4-trans-propenylveratrole

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Ames Test (OECD 471, GLP, K, rel. 1): non mutagenic in S. typhimurium TA 97, TA 98, , TA 100, TA 102 & TA 1535.
- CHO/HPRT Mammalian Cell Gene Mutation Assay (OECD 476, GLP, K, rel. 1): not mutagenic.
- HL/CAT Chromosomal aberration test (OECD 473, GLP, K, rel. 1): clastogenic and polypoidigenic.

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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

Target gene:

None

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.

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)

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).

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).

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

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.

Reference 2

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:

From December 31, 2014 to February 27, 2015

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)

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:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation 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:

mammalian cell gene mutation assay

Target gene:

hemizygous hypoxanthine phosphoribosyl transferase (HPRT) gene

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Source: European Collection of Cell Cultures
- CHO-KI cells are functionally hemizygous at the HPRT locus.
- Type and identity of media: Ham’s Nutrient Mixture F12 medium
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes
- Periodically checked for karyotype stability: No; karyotype was assumed to be stable.
- Other details: Prior to exposure to test item, spontaneous mutants were eliminated from the stock cultures by incubating the cells in H10 containing 15 μg/mL hypoxanthine, 0.3 μg/mL amethopterin and 4 μg/mL thymidine for three days. All cell cultures were maintained at 37 °C in an atmosphere of 5 % CO2 in air.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction (25% v/v); S9 fraction was prepared from liver homogenates of male Sprague Dawley rats treated with phenobarbital and 5,6-benzoflavone

Test concentrations with justification for top dose:

Preliminary toxicity test: 13.9, 27.8, 55.7, 111.4, 222.8, 445.5, 891 and 1782 μg/mL. The upper concentration level was selected as being equivalent to 10mM, which is the maximum recommended level to be used in mammalian cells assays.
Mutation tests: The upper concentration levels were selected based on cytotoxicity.
-S9 mix Test 1 (4 hours) 7.8, 15.6, 31.25, 62.5, 125, 250, 300, 350, 400 and 450 μg/mL.
+S9 mix Test 1 (4 hours) 7.8, 15.6, 31.25, 62.5, 125, 250, 300, 350, 400 and 450 μg/mL
-S9 mix Test 2 (4 hours) 3.13, 6.25, 12.5, 25, 50, 75, 100, 125, 150, 200, 250, 300, 350 and 400 μg/mL
+S9 mix Test 2 (4 hours) 100, 125, 150, 200, 250, 300, 350 and 400 μg/mL

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

250 μg/mL; without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

Remarks:

5 μg/mL; with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: Ham’s Nutrient Mixture F12 medium
- Ham’s Nutrient Mixture F12, supplemented with 2 mM L-glutamine and 50 μg/mL gentamicin. The resulting medium is referred to as H0.
- H0 medium supplemented with 10 % HiFCS referred to as H10, is used for general cell culture, e.g. when growing cells up from frozen stocks.

DURATION
- Exposure duration: 4 h
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 7 days
- All incubations were performed at 37 °C in a humidified atmosphere of 5 % CO2 in air.

SELECTION AGENT (mutation assays): Selective medium, in which only HPRT deficient cells will grow, consisted of H10 supplemented with 6-thioguanine (6-TG) at a final concentration of 10 μg/mL.

NUMBER OF REPLICATIONS:
- Preliminary toxicity test: Single culture/dose for test item and 2 cultures for vehicle control
- Main test: 4 cultures for vehicle control, 2 cultures/dose for test item and positive controls

NUMBER OF CELLS EVALUATED: 200 cells/plate were seeded for cloning efficiency and 10^6 cells were analyzed for mutant frequencies.

DETERMINATION OF CYTOTOXICITY
- Method: Cloning efficiency, Survival and Relative Survival
Cloning efficiency: Total no of colonies for each culture / (Number of plates scored for colony formation x 200)
Survival: Cloning efficiency x Cell count Correction Factor
Relative Survival (RS): (Individual survival value x100) / Mean control survival value
Following the expression period, three plates were scored for the presence of colonies from each culture and the CE was calculated.
Relative Cloning Efficiency (RCE): (Individual CE x100) / Mean control CE

OTHER:
Mutant Frequency (MF) per 10^6 viable cells for each set of plates was calculated as: (Total no. of mutant colonies x 5) / (CE x no. of uncontaminated plates)

Rationale for test conditions:

Mutation tests: The upper concentration levels were selected based on cytotoxicity.

Evaluation criteria:

The criteria for a positive response will be:
- The demonstration of a statistically significant increase in mutant frequency following exposure to the test substance;
- Evidence of a dose-response relationship, over at least two dose levels, when any single dose level shows a significant increase in mutant frequency;
- Demonstration of reproducibility in any increase in mutant frequency;
- The mean mutant frequency should fall outside the upper limit of the historical vehicle control with a corresponding survival rate of 20% or greater.

Statistics:

The statistical significance of the data was analysed by weighted analysis of variance, weighting assuming a Poisson distribution following the methods described by Arlett et al. (1989). Tests were conducted for a linear concentration-response relationship of the test substance, for non-linearity and for the comparison of positive control and treated groups to solvent control. Data was analysed using SAS (SAS Institute Inc., 2002).

Key result

Species / strain:

Chinese hamster Ovary (CHO)

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:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No fluctuations in pH of the medium of more than 1.0 unit compared with the vehicle control were observed at 1782 μg/mL.
- Effects of osmolality: No fluctuations in osmolality of the medium of more than 50 mOsm/kg were observed when compared with the vehicle control at 1782 µg/mL.
- Precipitation: Precipitate was seen at 891 μg/mL and above at the end of treatment.

PRELIMINARY TOXICITY TEST:
- In the preliminary toxicity test, a four-hour exposure at concentrations from 13.9 to 1782 µg/mL of test item, in the absence or the presence of S9 mix, resulted in Day 1 relative survivals of 67 to 0 % and 121 to 0 % respectively. Precipitate was seen at 891 μg/mL and above at the end of treatment.
Concentrations for the main test were based upon these data and toxicity was the primary determinant for the dose selection.

MAIN MUTATION TEST 1
4-hour treatment in the absence of S9 mix: No precipitate was seen at the end of treatment. Exposure to test item resulted in Day 1 relative survivals of between 92 and 25 %. Cultures exposed to 7.8, 15.6, 31.25 and 62.5 μg/mL test item were plated out for determination of cloning efficiency and mutation induction. The Day 8 cloning efficiencies were 95 to 86 % relative to the vehicle controls. No significant increases in mutant frequency were observed after exposure to test item.
4-hour treatment in the presence of S9 mix: No precipitate was seen at the end of treatment. Exposure to test item resulted in Day 1 relative survivals of between 111 and 6 %. Cultures exposed to 7.8, 15.6, 31.25, 62.5, 125, 250, 300 and 350 μg/mL were plated out for determination of cloning efficiency and mutation induction. The Day 8 cloning efficiencies were 100 to 83 % relative to the vehicle control. No significant increases in mutant frequency were observed after exposure to test item.

MAIN MUTATION TEST 2
4-hour treatment in the absence of S9 mix: No precipitate was seen at the end of treatment. Exposure to test item resulted in Day 1 relative survivals of between 104 and 17 %. Cultures exposed to 3.13, 6.25, 12.5, 25, 50, 75, 100, 125, 150, 200 and 250 μg/mL were plated out for determination of cloning efficiency and mutation induction. The Day 8 cloning efficiencies were 92 to 70 % relative to the vehicle control. No significant increases in mutant frequency were observed after exposure to test item.
4-hour treatment in the presence of S9 mix: No precipitate was seen at the end of treatment. Exposure to test item resulted in Day 1 relative survivals of between 122 and 12 %. All cultures exposed were plated out for determination of cloning efficiency and mutation induction. The Day 8 cloning efficiencies were 96 to 78 % relative to the vehicle control. No significant increases in mutant frequency were observed after exposure to test item.

COMPARISON WITH HISTORICAL CONTROL DATA:
- Results were compared with historical control data (11 June 2012 - 16 September 2013)

Table 7.6.1/1: Summary results

Test Article	Dose level (µg/mL)	Test 1				Dose level (µg/mL)	Test 2
		4-h Treatment ‑S9‑mix		4-h Treatment +S9‑mix			4-h Treatment ‑S9‑mix		4-h Treatment +S9‑mix
		Mean Relative Survival (%)	Mean Mutant Frequency (x10^-6)	Mean Relative Survival (%)	Mean Mutant Frequency (x10^-6)		Mean Relative Survival (%)	Mean Mutant Frequency (x10^-6)	Mean Relative Survival (%)	Mean Mutant Frequency (x10^-6)
Ethanol	0	100	4.46	100	3.08	0	100	1.50	NA
Test item	7.8	85	0.00	104	3.18	3.13	99	8.04
	15.6	92	1.32	100	1.24	6.25	99	8.74
	31.25	39	10.97	83	0.64	12.5	100	3.45
	62.5	25	4.27	103	5.87	25	86	6.69
	125			111	4.09	50	104	1.77
	250			87	3.46	75	69	1.76
	300			36	5.22	100	72	5.21	116	1.60
	350			15	5.26	125	85	7.10	122	11.73
	NA					150	78	3.74	90	12.83
						200	52	3.73	59	18.83
						250	17	10.48	66	7.25
						300			62	15.85
						350			36	17.14
						400			12	8.66
Ethyl methanesulphonate	250	31	191.73***			250	79	412.21***
3-methylcholanthrene	5			93	428.80***	5			59	929.83***

 

*** Statistically significant: p < 0.001

Conclusions:

Under the test conditions, test item did not demonstrate mutagenic potential in this in vitro HPRT cell mutation assay, under the experimental conditions described, in which adequate toxicity was achieved in all tests.

Executive summary:

In an in vitro mammalian cell gene mutation test performed according to OECD Guideline 476 and in compliance with GLP, Chinese hamster Ovary (CHO-K1) cells were exposed to test item for 4-h, with and without metabolic activation (25 % S9 [v/v] fraction of male Sprague Dawley rats liver induced with phenobarbital and 5,6-benzoflavone, at the following concentrations: 

Preliminary toxicity test: 13.9, 27.8, 55.7, 111.4, 222.8, 445.5, 891 and 1782 μg/mL

 

Mutation tests:

-S9 mix Test 1 (4 hours) 7.8, 15.6, 31.25, 62.5, 125, 250, 300, 350, 400 and 450 μg/mL

+S9 mix Test 1 (4 hours) 7.8, 15.6, 31.25, 62.5, 125, 250, 300, 350, 400 and 450 μg/mL

-S9 mix Test 2 (4 hours) 3.13, 6.25, 12.5, 25, 50, 75, 100, 125, 150, 200, 250, 300, 350 and 400 μg/mL

+S9 mix Test 2 (4 hours) 100, 125, 150, 200, 250, 300, 350 and 400 μg/mL

 

Cytotoxicity was measured as Day 1 relative survival; values were from 67% to 0%, and 121% to 0%, after exposure to concentrations from 13.9 to 1782 μg/mL in the absence and presence of S9 mix respectively.

 

In main test 1, Day 1 survival values ranged from 92 to 25% relative to the vehicle control (without S9 mix); 104 to 15% for relative to the vehicle control (with S9 mix). In main test 2, Day 1 survival values ranged from 104 to 17% relative to the vehicle control (without S9 mix); 122 to 12% for relative to the vehicle control (with S9 mix). Test item did not induce a statistically significant increase in the mutant frequency in any of the experiments.

 

Positive control chemicals [ethyl methanesulfonate at 250 µg/mL (without S9 mix) and 3- methylcholanthrene at 5 µg/mL (with S9 mix)] induced significant increase in the mutant frequencies indicating the validity of the study.

 

Under the test conditions, test item did not demonstrate mutagenic potential in this in vitro HPRT cell mutation assay, under the experimental conditions described, in which adequate toxicity was achieved in all tests.

Reference 3

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:

From November 22 to December 15, 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

(inspected on March 24-25, 1997 / Signed on July 07, 1997)

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine

Species / strain / cell type:

S. typhimurium, other: TA97a, TA98, TA100, TA102 and TA1535

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-mix: S9 fraction from livers of male Wistar rats induced with Phenobarbital intraperitoneally and β-Naphtoflavone orally.

Test concentrations with justification for top dose:

Experiment-1: 0.016, 0.05, 0.16, 0.5 and 1.6 mg/plate in TA97a, TA98, TA100, TA102 and TA1535, with and without S9-mix based on the lowest cytotoxic concentration.
Experiment-2: 0.016, 0.05, 0.16, 0.5 and 1.6 mg/plate in TA97a, TA100, TA102 and TA1535, with and without S9-mix as well as in TA98 without S9-mix; 0.05, 0.16, 0.5, 1.6 and 5 mg/plate in TA98 with S9-mix based on the lowest cytotoxic concentration.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

cumene hydroperoxide

other: Nitrofurantoine, 4-nitro-1,2-phenylenediamine, ICR 191

Remarks:

Without S9-mix.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene, Danthron

Remarks:

With S9-mix.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h at 37 ± 1 °C

NUMBER OF REPLICATIONS: Three plates/concentration level

DETERMINATION OF CYTOTOXICITY
- Method: Evaluation of the toxicity was performed on the basis of growth of the bacterial background lawn.

Rationale for test conditions:

Experiment 1 & 2- Maximum concentration was limited by cytotoxicity.

Evaluation criteria:

- Plates with reduced background lawn were not included into evaluation procedures.
- Arithmetic mean values and standard deviations were calculated from colonies per plate of three replicates.
- For evaluation of the results the induction rate of the mean values was calculated:
Induction rate = Revertant colonies of test item/ revertant colonies of the corresponding control
- The test item is to be considered mutagenic if there is a concentration effect relationship and the induction rate is ≥ 2.

Statistics:

None

Key result

Species / strain:

S. typhimurium, other: TA97a, TA98, TA100, TA102 and TA1535

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:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not applicable
- Effects of osmolality: Not applicable
- Evaporation from medium: No data
- Water solubility: Test material was soluble in water at 297 mg/L.
- Precipitation: No
- Other confounding effects: None

COMPARISON WITH HISTORICAL CONTROL DATA: No

ADDITIONAL INFORMATION ON CYTOTOXICITY: Test material was cytotoxic at 1.6 mg/plate to all the tester strains except TA98 with S9-mix and at 5 mg/plate to TA98 with S9-mix.

Table 7.6.1/2. Mutagenic and cytotoxic effect of the test material.

Strain	S9-mix	Test concentration range (mg/plate)	Lowest mutagenic concentration (mg/plate)	Lowest cytotoxic concentration (mg/plate)
TA97a	-	0.016-1.6	None	1.6
	+	0.016-1.6	None	1.6
TA98	-	0.016-1.6	None	1.6
	+	0.016- 5	None	5
TA100	-	0.016-1.6	None	1.6
	+	0.016-1.6	None	1.6
TA102	-	0.016-1.6	None	1.6
	+	0.016-1.6	None	1.6
TA1535	-	0.016-1.6	None	1.6
	+	0.016-1.6	None	1.6

Conclusions:

Under the test condition, test material is not mutagenic with and without metabolic activation in S. typhimurium strains TA97a, TA98, TA100, TA102 and TA1535 according to the criteria of the Annex VI of the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

Executive summary:

In a reverse gene mutation assay performed according to the OECD test guideline No. 471 and in compliance with GLP, S. typhimurium strains TA97a, TA98, TA100, TA102 and TA1535 were exposed to test material both in the presence and absence of metabolic activation system (rat liver S9 in standard co-factors) using the plate incorporation method in two independent experiments. In the Experiment-1, dose ranged between 0.016 to 1.6 mg/plate in all the tester strains, with and without S9-mix. In the Experiment-2, dose ranged between 0.016 to 1.6 mg/plate in TA97a, TA100, TA102 and TA1535, with and without S9-mix as well as in TA98 without S9-mix and for TA98 with S9-mix, dose ranged between 0.05 to 5 mg/plate.

Vehicle (dimethyl sulphoxide) and positive control groups were also included in mutagenicity tests.

Test material was cytotoxic at 1.6 mg/plate to all the tester strains except TA98 with S9-mix and at 5 mg/plate to TA98 with S9-mix. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, at any dose level either with or without metabolic activation. The vehicle control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

Under the test condition, test material is not mutagenic with and without metabolic activation in S. typhimurium strains TA97a, TA98, TA100, TA102 and TA1535 according to the criteria of the Annex VI of the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

This study is considered as acceptable and satisfies the requirement for reverse gene mutation endpoint.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Micronucleus test in vivo (OECD 474, GLP, K, Rel.1): not clastogenic and not aneugenic.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From January 19 to June 08, 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ICH (2011) EMA/CHMP/ICH/126642/2008. Guideline S2(R1): Guidance on Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP Compliance Programme (inspected on February 05-07, 2014/ signed on April 24, 2014)

Type of assay:

micronucleus assay

Species:

rat

Strain:

other: Crl:CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Age at study initiation: 71 to 78 days
- Weight at study initiation: 331-393 g for males; 225-276 g for females
- Housing: Pre-pairing - 5/sex/cage; Pairing – 1 male:1 female per cage; Males after mating – 5 males/cage; Gestation – 1 female /cage; Lactation – 1 female/cage (+ litter); Solid (polycarbonate) bottom cages were used during the acclimatisation, gestation, littering and lactation periods; Grid bottomed cages were used during pairing.
- Diet: SDS VRF1 Certified powdered diet, ad libitum
- Water: Potable water taken from the public supply, ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19-23 °C
- Humidity: 40-70 %
- Air changes: Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod: 12 h dark / 12 h light

IN-LIFE DATES: From January 19 to June 08, 2015

Route of administration:

oral: feed

Details on exposure:

DIET PREPARATION
- Method of preparation: On each occasion of the preparation of the premix, the required amount of test substance was added to an equal amount of plain diet and stirred until visibly homogenous. This doubling up process was repeated until half of the final weight of premix was achieved. This mixture was then ground using a mechanical grinder after which it was made up to the final weight of the premix with plain diet. This premix was mixed in a Turbula mixer for 200 cycles to ensure the test substance was dispersed in the diet.
Aliquots of the premix were then diluted with further quantities of plain diet to produce the required dietary concentrations. Each batch of treated diet was mixed for a further 200 cycles in a Turbula mixer.
- Frequency of preparation: Weekly
- Storage of formulation: Ambient temperature.

Duration of treatment / exposure:

Males were treated for a minimum of four consecutive weeks, including two weeks prior to pairing.
Females were treated for two weeks prior to pairing, throughout mating and gestation and until Day 7 of lactation.

Frequency of treatment:

Continuously

Post exposure period:

None

Dose / conc.:

1 500 ppm (nominal)

Dose / conc.:

4 500 ppm (nominal)

Dose / conc.:

15 000 ppm (nominal)

No. of animals per sex per dose:

Samples of one femur were taken at necropsy from five male and female animals per group.

Control animals:

yes, plain diet

Positive control(s):

- Positive control: Cyclophosphamide
- Doses / concentrations: 20 mg/kg bw
- Route of administration: Oral

Tissues and cell types examined:

Coded slides were examined by fluorescence microscopy and 4000 polychromatic erythrocytes per animal were examined for the presence of micronuclei. At least one smear was examined per animal, any remaining smears being held temporarily in reserve in case of technical problems with the first smear.
The proportion of polychromatic erythrocytes was assessed by examination of a total of at least 1000 erythrocytes per animal and the number of micronucleated normochromatic erythrocytes was recorded.

Details of tissue and slide preparation:

DETAILS OF SLIDE PREPARATION:
- The animals were killed by exposure to rising levels of carbon dioxide and one femur dissected out from each animal. The femurs were cleaned of all excess tissue and blood and the proximal epiphysis removed from each bone. The bone marrow of one femur from each animal was flushed out and pooled in a total volume of 3 mL of filtered foetal calf serum by aspiration. The resulting cell suspensions were centrifuged at 1000 rpm (150 x g) for 5 minutes and the supernatant discarded. The final cell pellet was resuspended in a small volume of foetal calf serum to facilitate smearing in the conventional manner on glass microscope slides (Schmid 1976).

FIXATION AND SLIDE STAINING
1. Fixed for a minimum of 10 minutes in methanol and allowed to air-dry
2. Rinsed in purified water
3. Stained in acridine orange solution (0.0125 mg/mL using purified water) for 4 minutes
4. Washed in purified water for 5 minutes
5. Rinsed in cold tap water for 2 minutes
6. Stored at room temperature until required
7. Immediately prior to scoring, slides are wet mounted with coverslips using purified water

Evaluation criteria:

See section “Any other information on materials and methods incl. tables”

Statistics:

Each sex was analysed separately.
For the proportion of polychromatic erythrocytes, an asymptotic one-tailed Jonckheere’s test for trend (Jonckheere 1954) with “step-down” was used on Groups 1 to 4 for a decrease from control. If significant, then the analysis was carried out on Groups 1 to 3. Exact one-tailed Wilcoxon pairwise tests (Wilcoxon 1945), for a decrease from control, were also carried out on Group 1 (control) versus Groups 2, 3, 4 and 5.
For incidences of micronucleated polychromatic erythrocytes, an exact one-tailed Linear-by-Linear association test (Cytel 1995) with “step-down” was used on Groups 1 to 4 for an increase from control. If significant, then the analysis was carried out on Groups 1 to 3. Exact one-tailed pairwise Permutation tests (Cytel 1995), for an increase from control, were also carried out on Group 1 (control) versus Groups 2, 3, 4 and 5.
If the exact version of a test could not be calculated (due to the amount of data), then the asymptotic version was used instead.
Statistical significance was declared at the 5% level for all tests.
The data were received in an Excel document and analysed using SAS 9.1.3 (SAS Institute Inc., 2002) (Jonckheere's and Wilcoxon tests) and StatXact 3 (Cytel 1995) (Linear-by-Linear and Permutation tests).

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

- Test item did not cause any statistically significant increases in the number of micronucleated polychromatic erythrocytes (MPCE) or micronucleated normochromatic erythrocytes (MNCE) in male or female rats.
- Proportion of polychromatic erythrocytes (%PCE): Test item did not cause any statistically significant decreases in the proportion of polychromatic erythrocytes in male or female rats.

- Positive control: The coded positive control slides prepared from study HLS1035 demonstrated the ability of the scorer to detect increases in micronucleated polychromatic erythrocytes.

Table 7.6.2/1: Summary of results and statistical analysis

Treatment	Dosage	Proportion of PCE % # (SD)	Incidence MPCE mean # (SD)	Group mean % MPCE #
Male data
Vehicle	0	49.5 (3.5)	5.6 (1.7)	0.14
Test item	1500 ppm	49.6 (2.9)	7.0 (2.9)	0.18
	4500 ppm	49.6 (2.3)	4.8 (3.1)	0.12
	15000 ppm	48.9 (2.7)	4.8 (3.0)	0.12
Cyclophosphamide^a	20 mg/kg	44.3* (3.1)	80.8** (11.6)	2.02
Female data
Vehicle	0	61.2 (4.9)	8.8 (2.7)	0.22
Test item	1500 ppm	60.3 (6.8)	9.2 (1.8)	0.23
	4500 ppm	68.7 (3.7)	6.2 (3.7)	0.16
	15000 ppm	63.0 (5.6)	7.0 (1.4)	0.18
Cyclophosphamide^a	20 mg/kg	44.1** (2.6)	64.4** (12.6)	1.61

 

Vehicle: Basal diet

PCE: Polychromatic erythrocytes

MPCE: Number of micronucleated polychromatic erythrocytes observed per 4000 polychromatic erythrocytes examined

SD: Standard deviation

^a Positive control slides from HLS1035

# Occasional apparent errors of ± 1% may occur due to rounding of values for presentation in the table.

Results of statistical analysis using the appropriate nonparametric method of analysis based on permutation (one-sided probabilities):

*p< 0.05 (significant)

**p< 0.01 (significant)

otherwise p> 0.05 (not significant)

Conclusions:

Under the test conditions, test item did not show any evidence of causing an increase in the induction of micronucleated polychromatic erythrocytes or bone marrow cell toxicity in male or female Crl:CD(SD) rats.

Executive summary:

In micronucleus test conducted according to OECD 474 guideline and in compliance with GLP, induction of micronuclei by test item in bone marrow cells of male and female Crl:CD(SD) rats was assessed.

 

Bone marrow smears were obtained from 5 male and 5 female animals in the vehicle control group and in each of the test substance groups at the appropriate time. At least one smear from each animal was examined for the presence of micronuclei in 4000 polychromatic erythrocytes. The proportion of polychromatic erythrocytes was assessed by examination of at least 1000 erythrocytes from each animal. A record of the incidence of micronucleated normochromatic erythrocytes was also kept.

 

No statistically significant increases in the frequency of micronucleated polychromatic erythrocytes and no statistically significant decreases in the proportion of polychromatic erythrocytes were observed in Crl:CD(SD) rats at any dose level, compared to vehicle control values. The coded positive control slides prepared from study HLS1035 demonstrated the ability of the scorer to detect increases in micronucleated polychromatic erythrocytes.

 

Under the test conditions, test item did not show any evidence of causing an increase in the induction of micronucleated polychromatic erythrocytes or bone marrow cell toxicity in male or female rats.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Table 7.6.1/1: Summary of Genetic Toxicity Tests:

 

Test n°	Test guideline / reliability	Focus	Strains tested	Metabolic activation	Test concentration	Statement
1 (Noack, 2001)	Ames Test (OECD 471) K, rel.1	Gene mutation	TA 97a, TA 98, TA 100, TA 102, TA 1535	-S9 +S9	Up to cytotoxic or limit concentrations	-S9 : non mutagenic +S9 : non mutagenic
2 (HLS, 2015)	CHO/hprt test (OECD 476) K, rel.1	Gene mutation	Chinese Hamster Ovary cells	-S9 +S9	Up to cytotoxic concentrations	-S9 : non mutagenic +S9 : non mutagenic
3 (HLS, 2015)	HL/CAT (OECD 473) K, rel.1	Chromosomal aberration	Human Lymphocytes	-S9 +S9	Up to cytotoxic concentrations	-S9: positive +/-S9: increase in the number of polyploid cells
4 (Envigo, 2015	MNT vivo (OECD 474) K, rel.1	Chromosomal aberration	Rats	N.A	Up to 15000 ppm	Not clastogenic Not aneugenic
5 (BioReliance, 2002)	UDS Assay (OECD 482) D, rel.3	DNA damage repaired by unscheduled DNA synthesis	Rat hepatocytes	N.A.	Up to cytotoxic concentrations	Equivocal

Gene mutation Assays (Tests n° 1-2):

- A Bacterial Reverse mutation Assay (Ames test) was performed according to OECD guideline No. 471 with the substance (See Table 7.6/1). No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains under the test condition, with any dose of the substance, either in the presence or absence of metabolic activation. The substance does not induce gene mutations in bacteria whereas all positive control chemicals (with and without metabolic activation) induced significant increase of colonies. The substance is therefore considered as non-mutagenic according to the Ames test.

- Inability to produce gene mutation was confirmed in mammalian cells using an in vitro forward mutation assay in Chinese hamster ovary cells (CHO/hprt test) (Test n°2). None of the dose levels up to the cytotoxicity limit with the substance, either in the presence or absence of metabolic activation, induced significant mutant frequency increases in the initial or repeat tests. The substance does not induce forward mutations at the hprt locus in CHO cells under activation and non-activation conditions whereas both positive control chemicals (with and without metabolic activation) induced significant mutant frequency increases. The substance is therefore considered as negative for inducing forward mutations at the hprt locus in CHO cells under activation and non-activation conditions used in this assay. This result confirms the results of the Ames test and extends the non-mutagenic effect of the substance to mammalian cells.

Chromosomal aberration (test n°3-4)

The clastogenic potential of the substance was determined using an in vitro chromosome aberration test in human lymphocytes (OECD 473), which measures the potential of a substance to increase the incidence the of structural chromosome aberrations in cultured human lymphocytes.

Exposure to the substance 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 the substance, 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. 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. However, further testing was conducted to investigate the potential for chromosome aberrations in vivo.

For this purpose, a micronucleus add-in (following OECD TG 474) was included in an OECD 421 Reproductive/Developmental Toxicity Screening Study performed on the substance. Bone marrow smears were obtained from 5 male and 5 female animals in the vehicle control group and in each of the test substance groups at the appropriate time. At least one smear from each animal was examined for the presence of micronuclei in 4000 polychromatic erythrocytes. The proportion of polychromatic erythrocytes was assessed by examination of at least 1000 erythrocytes from each animal. A record of the incidence of micronucleated normochromatic erythrocytes was also kept. No statistically significant increases in the frequency of micronucleated polychromatic erythrocytes and no statistically significant decreases in the proportion of polychromatic erythrocytes were observed in Crl:CD(SD) rats at any dose level, compared to vehicle control values. The coded positive control slides prepared from parallel study demonstrated the ability of the scorer to detect increases in micronucleated polychromatic erythrocytes.

The test substance is therefore considered as non-clastogenic and non-aneugenic in vivo.

DNA damage repaired by unscheduled DNA synthesis (test n°5)

The ability of the substance to induce DNA damage (i.e. short-patch repair) in vitro was assessed using an UDS Assay (Test n°5) in rat hepatocytes.

The results of preliminary toxicity assay indicated that test material elicited a significant increase in the mean net nuclear counts of 7.2 and 6.5 (i.e., an increase of at least 5 counts over the negative control) at 60 and 70 µg/mL, respectively. In a confirmatory assay, the results indicate that test material induced a significant increase in the mean number of net nuclear grain counts (i.e., an increase of at least 5 counts over the negative control) in isolated rat hepatocytes, at the lowest dose level only. However, the increase of 5.2 (i.e., 2.7 over -2.5) at the lowest dose level (10 µg/mL) was only marginally above the minimum for a positive response (i.e., an increase of at least 5 counts over the negative control). In the absence of a dose response, this marginal increase in only 25 % of the cells examined is considered equivocal. The positive response observed in the preliminary UDS assay was not entirely reproduced in confirmatory assay, therefore no clear conclusion can be drawn and the test result is considered to be equivocal. It is noted that the OECD withdrew this test guideline in April 2014 because it is no longer considered to be a sufficiently reliable test method for routine use. In this case there is a fully reliable study for the mammalian cell gene mutation endpoint (HPRT assay) and consequently, the results of the UDS study are disregarded for the evaluation of the genotoxic potential of this substance.

CONCLUSIONS:

All the available studies are complementary as they focus on different kind of damage a chemical or its metabolites can induce to DNA. This ensemble of studies investigating gene mutation (both in bacterial and mammalian cells), and also mammalian chromosome aberration, both in vitro and in vivo. The studies provide evidence that the substance has no concerns for genotoxicity in mammals.

Justification for classification or non-classification

Harmonized classification:

The test material has no harmonized classification for human health according to the Regulation (EC) No. 1272/2008.

Self-classification:

Based on the available data, no additional classification is proposed regarding germ cell mutagenicity according to the Annex VI of the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.