according to OECD 490 and EC Test Method B.17 under GLP conditions.

The selection of the concentrations used in the main experiment was based on data from the pre-experiment. In the experiment 2.0 mg/mL (without and with metabolic activation) was selected as the highest concentration. The experiment was performed as a 4 h short-term exposure assay. The test item was investigated at the following concentrations:

without and with metabolic activation:

0.075, 0.10, 0.25, 0.50, 0.75, 1.0, 1.5 and 2.0 mg/mL

No precipitation of the test item was noted in the experiments.

No growth inhibition was observed in the experiment without and with metabolic activation.

In the experiment without metabolic activation the relative total growth (RTG) was 91.7% for the highest concentration (2.0 mg/mL) evaluated. The highest concentration evaluated with metabolic activation was 2.0 mg/mL with a RTG of 110.5%.

In the experiment no biologically relevant increase of mutants was found after treatment with the test item (without and with metabolic activation).The Global Evaluation Factor (GEF; defined as the mean of the negative/vehicle mutant frequency plus one standard deviation; data gathered from ten laboratories) was not exceeded by the induced mutant frequency at any concentration.

No dose-response relationship was observed.

Additionally, in the experiment colony sizing showed no clastogenic effects induced by the test item under the experimental conditions (without and with metabolic activation The positive controls did induce the appropriate response.  There was no evidence of a concentration related positive response of induced mutant colonies over background. This study is classified as acceptable.  This study satisfies the requirement for OECD 490 for in vitro mutagenicity (mammalian forward gene mutation) data.  

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

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.

Justification for selection of genetic toxicity endpoint
One study available performed according to OECD/EC guidelines and according to GLP principles.

Short description of key information:
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.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the available data, pyridoxine hydrochloride is not classified for mutagenic properties according to CLP Regulation (EC) No. 1272/2008.