4-(3-phenylpropyl)pyridine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

not mutagenic or genotoxic

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

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

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

GLP compliance:

yes

Type of assay:

other: chromosomal aberrations

Specific details on test material used for the study:

Sponsor's identification 4-(3-phenylpropyl)pyridine
CAS No: 2057-49-0
Description: Yellow liquid
Purity: 98.31%

Target gene:

chromosomes

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CHL/IU cells derived from Chinese hamster, obtained from Research · Resource Bank (JCRB) (February 1988, at passage: 4th passage, now 12th) were used in the test within 10 years of thawing succession age.

Cytokinesis block (if used):

colcemid

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction from livers of rats induced with phenobarbital and 5,6-benzoflavone

Test concentrations with justification for top dose:

A preliminary toxicity test was conducted based on cell proliferation rates. The proliferation inhibitory action of the test substance on CHL/IU cells was determined by measuring the proliferation of each group using a monolayer culture cell densitometer (Monocellater ™ , Olympus Optical Co., Ltd.), and the solvent control group as baseline.

Vehicle / solvent:

acetone

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

DURATION
-Exposure duration:
a) Continuous treatment: 24 hrs, 48 hrs
b) Short-term treatment: 6 hrs; 18 hrs for recovery
-Fixation time (start of exposure up to fixation or harvest of cells):
a) Continuous treatment: 24 hrs, 48 hrs
b) Short-term treatment: 24 hrs
Vehicle: DMSO
SPINDLE INHIBITOR: Colcemid
STAIN: Giemsa stain
NUMBER OF CELLS EVALUATED:
Stractural aberrations: 200 cells/group
Polyploidy: 800 cells/group
DETERMINATION OF CYTOTOXICITY:
-Method: relative total growth

Rationale for test conditions:

guideline

Evaluation criteria:

Criteria for a positive call: a statistically significant increase in the frequency of cells with chromosomal aberrations in the treated group compared with that of the solvent control group, and a statistically significant difference in the dose trend test. An inconclusive result will be designated when there is a significant increase in the frequency of cells with aberrations without a postive dose response trend test.
The test will be considered negative when there is no significant difference in frequency of cells with chromosomal aberrations.
The test is inconclusive when the number of cells observed has fewer than 100 structural aberrations, and less than 400 polyploidy due to cytotoxicity.

Statistics:

Fisher's direct probability method is used to distinguish between the solvent background chromosomal aberration levels and those of the test substance- treated group, with a significance level of p <0.05. In addition, when significant differences were found by Fisher's exact stochastic method, the Cochran-Armitage's trend test is applied (p <0.05) to test for significance of the dose dependency.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

In the dose-range finding studies, concentrations were up to 1970 μg /mL, with precipitate seen above 246 μg /mL in the continuous assay and the short term assay with S9. In the short term assay without S9, precipitate was seen at and above 492.5 µg/ml. In all assays the test material showed evidence of cell toxicity.

In the main assay, the cell count data show that an approximate 50% growth inhibition was achieved at 60 µg/ml in the absence of S9, whilst in the presence of S9 21% growth inhibition was achieved at 80 µg/ml. No precipitate of the test material was observed at the end of the exposure period in either the absence or presence of S9. The test material did not induce any statistically significant increases in the frequency of cells with structural chromosome aberrations or polyploidy either in the presence or absence of metabolic activation. All controls behaved within historical ranges.

Conclusions:

A guideline OECD 473-compliant chromosomal aberration test was undertaken with the test material in DMSO, with and without rat liver S9 fraction, with valid positive controls. Cytotoxicity was seen at the higher doses in a preliminary dose-range finding study. There were no increase in the number of chromosome aberrations or polyploidy observed. The test substance does not induce chromosomal aberration in CHL/IU cells under the conditions of this study.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

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

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Target gene:

HPRT (hypoxanthine-guanine phosphoribosyl transferase)

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Laboratory for Mutagenicity Testing; Technical University, 64287 Darmstadt, Germany
- Suitability of cells: Highly suitable, used successfully in in vitro experiments for many years
- Cell cycle length, doubling time or proliferation index: 12-16 hours
- Sex, age and number of blood donors if applicable: N/A
- Whether whole blood or separated lymphocytes were used if applicable: N/A
- Number of passages if applicable: 2
- Methods for maintenance in cell culture if applicable: Thawed stock cultures are propagated at 37 °C in 75 cm2 plastic flasks; about 2-3×106 cells are seeded into each flask with 15 mL of media
- Modal number of chromosomes: 22
- Normal (negative control) cell cycle time:

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: MEM (minimal essential medium) containing Hank’s salts supplemented with 10% foetal bovine serum (FBS), neomycin (5 µg/mL) and amphotericin B (1%), cultured in 1.5% CO2
- Properly maintained: yes
- 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/β-naphthoflavone induced rat liver S9 mix containing MgCl2 (8 mM), KCl (33 mM), glucose-6-phosphate (5 mM) and NADP (4 mM) in sodium-ortho-phosphate-buffer (100 mM, pH 7.4)

Test concentrations with justification for top dose:

up to 2000 µg/mL (maxiumum concentration for OECD guideline 476)

Vehicle / solvent:

dimethyl sulphoxide (DMSO)

Untreated negative controls:

yes

Remarks:

Test material without S9 mix

Negative solvent / vehicle controls:

yes

Remarks:

No test material

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

Metabolically activated V79 cells are exposed to the test substance and evaluated for the ability to proliferate in media containing 6-TG.

METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk
- Cell density at seeding (if applicable): 2-3×10^6 cells in MEM with Hanks salts and 10% FBS, neomycin (5 μg/mL) and amphotericin B (1%). No FBS is present in the exposure medium.

DURATION
- Preincubation period: at least 24 h
- Exposure duration: 4 h, in serum-free medium either without S9 mix or with 50 μl/mL S9 mix. Concurrent solvent and positive controls are treated in parallel.
- Expression time (cells in growth medium): 6 to 8 days after treatment. The exposure medium is replaced with complete medium following two washing steps with PBS.
- Selection time (if incubation with a selection agent): 7 days. For the selection of mutant cells the complete medium will be supplemented with 11 μg/mL 6-thioguanine. All cultures will be incubated at 37 °C in a humidified atmosphere with 1.5% CO2 (98.5% air).
- Fixation time (start of exposure up to fixation or harvest of cells): After 7 – 10 days of selection,the colonies are stained with 10% methylene blue in 0.01% KOH solution.

SELECTION AGENT (mutation assays): 6-TG


NUMBER OF CELLS EVALUATED: Colonies with more than 50 cells are counted. In doubt the colony size will be checked with a preparation microscope.


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: At least four analysable concentrations will be used in two parallel cultures. For freely-soluble and non-cytotoxic test items the maximum concentration will be 2 mg/mL, 2 μL/mL or 10 mM, whichever is the lowest. For cytotoxic test items the maximum concentration should result in approximately 10 to 20% relative survival or cell density at sub-cultivation and the analysed concentrations should cover a range from the maximum to little or no cytotoxicity. Relatively insoluble test items will be tested up to the highest concentration that can be formulated in an appropriate solvent as solution or homogenous suspension. These test items will be tested up or beyond their limit of solubility. Precipitation or phase separation will be evaluated at the beginning and at the end of treatment by the unaided eye. Furthermore, test item induced changes in the osmolarity will influence dose selection.


OTHER EXAMINATIONS:
- Determination of polyploidy:
- Determination of endoreplication:
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable):

- OTHER:

Rationale for test conditions:

HPRT (hypoxanthine-guanine phosphoribosyl transferase) catalyzes the conversion of the nontoxic 6-TG (6-thioguanine) to its toxic ribophosphorylated derivative. Therefore, cells deficient in HPRT due to a forward mutation are resistant to 6-TG. These cells are able to proliferate in the presence of 6-TG whereas the non-mutated cells die. However, the mutant phenotype requires a certain period of time before it is completely expressed. The phenotypic expression is achieved by allowing exponential growth of the cells for 7 - 9 days. The expression period is terminated by adding 6-TG to the culture medium.

Evaluation criteria:

A test item is classified as clearly mutagenic if, in any of the experimental conditions examined, all of the following criteria are met:

a) at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) the increase is dose-related when evaluated with an appropriate trend test,
c) any of the results are outside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits).

A test item is classified as clearly non-mutagenic if, in all experimental conditions examined, all of the following criteria are met:

a) none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) there is no concentration-related increase when evaluated with an appropriate trend test,
c) all results are inside the distribution of the historical negative control data (based 95% control limits).

In cases when the response is neither clearly negative nor clearly positive as described above, or in order to judge the biological relevance of a result, the data should be evaluated by expert judgement or further investigations.

Statistics:

A linear regression (least squares, calculated using a validated excel spreadsheet) will be performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item will be compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05.
A t-Test will be performed using a validated test script of “R”, a language and environment for statistical computing and graphics, to evaluate a significant increase of the mutation frequency at test points exceeding the 95% confidence interval. Again a t-test is judged as significant if the p-value (probability value) is below 0.05. However, both, biological and statistical significance will be considered together.

Key result

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:

not valid

Positive controls validity:

valid

Conclusions:

The test substance was evaluated for in vitro genetic toxicity in V79 cells using the HPRT mutation evaluation technique. The test substance was observed to be non-mutagenic at concentrations equal to or less than 2000 µg/mL.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

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

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

histidine, tryptophan

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Metabolic activation system:

Rat liver induced with phenobarbital and 5,6-benzoflavone

Test concentrations with justification for top dose:

Dose-determination test: 0, 2.29, 6.86, 10.6, 61.7, 185, 556, 1667, 5000 µg/plate for all strains. Main assay: 0, 313, 625, 1250 and 5000 µg/plate for TA98, TA100, TA 1535 and WP2uvrA/pKM101. For Main assay for strain TA1537: 0, 2.44, 4.88, 9.77, 19.5, 39.1, 78.1, 156, 313. The high dose of 5000 µg/plate is according to OECD TG471 guideline.

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

other: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamine; 2-aminoanthracene

Details on test system and experimental conditions:

The pre-incubation method was used according to OECD TG471. Standard strains, dosing, metabolic activation, incubation conditions, criteria for acceptance and criteria for evaluation of positive results were adopted from the guideline. Cultures were plated in triplicate, and a replicate assay was conducted. All testing was performed under the auspices of Good Laboratory Practices.

Rationale for test conditions:

The main test was performed with several doses up to the growth inhibition doses or the maxiumum recommended dose of 5000 µg/plate. Growth inhibition of the bacterial lawn was an indication of toxicity of the test substance.

Evaluation criteria:

A finding of an increase in the number of revertant colonies more than twice that of the negative control (solvent control) in any tester strain with or without metabolic activation was evaluated as positive.

Statistics:

none

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

not examined

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

The dose-determination test was performed at eight doses of common ratio up to 5000 µg/plate. The test substance did not increase (more than twofold) the number of revertant colonies. Gorwth inhibition of the bacterial lawn with the chemical was observed in strain___. The main test was performed with at least 4 doses up to the growth inhibiton doses, or the maxumum recommended dose of 5000 µg/plate. The test substance did not increase (more than twofold) the number of revertant colonies with or without metabolic activation. The positive control substances increased the number of revertant colonies more than twice that of the solvent control. The number of revertant colonies for the negative and positive control were within the range of standard values derived from historical control data in this laboratory.

Conclusions:

The test substance was assayed in the reverse mutation test (OECD TG471, Ames Assay) in bacteria under standard conditions and found to be non-mutagenic. The criteria for classification of mutagenicity according to Regulation EC No. 1272/2008 are not met.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

Not applicable

Justification for classification or non-classification

No evidence of genotoxicity was observed in testing of the registered substance. The criteria for classification according to Regulation EC No. 1272/2008 are not met.