4-trans-propenylveratrole

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From January 8 to March 13, 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 guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP Compliance Programme (inspected on July 01-03, 2014/ signed on September 15, 2014)

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

4-trans-propenylveratrole

EC Number:

228-958-7

EC Name:

4-trans-propenylveratrole

Cas Number:

6379-72-2

Molecular formula:

C11H14O2

IUPAC Name:

(E)-1,2-Dimethoxy-4-prop-1-en-1-ylbenzene

Test material form:

liquid

Details on test material:

- Physical state: Pale yellow, oily liquid
- Storage condition of test material: Room temperature

Method

Target gene:

None

Species / strain

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction (10% v/v); S9 fraction, prepared from male Sprague-Dawley derived rats, dosed with phenobarbital and 5,6-benzoflavone

Test concentrations with justification for top dose:

Preliminary toxicity test: 17.96, 29.93, 49.88, 83.14, 138.57, 230.95, 384.91, 641.52, 1069.2, 1782 µg/mL
Main tests: The upper concentration levels were selected based on cytotoxicity.
-S9 mix (3 hours): 50, 100, 125, 150, 175, 200, 225, 250, 275, 300 µg/mL
+S9 mix (3 hours): 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350 µg/mL
-S9 mix (21 hours): 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150 µg/mL
-S9 mix (21 hours) (additional test): 0.63, 1.25, 2.5, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Ethanol
- Test item was dissolved and diluted in ethanol (absolute), shortly before dosing. The final volume of ethanol added to the cultures was 1% v/v.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without S9 mix: 0.2 μg/mL (3-hour treatment); 0.1 μg/mL (20-hour continuous treatment)

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9 mix: 7.5 μg/mL

Details on test system and experimental conditions:

PREPARATION OF CULTURES:
- Human blood was collected aseptically from two healthy, non-smoking, adult donors, pooled (in equal volumes from each donor) and diluted with HML media. As lymphocytes do not normally undergo cell division, they were stimulated to do so by the addition of phytohaemagglutinin (PHA), a naturally occurring mitogen. Cultures were established from the prepared (pooled) sample and dispensed as 5 mL aliquots (in sterile universal containers) so that each contained blood (0.4 mL), HML media (4.5 mL) and PHA solution (0.1 mL). All cultures were then incubated at 37 °C, and the cells resuspended (twice daily) by gentle inversion.

METHOD OF APPLICATION: in medium
HML media: RPMI 1640, supplemented with 10% foetal calf serum, 0.2 IU/mL sodium heparin, 20 IU/mL penicillin / 20 μg/mL streptomycin and 2.0 mM L-glutamine.

DURATION
- Exposure duration: Preliminary toxicity test: 3 h (±S9); 21 h continuous treatment (-S9); Main test: 3 h (±S9); 21 h continuous treatment (-S9)
- Fixation time (start of exposure up to harvest of cells): 21 h, with and without S9 mix in preliminary toxicity and main tests

SPINDLE INHIBITOR (cytogenetic assays): Two hours before the cells were harvested, mitotic activity was arrested by addition of Colcemid to each culture at a final concentration of 0.1 µg/mL.

STAIN (for cytogenetic assays): Giemsa staining (10% in buffered water (pH 6.8))

NUMBER OF REPLICATIONS:
- Preliminary toxicity test: Duplicate cultures were used for treatment with the vehicle, and single cultures for treatment with test substance for each test condition.
- Main test: Duplicate cultures were used for treatment with the vehicle, test substance and positive controls.

NUMBER OF CELLS EVALUATED:
- The proportion of mitotic cells per 1000 cells in each culture was recorded (except for when clear evidence of overt toxicity was observed, or in cultures where there were no signs of cytotoxicity).
- One hundred and fifty metaphase figures were examined from each culture, however, this number was reduced in cultures showing a high level of aberrant cells, where 15 metaphases with structural aberrations (excluding gaps) were observed. In this study scoring was truncated only for the positive control cultures. Chromosome aberrations were scored according to the classification of the ISCN (2009). Only cells with 44 - 48 chromosomes were analysed.

DETERMINATION OF CYTOTOXICITY
- Method: Mitotic index

OTHER EXAMINATIONS:
- The incidence of polyploid and endoreduplicated cells (i.e. the ploidy status) were each recorded as a percentage of 150 metaphases analysed per slide, independently from the analysis for chromosome aberrations.

Rationale for test conditions:

The upper concentration levels were selected based on cytotoxicity

Evaluation criteria:

An assay is considered to be acceptable if the negative and positive control values lie within the current historical control range.
The test substance is considered to cause a positive response if the following conditions are met:
Statistically significant increases (p<0.01) in the frequency of metaphases with aberrant chromosomes (excluding gaps) are observed at one or more test concentration.
The increases exceed the vehicle control range of this laboratory, taken at the 95% confidence limit.
The increases are reproducible between replicate cultures.
The increases are not associated with large changes in pH, osmolality of the treatment medium or extreme toxicity.
Evidence of a concentration-related response is considered to support the conclusion.
A negative response is claimed if no statistically significant increases in the number of aberrant cells above concurrent control frequencies are observed, at any concentration. A further evaluation may be carried out if the above criteria for a positive or a negative response are not met.

Statistics:

The number of aberrant metaphase cells in each test substance group was compared with the vehicle control value using the one-tailed Fisher exact test (Fisher 1973). Statistical significance was declared at the 1% level.

A Cochran-Armitage test for trend (Armitage, 1955) was applied to the control and all test substance groups. If this is significant at the 1% level, the test is reiterated excluding the highest concentration group - this process continues until the trend test is no longer significant.

D20’s (the minimum concentration (mg/mL) at which aberrations were found in 20% of metaphases) were estimated (where possible) using logistic regression on a log(concentration) scale, allowing the number of control aberrations to be non-zero (Armitage et al., 2002).

The data was analysed using the SAFEStat (SAS statistical applications for end users, version 1.1) Chromosome Aberrations application (version 1.1) which was developed in SAS (SAS INSTITUTE 2002).

Results and discussion

Test results

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

3 hours in the absence or presence of metabolic activation

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolality: No fluctuation in osmolality of more than 50 mOsm/kg and no change in pH of more than 1.0 unit were observed at 1782 μg/mL, when compared to the vehicle control.

PRELIMINARY TOXICITY TEST:
- In the absence of S9 mix following 3-hour treatment, test item caused a reduction in the mitotic index to 52% of the vehicle control value at 230.95 μg/mL. At higher tested concentrations excessive toxicity was observed. No notable culture medium changes were observed by eye, when compared to the vehicle control. Fine precipitate may not have been detected.
- In the presence of S9 mix following 3-hour treatment, test item caused a reduction in the mitotic index to 82% of the vehicle control value at 230.95 μg/mL. At higher tested concentrations excessive toxicity was observed. No notable culture medium changes were observed, when compared to the vehicle control.
- In the absence of S9 mix following 21-hour continuous treatment, test item caused a reduction in the mitotic index to 59% of the vehicle control value at 83.14 μg/mL. At higher tested concentrations excessive toxicity was observed. No notable culture medium changes were observed, when compared to the vehicle control.

MAIN TEST:

Cytotoxicity:
- In the absence of S9 mix, 3-hour treatment: Test item caused a reduction in the mitotic index to 48% of the vehicle control value at 225 μg/mL. The concentrations selected for metaphase analysis were 50, 200 and 225 μg/mL. The mitotic index at the two lower dose levels was 101 and 70%, respectively. No notable culture medium changes were observed by eye, when compared to the vehicle control. Fine precipitate may not have been detected.
- In the presence of S9 mix, 3-hour treatment: Test item caused a reduction in the mitotic index to 47% of the vehicle control value at 275 μg/mL. The concentrations selected for metaphase analysis were 225, 250 and 275 μg/mL. The mitotic index at the two lower dose levels was 106 and 78% respectively. No notable culture medium changes were observed by eye, when compared to the vehicle control. Fine precipitate may not have been detected.
- In the absence of S9 mix, 21-hour continuous treatment: A suitable toxicity range including a non-toxic concentration was not achieved therefore no metaphase analysis was conducted. No notable culture medium changes were observed, when compared to the vehicle control.
- Additional main test: 21-hour continuous treatment in the absence of S9 mix: Test item caused a reduction in the mitotic index to 46% of the vehicle control value at 80 μg/mL. The concentrations selected for metaphase analysis were 20, 60 and 80 μg/mL. The mitotic index at the two lower dose levels was 110 and 74% respectively. No notable culture medium changes were observed, when compared to the vehicle control.

Metaphase analysis:
3-hour treatment in the absence of S9 mix:
- No statistically significant increases in the proportion of cells with chromosomal aberrations were observed at any analysed concentration, when compared to the vehicle control.
- A statistically significant increase (p<0.01) in polyploidy cell frequency was seen at 225 μg/mL. The individual and mean incidence values of polyploidy were outside of the laboratory historical control range.
- No statistically significant increases in endoreduplicated metaphases were observed during metaphase analysis, when compared to the vehicle control.

3-hour treatment in the presence of S9 mix
- Statistically significant increases in the proportion of cells with chromosomal aberrations were observed at 275 μg/mL excluding gaps (p<0.01). No other statistically significant increases were observed. All mean values for the vehicle control (ethanol), and all test item treatment concentrations were within laboratory historical control range, when taken at the 95% confidence limit. The statistically significant increase seen in the proportion of cells with chromosomal aberrations at 275 μg/mL was therefore considered to be of questionable biological relevance.
- A statistically significant increase (p<0.01) in polyploidy cell frequency was seen at 275 μg/mL. The individual and mean incidence values of polyploidy were outside of the laboratory historical control range.
- No statistically significant increases in endoreduplicated metaphases were observed during metaphase analysis, when compared to the vehicle control.

21-hour continuous treatment in the absence of S9 mix
- A statistically significant increase in the proportion of cells with chromosomal aberrations was observed at 80 μg/mL (excluding gaps (p<0.001)). No other statistically significant increases were observed. However in there was no observation of chromatid exchange aberrations, which are rare in control cultures and considered to be strong evidence of clastogenicity when present at elevated frequencies.
- No statistically significant increases in polyploid or endoreduplicated metaphases were observed during metaphase analysis, when compared to the vehicle control.

COMPARISON WITH HISTORICAL CONTROL DATA:
- Results were compared with historical data of the laboratory (October 2012 – September 2014).

Any other information on results incl. tables

Table 7.6.1/1: Summary of results

Exposure period (hours)	S9 mix	Nominal concentration of test item (µg/mL)	Cells with aberrations excluding gaps			Cells with aberrations including gaps			Relative Mitotic
			Individual values (%)		Mean (%)	Individual values (%)		Mean (%)	Index (%)
3	-	0 (Ethanol)	0.0	0.7	0.3	2.0	0.7	1.3	100
		50	0.0	1.3	0.7	0.7	1.3	1.0	101
		200	3.3	2.0	2.7	4.0	2.7	3.3	70
		225	2.0	0.7	1.3	2.0	1.3	1.7	48
		0.2 (Mitomycin C)	12.0	12.9	12.4***	18.4	19.0	18.7	100
3	+	0 (Ethanol)	0.0	0.7	0.3	0.7	0.7	0.7	100
		225	1.3	0.7	1.0	2.0	2.7	2.3	106
		250	1.3	1.3	0.3	2.7	3.3	3.0	78
		275	2.0	4.7	3.3**	4.0	6.7	5.3	47
		7.5 (Cyclophosphamide)	39.5	22.7	28.8***	39.5	25.8	30.8	98
21	-	0 (Ethanol)	1.3	2.7	2.0	1.3	4.0	2.7	100
		20	1.3	1.3	1.3	2.0	4.0	3.0	110
		60	6.7	4.0	5.3	10.7	7.3	9.0	74
		80	8.0	8.7	8.3***	13.3	10.0	11.7	46
		0.1 (Mitomycin C)	32.6	35.7	34.1***	32.6	40.5	36.4	130

 

One-tailed Fisher's exact test

***                p<0.001

**                 p<0.01

Otherwise        p>0.01

Applicant's summary and conclusion

Conclusions:

Exposure to the test item has been shown to have the potential to cause an increase in the frequency of structural chromosome aberrations in this in vitro cytogenetic test system, under the experimental conditions described for the 21-hour exposure in the absence of S9-mix only
Exposure to test item for 3 hours in the absence or presence of metabolic activation, has been shown to induce statistically significant increases in numerical aberrations in the form of polyploidy in this in vitro cytogenetic test system, under the experimental conditions described.

Executive summary:

In an in vitro chromosome aberration test performed according to OECD Guideline 473 and in compliance with GLP, cultured human lymphocytes were exposed to test item at the following concentrations:

Preliminary toxicity test: 17.96, 29.93, 49.88, 83.14, 138.57, 230.95, 384.91, 641.52, 1069.2, 1782 µg/mL

Main tests:

-S9 mix (3 hours): 50, 100, 125, 150, 175, 200, 225, 250, 275, 300 µg/mL

+S9 mix (3 hours): 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350 µg/mL

-S9 mix (21 hours): 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150 µg/mL

-S9 mix (21 hours) (additional test): 0.63, 1.25, 2.5, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 µg/mL 

 

Two hours before the cells were harvested, mitotic activity was arrested by addition of Colcemid to each culture at a final concentration of 0.1 µg/mL. The cells were then treated with a hypotonic solution, fixed, stained and examined for mitotic indices and chromosomal aberrations. Metabolic activation system used in this test was S9 fraction (10% v/v); S9 fraction, prepared from male Sprague-Dawley derived rats, dosed with phenobarbital and 5,6-benzoflavone.

A preliminary toxicity test was performed using a dose range of 17.96 to 1782 μg/mL. Cells were exposed for a 3-hour treatment in the absence and presence of S9 mix, and a 21-hour continuous treatment in the absence of S9 mix and mitotic index data were used to determine toxicity. Based on the mitotic index data, concentrations were selected for the main test.

 

In the main test, the mitotic index was assessed for all cultures treated with test item and the vehicle control, Ethanol. Justification for the highest analysed concentration was determined by cytotoxicity. On the basis of these data, the following concentrations were selected for metaphase analysis:

In the absence of S9 mix, 3-hour treatment: 50, 200 and 225 μg/mL.

In the presence of S9 mix, 3-hour treatment: 225, 250 and 275 μg/mL.

In the absence of S9 mix, 21-hour continuous treatment: 20, 60 and 80 μg/mL.

 

In the absence of S9 mix following a 3-hour treatment with test item, an increase in the proportion of metaphase figures containing chromosomal aberrations was observed at the mid-dose level of 200 μg/mL (excluding gaps) but it was not statistically significant at the 0.01 level. All mean values for the test item treatment concentrations were within the laboratory historical control range, when taken at the 95% confidence limit. Therefore the increase in the proportion of cells with chromosomal aberrations was considered to be of questionable biological relevance.

 

In the presence of S9 mix following a 3-hour treatment with test item, statistically significant increases in the proportion of cells with chromosomal aberrations were observed at the high dose level of 275 μg/mL (excluding gaps). No other statistically significant increases were observed. All mean values for the test item treatment concentrations were within the laboratory historical control range, when taken at the 95% confidence limit. Therefore the increase in the proportion of cells with chromosomal aberrations was considered to be of questionable biological relevance.

 

A statistically significant increase in the proportion of polyploid metaphase cells (p<0.01) was observed during metaphase analysis at the maximum concentration only, when examined following the 3-hour treatments, when compared to the vehicle control. No statistically significant increases in the proportion of endoreduplicated metaphase cells were observed during metaphase analysis, under the 3-hour treatment condition, when compared to the vehicle control.

 

In the absence of S9 mix following a 21-hour continuous treatment, statistically significant increases in the proportion of metaphase figures containing chromosomal aberrations were observed at 60 and 80 μg/mL (including and excluding gaps), when compared to the vehicle control. The increases in the proportion of metaphase figures containing chromosomal aberrations were outside of the laboratory historical control.

 

No statistically significant increases in the proportion of polyploid or endoreduplicated metaphase cells were observed during metaphase analysis, following a 21-hour continuous treatment, when compared to the vehicle control range, when taken at the 95% confidence limit.

 

Both positive control compounds caused statistically significant increases in the proportion of aberrant cells, demonstrating the sensitivity of the test system and the efficacy of the S9 mix.

 

Exposure to test item has been shown to have the potential to cause an increase in the frequency of structural chromosome aberrations in this in vitro cytogenetic test system, under the experimental conditions described for the 21-hour exposure in the absence of S9-mix only.

 

Under the test conditions, exposure to test item for 3 hours in the absence or presence of metabolic activation, has been shown to induce statistically significant increases in numerical aberrations in the form of polyploidy in this in vitro cytogenetic test system. The absence of a response in the 3-hour exposure without S9 suggests a doubt about the validity of the response seen after 21 hours of exposure. In addition the absence of chromatid exchange aberrations in the exposed cells further undermines the toxicological significance of the observations.