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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
July 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guidelineopen allclose all
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
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
p-vinylphenol
EC Number:
220-103-6
EC Name:
p-vinylphenol
Cas Number:
2628-17-3
Molecular formula:
C8H8O
IUPAC Name:
4-vinylphenol
Test material form:
liquid
Details on test material:
- Name as cited in the report: 4-Vinylphenol
- name: p-Vinylphenol
- CAS: 2628-17-3
- Batch No.: STBD7044V
- Storage Conditions: room temperature
- Expiration date: November 2018
- Description: yellow liquid
- Purity: 10.2% in propylene glycol, FG
Specific details on test material used for the study:
The test item was dissolved in propylene glycol (50%) and diluted prior to treatment. The solvent was compatible with the survival of the bacteria and the S9 activity. A correction factor of 9.8 was applied to consider purity of the test item.

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
- Pre-experiment: 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate
- Main experiment: 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate
Vehicle / solvent:
Propylene glycol
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
other: Sodium azide (TA100, TA1535), 4-nitro-o-phenylene-diamine (TA98, TA1537)
Remarks:
Without S9-mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (all strains)
Remarks:
With S9-mix
Details on test system and experimental conditions:
Samples of each tester strain were grown by culturing for 12 h at 37°C in Nutrient Broth to the late exponential or early stationary phase of growth (approx. 10E9 cells/mL). The nutrient medium consists per litre:
- 8 g Nutrient Broth
- 5 g NaCI
A solution of 125 µL ampicillin (10 mg/mL) (TA 98, TA 100, TA 102) was added in order to retain the phenotypic characteristics of the strain.

The Vogel-Bonner Medium E agar plates with 2 % glucose used in the Ames Test were prepared by Eurofins Munich or provided by an appropriate supplier. Quality controls were performed. Sterilisation was performed for 20 min at 121°C in an autoclave.

The overlay agar contains per litre:
- 7.0 g Agar Agar
- 6.0 g NaCI
- 10.5 mg L-histidine x HCI x H2O
- 12.2 mg biotin
Sterilisation was performed for 20 min at 121°C in an autoclave.
Evaluation criteria:
CYTOTOXICITY:
Cytotoxicity can be detected by a clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately <= 0.5 in relation to the solvent control.

VALIDITY:
A test is considered acceptable if for each strain:
- the bacteria demonstrate their typical responses to ampicillin (TA 98, TA 100, TA 102)
- the mean values of the spontaneous reversion frequency of the control plates with and without S9 mix are within the historical control data range
- corresponding background growth on negative control, solvent control and test plates is observed
- the positive controls show a distinct enhancement of revertant rates over the control plate
- at least five different concentrations of each tester strain are analysable.

MUTAGENICITY
The Mutation Factor is calculated by dividing the mean value of the revertant counts by the mean values of the solvent control (exact values).
A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as folIows:
- if in tester strains TA 98, TA 100 and TA 102 the number of reversions is at least twice as high
- if in tester strains TA 1535 and TA 1537 the number of reversions is at least three times higher than the reversion rate of the solvent control.
According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary. A test item producing neither a dose related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.

Results and discussion

Test results
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

Precipitation of the test item was observed in all tester strains used in experiment II at a concentration of 5000 µg/plate (with and without metabolic activation).

Toxic effects of the test item were noted in all tester strains evaluated in experiment l and II.

In experiment I toxic effects of the test item were observed in tester strains TA 98 and TA 102 at concentrations of 2500 µg/plate and higher (with and without metabolic activation). In tester strain TA 100 toxic effects of the test item were noted at concentrations of 1000 µg/plate and higher (without metabolic activation) and at concentrations of 316 µg/plate (with metabolic activation). In tester strains TA 1535 and TA 1537 toxic effects of the test item were observed at concentrations of 1000 µg/plate and higher (without metabolic activation) and at concentrations of 2500 µg/plate and higher (with metabolic activation).

In experiment II toxic effects of the test item were observed in tester strains TA 98 and TA 1535 at concentrations of 1000 µg/plate and higher (with and without metabolic activation). In tester strains TA 100 and TA 102 toxic effects of the test item were noted at concentrations of 1000 µg/plate and higher (without metabolic activation) and at concentrations of 2500 µg/plate and higher (with metabolic activation). In tester strain TA 1537 toxic effects of the test item were observed at concentrations of 316 µg/plate and higher (without metabolic activation) and at concentrations of 2500 µg/plate and higher (with metabolic activation). The reduction in the number of revertants down to a mutation faetor of 0.5 found in tester strain TA 1535 at a concentration of 100 µg/plate (without metabolic activation) was regarded as not biologieally relevant due to lack of a dose-response relationship.

No biologially relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with 4-Vinylphenol at any concentration level, neither in the presence nor absence of metabolic activation in experiment l and II.

The reference mutagens induced a distinct increase of revertant colonies indicating the validity of the experiments.

EXPERIMENT I (Plate-incorporation Test)

Mean revertant colonies per plate

   TA 98  TA98  TA 100  TA 100  TA 1535  TA 1535  TA 1537  TA 1537  TA 102  TA 102
   -S9  +S9  -S9  +S9  -S9  +S9  -S9  +S9  -S9  +S9
 neg. control  25  27  102  107  9  5  8  5  248  283
 solvent control  25  28  103  115  10  7  7  5  224  262
 test item 3.16 µg  30  39  99  102  15  9  8  11  255  391
 test item 10.0 µg  20  30  105  108  10  10 7  10  217  303
 test item 31.6 µg  28  26  97  115  7  6  6  7  197  304
 test item 100 µg  30 41  104  124  8  10  8  6  187  304
 test item 316 µg 34 30  96  112  11  14  6  9  217  309
 test item 1000 µg  20  31  47  100  14  11  8  8  206 318
 test item 2500 µg  12  15  0  0  0  4  0  6  21  84

 test item 5000 µg

 0

 0

 0

 0

 0

0

 0

 0

0

 0

 pos. control

 277

 1368

 471

 1934

 581

 94

 118

 222

 1549

 770

EXPERIMENT II (Pre-incubation Test)

Mean revertant colonies per plate

   TA 98  TA98  TA 100  TA 100  TA 1535  TA 1535  TA 1537  TA 1537  TA 102  TA 102
   -S9  +S9  -S9  +S9  -S9  +S9  -S9  +S9  -S9  +S9
 neg. control  21  27  78  82  9  14  8  8  177  241
 solvent control  22  31  80 90  14  12  9  13  188  264
 test item 3.16 µg  20 35  95  104  10  11 7  9  196  241
 test item 10.0 µg  19  28  84  99  8  14  8  14  181  238
 test item 31.6 µg  20  29 84  99  13  11  8  14  206  211
 test item 100 µg  24  31  78  83  6  11  6  12  188  229
 test item 316 µg 25  32  83  113  14  21  10  14  182  246
 test item 1000 µg  0  27  0  72  0  18  0  13  0 237
 test item 2500 µg  0  0  0  0  0  0  0  0  0  0

 test item 5000 µg

 0

 0

 0

 0

 0

 0

 0

 0

 0

 0

 pos. control

 295  1013  423  1037  554  86  103  137  1657  579

Applicant's summary and conclusion

Conclusions:
In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, 4-Vinylphenol did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used. Therefore, 4-Vinylphenol is considered to be non-mutagenic in this bacterial reverse mutation assay.
Executive summary:

In order to investigate the potential of 4-Vinylphenol for its ability to induce gene mutations the plate incorporation test (experiment I) and the pre-incubation test (experiment II) were performed with the Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 102,

In two independent experiments several concentrations of the test item were used. Each assay was conducted with and without metabolic activation. The concentrations, including the controls, were tested in triplicate. The following concentrations of the test item were prepared and used in the experiments:

3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate

Precipitation was observed in all tester strains used in experiment II (with and without metabolic activation).

Toxic effects of the test item were noted in all tester strains used in experiment l and II:

- In experiment I toxic effects of the test item were observed at concentrations of 1000 µg/plate and higher (without metabolic activation) and at concentrations of 316 µg/plate and higher (with metabolic activation), depending on the particular tester strain.

- In experiment II toxic effects of the test item were noted at concentrations of 316 µg/plate and higher (without metabolic activation) and at concentrations of 1000 µg/plate and higher (with metabolic activation), depending on the particular tester strain.

No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with 4-Vinylphenol at any concentration level, neither in the presence nor absence of metabolic activation in experiment l and II.

The reference mutagens induced a distinct increase of revertant colonies indicating the validity of the experiments,

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, 4-Vinylphenol did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used. Therefore, 4-Vinylphenol is considered to be non-mutagenic in this bacterial reverse mutation assay.