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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
The test item was investigated for genetic toxicity in vitro. A gene mutation test at the HPRT locus of Chinese Hamster V79 cells according to OECD guideline 476 revealed no mutagenic activity with and without metabolic activation. A chromosome aberration test in human lymphocytes according to OECD guideline 473 showed no clastogenic activity with and without metabolic activation. Bacterial reverse mutation testing with S. typhimurium according to OECD guideline 471 with the supporting substance DL-Panthenol revealed no mutagenic activity as well with and without metabolic activation. According to this available results, no classification for genetic toxicity is required.
Link to relevant study records
Reference
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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Principles of method if other than guideline:
first experiment 4 hours treatment with and without metabolic activation
second experiment 24 hours treatment without metabolic activation and 4 hours treatment with metabolic activation
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Target gene:
HPRT
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: MEM
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital/ß-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
Experiment I
without S9 mix: 65; 130; 260; 520; 1040; 2080 µg/mL (4 hours treatment)
with S9 mix: 65; 130; 260; 520; 1040; 2080 µg/mL (4 hours treatment)
Experiment II
without S9 mix: 65; 130; 260; 520; 1040; 2080 µg/mL (24 hours treatment)
with S9 mix: 65; 130; 260; 520; 1040; 2080 µg/mL (4 hours treatment)

In both experiments the cultures at the lowest concentration with and without metabolic activation (65 µg/mL) were not continued since a minimum of only four analysable concen-trations is required by the guidelines.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: Solubility properties
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours with and without metabolic activation in experiment 1, 24 hours without metaoblic activation and 4 hours treatment with metabolic activation in experiment 2
- Expression time (cells in growth medium): 72 hours
- Selection time: 10 days

SELECTION AGENT: Thioguanine

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: >1,5x10exp.6

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency


Evaluation criteria:
A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered to be non-mutagenic in this system.
A mutagenic response is described as follows:
The test item is classified as mutagenic if it induces reproducibly with one of the concen¬trations a mutation frequency that is three times higher than the spontaneous mutation fre¬quency in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
In a case by case evaluation this decision depends on the level of the correspon¬ding solvent control data.
Statistics:
A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT¿11 (SYSTAT Software, Inc., 501, Canal Boulevard, Suite C, Richmond, CA 94804, USA) statistics software. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance were considered together.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not effected
- Effects of osmolality: not increased
- Water solubility: well soluble in water
- Precipitation: no
- Other confounding effects: None


RANGE-FINDING/SCREENING STUDIES:
The highest concentration in the pre-experiment should be 2080 µg/mL (10 mM) regard-ing the solubility properties of the test item in water and taking in consideration the purity (99.2 %) and the molecular weight of the test item (205.25 g/mol). However, based on an error at the initial weight, test item concentrations between 16.6 and 2123 µg/mL were used to evaluate toxicity in the presence (4 h treatment) and absence (4 h and 24 h treatment) of metabolic activation. No relevant toxic effect occurred up to the maximum concentration tested with and without metabolic activation following 4 and 24 hours of treatment.
There was no relevant change of the pH value and the osmolarity even at the maximum concentration of the test item.
The test medium was checked for precipitation at the end of each treatment period (4 or 24 hours) before the test item was removed. No precipitation was observed by the unaided eye up to the maximum concentration.
Therefore, the maximum concentration of experiments I and II was 2080 µg/mL equal to approximately 10 mM. The lower concentrations were spaced by a factor of 2.


COMPARISON WITH HISTORICAL CONTROL DATA:
The highest solvent control (40.3 colonies per 106 cells) exceeded the historical range of solvent controls (2.2 31.5 colonies per 10 exp. 6 cells). However, this isolated effect was judged as irrelevant since the mean value of both parallel cultures (40.3 and 9.2 equal to 24.8 colonies per 10 exp. 6 cells) remained well within the range of historical controls.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No relevant toxic effects occurred up to the maximal concentration of 2080 µg/mL.
Remarks on result:
other: strain/cell type: Chinese hamster lung fibroblasts (V79)
Remarks:
Migrated from field 'Test system'.
Summary Table
      relative relative mutant   relative relative mutant  
  conc. µg S9 cloning cloning colonies/ induction cloning cloning colonies/ induction
  per mL mix efficiency I efficiency II 106cells factor efficiency I efficiency II 106cells factor
    % %   % %  
Column 1 2 3 4 5 6 7 8 9 10
Experiment I / 4 h treatment     culture I          culture II
Solvent control with water - 100.0 100.0  26.7 1.0 100.0 100.0  6.6 1.0
Positive control with EMS 150.0 -  95.3  88.1 134.0 5.0  94.4 104.0 119.2 18.1
Test item 65.0 -  97.4 culture was not continued# 100.9 culture was not continued#
Test item 130.0 -  97.1  91.1  10.8 0.4  98.6 103.1  25.1 3.8
Test item 260.0 -  96.8  95.9  9.5 0.4  98.1 100.6  12.8 1.9
Test item 520.0 -  96.3  69.7  19.4 0.7  99.1  77.6  30.3 4.6
Test item 1040.0 -  96.8  84.0  14.8 0.6  98.4  95.4  8.5 1.3
Test item 2080.0 - 102.2  57.1  18.5 0.7  94.0  59.2  15.6 2.4
Solvent control with water + 100.0 100.0  11.2 1.0 100.0 100.0  13.0 1.0
Positive control with DMBA  1.1 +  38.0  77.5 963.7 85.7  43.7  72.8 1039.6 79.9
Test item 65.0 +  99.3 culture was not continued# 106.8 culture was not continued#
Test item 130.0 + 100.1  99.1  16.1 1.4 101.8  98.6  19.3 1.5
Test item 260.0 +  97.6 110.4  15.3 1.4  90.7 103.3  27.6 2.1
Test item 520.0 + 100.4 102.3  13.8 1.2  96.0 107.4  18.9 1.5
Test item 1040.0 +  93.4 107.6  10.0 0.9  84.8 111.0  11.1 0.9
Test item 2080.0 +  97.0 107.1  17.6 1.6  86.6 108.3  9.7 0.7
Experiment II / 24 h treatment     culture I          culture II
Solvent control with water - 100.0 100.0  40.3 1.0 100.0 100.0  9.2 1.0
Positive control with EMS 75.0 -  73.0  96.9 188.4 4.7  65.4 102.7 172.7 18.7
Test item 65.0 - 110.0 culture was not continued# 105.9 culture was not continued#
Test item 130.0 - 104.8 105.0  9.5 0.2  99.3 110.8  15.2 1.6
Test item 260.0 - 104.5  91.2  17.5 0.4 101.0 106.0  3.3 0.4
Test item 520.0 -  98.1  89.4  9.6 0.2  94.9 106.2  6.7 0.7
Test item 1040.0 -  97.9 101.4  16.9 0.4  92.6  87.3  21.9 2.4
Test item 2080.0 -  94.8  98.8  14.2 0.4  91.9 104.3  13.3 1.4
Experiment II / 4 h treatment        
Solvent control with water + 100.0 100.0  10.8 1.0 100.0 100.0  20.9 1.0
Positive control with DMBA  1.1 +  69.6  76.3 948.0 87.6  63.7  93.6 696.9 33.3
Test item 65.0 + 100.0 culture was not continued# 102.6 culture was not continued#
Test item 130.0 +  95.8 106.4  7.5 0.7  99.4 100.0  23.9 1.1
Test item 260.0 +  94.6  98.9  9.8 0.9  97.6  97.7  16.6 0.8
Test item 520.0 +  93.8  93.9  21.0 1.9  98.6  90.4  18.7 0.9
Test item 1040.0 +  88.0  92.5  21.5 2.0  89.2 102.6  27.2 1.3
Test item 2080.0 +  86.8 102.1  8.5 0.8  92.2  97.8  10.6 0.5

#       culture was not continued since a minimum of only four analysable concentrations is required

Conclusions:
Interpretation of results (migrated information):
negative

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, D-PANTHENOL is considered to be non-mutagenic in this HPRT assay.
Executive summary:

The test item D-PANTHENOL was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster.

The study was performed in two independent experiments, using identical experimental procedures. In the first experiment the treatment period was 4 hours with and without metabolic activation. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.

The cell cultures were evaluated at the following concentrations:

Experiment I:

without S9 mix: 130; 260; 520; 1040; and 2080 µg/mL

with S9 mix:130; 260; 520; 1040; and 2080 µg/mL

Experiment II:

without S9 mix: 130; 260; 520; 1040; and 2080 µg/mL

with S9 mix: 130; 260; 520; 1040; and 2080 µg/mL

The maximal concentration of the test item in the main experiments equals a molar concentration of approximately 10 mM, with respect to the purity of the test item (99.2 %).

No precipitation of the test item was observed up to the maximal concentration in all experiments.

No relevant toxic effects occurred up to the maximal concentration of 2080 µg/mL.

No relevant and reproducible increase in mutant colony numbers/10E06 cells was observed in the main experiments up to the maximal concentration. All mutant frequencies remained well within the historical range of solvent controls. The induction factor exceeded the threshold of three times the corresponding solvent control in the second culture of experiment I without metabolic activation at 130 and 520 µg/mL. These effects however, were judged to be based upon the rather low solvent control of just 6.6 mutant colonies/10E06 cells. The isolated increases were not dose dependent as indicated by the lacking statistical significance.

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT statistics software. No significant dose dependent trend of the mutation frequency indicated by a probability value of < 0.05 was determined in any of the experimental groups.

In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 6.6 up to 40.3 mutants per 10E06 cells; the range of the groups treated with the test item was from 3.3 up to 30.3 mutants per 10E06 cells.

The highest solvent control (40.3 colonies per 10E06 cells) exceeded the historical range of solvent controls (2.2 - 31.5 colonies per 10E06 cells). However, this isolated effect was judged as irrelevant since the mean value of both parallel cultures (40.3 and 9.2 equal to 24.8 colonies per 10E06 cells) remained well within the range of historical controls.

EMS (150 µg/mL in experiment I and 75 µg/mL in experiment II) and DMBA (1.1 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Ames test

Key study:

A read across approach was performed on data available for a substance with similar physical-chemical properties. For justification of read across please refer to the attachment in IUCLID5 section 13. The substance DL-Panthenol was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay.

Standard plate test (SPT) and Preincubation Test (PIT) both with and without metabolic activation with liver homogenate of Aroclor 1254 -pretreated male Sprague-Dawley rats were applied. Two independent experiments were carried out:

1st Experiment

Strains: TA 1535, TA 100, TA 1537, TA 98, E. coli WP2 uvrA

Doses: 0; 20; 100; 500; 2500 and 5000 µg/plate

Vehicle: Water

Type of test: Standard plate test with and without S-9 mix

Number of plates: 3 test plates per dose or per control

2nd Experiment

Strains: TA 1535, TA 100, TA 1537, TA 98, E. coli WP2 uvrA

Doses: 0; 20; 100; 500; 2500 and 5000 µg/plate

Vehicle: Water

Type of test: Preincubation test with and without S-9 mix

Number of plates: 3 test plates per dose or per control

Negative controls treated with the vehicle (water) and positive controls treated with 2-aminoanthracene, N-methyl-N'-nitro-N-nitrosoguanidine, 4-nitro-o-phenylendiamine, 9-aminoacridine, or 4-nitroquinoline-N-oxide were included in each replicate.

According to the results of the present study, DL-Panthenol was considered to be not mutagenic.

Based on the close structural similarity of D-and DL-Panthenol it can be assumed that also D-Panthenol is neither genotoxic nor mutagenic.

Supporting study:

The results of the Ames-test were confirmed in a supporting non-GLP study performed in Salmonella typhimurium with D-Panthenol. The substance D-Panthenol was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several strains of Salmonella typhimurium in a modified version of the traditional Ames test, i.e. the Ames II Assay (microtiter version).

STRAINS: TA 98, TA Mix (Mixed strains TA 7001 - TA 7006)

DOSE RANGE: 2 µg - 2500 µg/mL. Higher concentrations of the test substance could not be formulated as a homogeneous suspension.

TEST CONDITIONS: Liquid fluctuation test both with and without metabolic activation (Aroclor-induced rat liver S-9 mix). SOLUBILITY: No precipitation of the test substance was found.

TOXICITY: No bacteriotoxic effect was observed.

MUTAGENICITY: An increase in the number of positive wells (his+ revertants) was not observed either without S-9 mix or after the addition of a metabolizing system.

CONCLUSION: According to the results of the present study, the test substance D-Panthenol is not mutagenic in the Ames Assay (Salmonella typhimurium reverse mutation assay) under the experimental conditions chosen here.

According to the results of the key study and supporting study, the test item was not cosidered mutagenic in the Ames test.

Gene mutation test

The test item D-PANTHENOL was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster.

The study was performed in two independent experiments, using identical experimental procedures. In the first experiment the treatment period was 4 hours with and without metabolic activation. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.

The cell cultures were evaluated at the following concentrations:

Experiment I:

without S9 mix: 130; 260; 520; 1040; and 2080 µg/mL

with S9 mix:130; 260; 520; 1040; and 2080 µg/mL

Experiment II:

without S9 mix: 130; 260; 520; 1040; and 2080 µg/mL

with S9 mix: 130; 260; 520; 1040; and 2080 µg/mL

The maximal concentration of the test item in the main experiments equals a molar concentration of approximately 10 mM, with respect to the purity of the test item (99.2 %).

No precipitation of the test item was observed up to the maximal concentration in all experiments.

No relevant toxic effects occurred up to the maximal concentration of 2080 µg/mL.

No relevant and reproducible increase in mutant colony numbers/10E06 cells was observed in the main experiments up to the maximal concentration. All mutant frequencies remained well within the historical range of solvent controls. The induction factor exceeded the threshold of three times the corresponding solvent control in the second culture of experiment I without metabolic activation at 130 and 520 µg/mL. These effects however, were judged to be based upon the rather low solvent control of just 6.6 mutant colonies/10E06 cells. The isolated increases were not dose dependent as indicated by the lacking statistical significance.

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT statistics software. No significant dose dependent trend of the mutation frequency indicated by a probability value of < 0.05 was determined in any of the experimental groups.

In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 6.6 up to 40.3 mutants per 10E06 cells; the range of the groups treated with the test item was from 3.3 up to 30.3 mutants per 10E06 cells.

The highest solvent control (40.3 colonies per 10E06 cells) exceeded the historical range of solvent controls (2.2 - 31.5 colonies per 10E06 cells). However, this isolated effect was judged as irrelevant since the mean value of both parallel cultures (40.3 and 9.2 equal to 24.8 colonies per 10E06 cells) remained well within the range of historical controls.

EMS (150 µg/mL in experiment I and 75 µg/mL in experiment II) and DMBA (1.1 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.

Chromosome aberration test

The test item D-Panthenol, dissolved in deionised water, was assessed for its potential to induce structural chromosomal aberrations in human lymphocytes in vitro in two independent experiments. The highest applied concentration in this study (2080.0 µg/mL of the test item, approx. 10 mM) was chosen with regard to the molecular weight of the test item and with respect to the current OECD Guideline 473.

Dose selection of the cytogenetic experiment was performed considering the toxicity data in accordance with OECD Guideline 473. The chosen treatment concentrations in both experiments ranged downwards from 2080.0 µg/mL (approx. 10 mM), with a factor of 1.75 between the concentrations. Mitotic indices, as percentage of control, and percentages of aberrant cells were determined. In the absence and presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration.

Either with or without metabolic activation, no clastogenicity was observed at the concentrations evaluated. However, in Experiment II, in the absence of S9 mix, one single statistically significant increase in chromosomal aberrations (2.0 %) was observed after treatment with 679.2 µg/mL of the test item. Since the value is within the range of the laboratorys historical solvent control data and no dose-dependency was observed, this finding was regarded as being biologically irrelevant. No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures. Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations.


Justification for selection of genetic toxicity endpoint
The most reliable in vitro mutation study with mammalian cells according to GLP and OECD guideline was considered.

Justification for classification or non-classification

Based on the negative in vitro results of the Ames test, the HPRT genome mutation assay and the CA assay, the test item has not to be classified and labelled mutagenic according to Regulation (EC) No 1272/2008 and EU Directive 67/548/EEC.