rac-(1R,4S,4aR,8R,8aS)-9-(dichloromethylidene)-8-hydroxyoctahydro-1,4-methanonaphthalen-5(1H)-one

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Ames test, +S9 negative, -S9 negative, S. typhimurium TA1535, TA1537, TA100, TA98 and E. coli WP2 uvrA pKM 101, WP2 pKM101, OECD 471, Sokolowski 2014

- Chromosome aberration test - in vitro, -S9 positive, +S9 negative, human lymphocytes, OECD 473, Bohnenberger 2014

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 Dec 2013 to 13 Jan 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

August 1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

30 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

His-locus and Trp-locus

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

Species / strain / cell type:

E. coli, other: WP2 pKM 101

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/ß-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Range in plate incorporation test (experiment I) and the pre-incubation test (experiment II): 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate
Justification for top dose: 5000 μg/plate is the maximum recommended dose according to the test guideline

Vehicle / solvent:

- Solvent used: DMSO (purity 99%)
- Justification for choice of solvent: The solvent was chosen because of its solubilisation properties and its relative non-toxicity to the bacteria.

Untreated negative controls:

yes

Remarks:

DMSO

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine, 4-NOPD and 2-aminoanthracene, 2-AA

Details on test system and experimental conditions:

METHOD OF APPLICATION:
Experiment I: in agar (plate incorporation)
Experiment II: preincubation

DURATION
- Preincubation period: 60 min (experiment II only)
- Exposure duration: 72 hours

NUMBER OF REPLICATIONS: 2 independent experiments, each using 3 replicates

DETERMINATION OF CYTOTOXICITY
- Method: a reduction in the number of spontaneous revertants (below a factor of 0.5) or a clearing of the bacterial background lawn

Evaluation criteria:

ACCEPTABILITY OF THE ASSAY
The Salmonella typhimurium and Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
- regular background growth in the negative and solvent control
- the spontaneous reversion rates in the negative and solvent control are in the range of the historical data
- the positive control substances should produce a significant increase in mutant colony frequencies
- a minimum of five analysable concentrations should be present with at least four showing no signs of toxic effects, evident as a reduction in the number of revertants below an induction factor of 0.5.

EVALUATION OF RESULTS
A test substance is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice the colony count of the corresponding solvent control is observed.
A concentration-dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A concentration-dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Key result

Species / strain:

S. typhimurium, other: TA1537, TA1535, TA100, TA98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli, other: WP2 uvrA pKM101, WP2 pKM101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST SPECIFIC CONFOUNDING FACTORS
Precipitation: The test substance precipitated in the overlay agar of the test tubes from 2500 to 5000 μg/plate with and without S9 mix in both experiments. Precipitation of the test substance in the overlay agar on the incubated agar plates was observed at 2500 and 5000 μg/plate in experiment I and II. The undissolved particles had no influence on data recording.

NUMBER OF REVERTANT COLONIES
No increase in revertant colony numbers of any of the six tester strains was observed following treatment with the test substance at any concentration, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

(HISTORICAL) CONTROL DATA
- The number of colonies did not quite reach the lower limit of the laboratories historical control range in the untreated and solvent control of strain WP2 uvrA pKM101 with S9 mix in experiment II. Since these deviations are rather small, they are judged to be based on biologically irrelevant fluctuations in the number of colonies and have no impact on the outcome of the study.
- Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Cytotoxicity: Reduced background growth and toxic effects were observed at concentrations (μg/plate) shown in Table 1 and 2 in 'Any other information on results incl. tables', respectively. The pre-experiment is reported as experiment I as at least five concentrations were analysable and the criteria under "Acceptability of the assay" were met.

Table 1. Reduced background growth

Strain	Experiment I		Experiment II
	without S9 mix	with S9 mix	without S9 mix	with S9 mix
TA 1535	2500, 5000	2500, 5000	/	/
TA 1537	2500, 5000	2500, 5000	/	1000 - 5000
TA 98	2500, 5000	2500, 5000	/	2500, 5000
TA 100	2500, 5000	2500, 5000	1000 - 5000	1000 - 5000
WP2 pKM101	2500, 5000	2500, 5000	2500, 5000	5000
WP2 uvrA pKM101	2500, 5000	2500, 5000	/	/

/ = no reduction of the background growth observed

 

Table 2. Toxic effects, evident as a reduction in the number of revertants (below an induction factor of 0.5)

Strain	Experiment I		Experiment II
	without S9 mix	with S9 mix	without S9 mix	with S9 mix
TA 1535	5000	/	5000	2500, 5000
TA 1537	/	5000	5000	5000
TA 98	5000	2500, 5000	5000	5000
TA 100	5000	5000	2500, 5000	1000 - 5000
WP2 pKM101	5000	5000	2500, 5000	2500, 5000
WP2 uvrA pKM101	5000	/	/	5000

/ = no toxic effects observed

Conclusions:

During a GLP-compliant OECD 471 mutagenicity tests and under the experimental conditions reported, the test substance did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. The test substance is considered to be non-mutagenic in the Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

This GLP-compliant OECD 471 study was performed to investigate the potential of the test substance to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA1535, TA1537, TA98, and TA100, and the Escherichia coli strains WP2 uvrA pKM101 and WP2 pKM101. The assay was performed with and without Phenobarbital/ß-naphthoflavone induced rat liver S9 metabolic activation. Each concentration, including the controls, was tested in triplicate. The test substance was tested at the following concentrations: Experiment I and II: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate.

The plates incubated with the test substance showed reduced background growth at higher concentrations with all strains used in the first experiment and some of the strains in the second experiment. Cytotoxic effects, evident as a reduction in the number of revertants (below an induction factor of 0.5), were observed at high concentrations in nearly all strains used. No increase in revertant colony numbers of any of the six tester strains was observed following treatment with the test substance at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies.

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

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 Nov 2013 to 24 Feb 2014

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)

Version / remarks:

21 July 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Version / remarks:

August 1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

30 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: Human

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Blood samples were obtained from healthy donors not receiving medication.
- Suitability of cells: This in vitro test is an assay for the detection of structural chromosomal aberrations.
- Sex, age and number of blood donors if applicable: For this study, blood was collected from a female donor (37 years old) for Experiment I, from a female donor (36 years old) for Experiment IIA, from a female donor (29 years old) for Experiment IIB and from a female donor (25 years old) for Experiment IIC.
- Whether whole blood or separated lymphocytes were used if applicable: whole blood

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: The culture medium was Dulbecco's Modified Eagles Medium/Ham's F12 (DMEM/F12, mixture 1:1) already supplemented with 200 mM GlutaMAX™. Additionally, the medium was supplemented with penicillin/streptomycin (100 U/mL/100 Kg/mL), the mitogen PHA (phytohemagglutinin) (3 Kg/mL), 10 % FBS (fetal bovine serum), 10 mM HEPES and the anticoagulant heparin (125 U.S.P.-U/mL). All incubations were done at 37 °C with 5.5 % CO2 in humidified air.

Cytokinesis block (if used):

Colcemid (final concentration: 0.2 µg/mL)

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/ß-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

JUSTIFICATION FOR TOP DOSE: With regard to the molecular weight and the purity of the test substance, 2760.0 µg/mL of the test substance (approx. 10 mM) was applied as top concentration for treatment of the cultures in experiment I. In the absence of cytotoxicity in Experiment I, 2760.0 µg/mL was chosen as top treatment concentration for Experiment IIA. The experimental part without S9 mix was repeated (Exp. IIB) with a top concentration of 600.0 µg/mL, because the positive and solvent controls were not evaluable for cytogenetic damage. A second repeat of this experimental part was performed (Exp. IIC) with the same top concentration and narrow concentration spacing to obtain evaluable concentrations in a cytotoxic range.

CHROMOSOME ABBERATION STUDY
Experiment I: 168.2, 294.3 and 515.0 µg/mL (with and without metabolic activation)
Experiment IIA: 168.2, 294.3 and 515.0 µg/mL (with metabolic activation)
Experiment IIB: 112.0, 195.9 and 342.9 µg/mL (without metabolic activation)
Experiment IIC: 375.0, 400.0, 425.0 and 450.0 µg/mL (without metabolic activation)

Vehicle / solvent:

- Solvent used: DMSO
- Justification for choice of solvent: The solvent was chosen due to its solubilisation properties and its relative non-toxicity to the cell cultures.

Untreated negative controls:

yes

Remarks:

DMSO

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours (experiment I and IIA), 22 hours (experiment IIB and IIC)
- Fixation time: 22 hours for all experiments

SPINDLE INHIBITOR: Colcemid (final concentration: 0.2 µg/mL) approximately three hours before the requested harvest time.

STAIN: Giemsa

NUMBER OF REPLICATIONS: duplicate cultures

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: The supernatant was discarded and the cells were resuspended in hypotonic solution (0.0375 M KCl). Then the cell suspension was allowed to stand at 37 °C for 20 minutes. After removal of the hypotonic solution by centrifugation (approx. 900 x g) the cells were fixed with a mixture of methanol and glacial acetic acid (3+1 parts, respectively). A small amount of cell suspension was then dropped onto clean, wet microscope slides and allowed to dry. The slides were stained with Giemsa, mounted after drying and covered with a cover slip. All slides were labelled with a computer-generated random code to prevent scorer bias.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE: At least 100 well-spread metaphases per culture were scored for cytogenetic damage on coded slides, except for the positive control in Experiment IIC without S9 mix, where only 50 metaphases were evaluated. Only metaphases with 46 ± 1 centromere regions were included in the analysis.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes

Rationale for test conditions:

The highest treatment concentration in this study, 2760.0 µg/mL (approx. 10 mM) was chosen with regard to the molecular weight and the purity of the test substance and with respect to the OECD Guideline 473 for in vitro mammalian cytogenetic tests.

Evaluation criteria:

ACCEPTABILITY OF THE ASSAY
The chromosomal aberration assay is considered acceptable if it meets the following criteria:
a) The number of aberrations found in the solvent controls falls within the range of the laboratory historical control data.
b) The rate of chromosomal aberrations in the positive controls is statistically significant
increased.

EVALUATION OF RESULTS
A test substance is classified as non-clastogenic if:
- the number of induced structural chromosomal aberrations in all evaluated concentration groups is in the range of the laboratory historical control data.
- no statistically significant increase of the rate of structural chromosomal aberrations is observed in comparison to the respective solvent control.

A test substance is classified as clastogenic if:
- the number of induced structural chromosomal aberrations is not in the range of the historical laboratory control data
and
- either a concentration-related or a statistically significant increase in the number of cells carrying structural chromosomal aberrations is observed.

A test substance not meeting the criteria for classification as non-mutagenic or mutagenic may be considered equivocal in this assay and may be subject to further investigation.

Statistical significance was confirmed by means of the Fisher´s exact test (p < 0.05).
However, both biological and statistical significance should be considered together. If the above mentioned criteria for the test substance are not clearly met, the classification with regard to the historical data and the biological relevance is discussed and/or a confirmatory experiment is performed.

An increase in the number of polyploid cells or endoreduplicated cells may indicate that the test item has the potential to inhibit mitotic processes and to induce numerical chromosomal aberrations or to inhibit cell cycle progression.

Statistics:

Statistical significance was confirmed by means of the Fisher´s exact test (p < 0.05).

Key result

Species / strain:

lymphocytes: Human

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolality: No relevant influence on the osmolarity or pH values was observed.
- Precipitation: In Experiment I in the absence of S9 mix precipitation of the test substance in the culture medium was observed at 515.0 µg/mL and above at the end of treatment. In Experiment IIB and IIC in the absence of S9 mix precipitation of the test substance in the culture medium was observed at 600.0 µg/mL at the end of treatment. In Experiment I and IIA in the presence of S9 mix precipitation of the test substance in the culture medium was observed at 515.0 µg/mL and above at the end of treatment.
- Definition of acceptable cells for analysis: Only metaphases with 46 ± 1 centromere regions were included in the analysis.

RANGE-FINDING/SCREENING STUDIES:
A preliminary cytotoxicity test was performed to determine the concentrations to be used in the mutagenicity assay. The pre-test phase was performed with 10 concentrations of the test substance and a solvent and positive control. All cell cultures were set up in duplicate. Exposure time was 4 h (with and without S9 mix). The preparation interval was 22 h after start of the exposure.

CHROMOSOME ABERRATION:
- Either EMS (770.0 or 660.0 µg/mL) or CPA (2.5 or 7.5 µg/mL) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.
- In Experiment I, IIA and IIB either with or without metabolic activation, no clastogenicity was observed at the concentrations evaluated. In Experiment IIC in the absence of S9 mix one statistically significant increase in chromosomal aberrations (6.8 % aberrant cells, excluding gaps) clearly exceeding the range of the laboratory solvent control data (0.0 – 2.5 % aberrant cells, excluding gaps) was observed after treatment with 450.0 µg/mL. In addition, at concentrations of 400.0 and 425.0 µg/mL, the percentage cells with aberrations (4.3 and 3.0 %) exceeded the range of the laboratory solvent control data, however these values did not reach statistical significance when compared to the concurrent control. A clear concentration-dependency was not observed. Results can be found in Table 2 in 'Any other information or results incl. tables'.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive and negative (DMSO) historical control data: an overview with the historical ranges for negative controls can be found in Table 1 in ‘Any other information on results incl. tables’.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In Experiment I in the absence and presence of S9 mix and in Experiment IIA in the presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration. In Experiment IIB and IIC in the absence of S9 mix, concentrations showing clear cytotoxic effects were not evaluable for cytogenetic damage. However, the mitotic index was markedly reduced to 66.1 % of control in Experiment IIB.

Table 1. Historical Control data: Percentage of aberrant cells in human lymphocyte cultures (2011-2012)

Without S9 mix: preparation interval 22 hrs, treatment 4 hrs

Without S9 mix: preparation interval 22 hrs, treatment 22 hrs

With S9 mix: preparation interval 22 hrs, treatment 4 hrs

Aberrant cells (%)

Aberrant cells (%)

Aberrant cells (%)

No. of exp.

Range

Mean

Standard deviation

No. of exp.

Range

Mean

Standard deviation

No. of exp.

Range

Mean

Standard deviation

Solvent control

Solvent control

Solvent control

Aqueous solv.^1

12

0.0 – 2.5

1.1

±0.6

Aqueous solv.^1

12

0.0 – 2.0

1.0

±0.5

Aqueous solv.^1

19

0.0 – 3.5

1.2

±0.7

Organic solv.^2

40

0.0 – 3.0

1.3

±0.6

Organic solv.^2

36

0.0 – 2.5

1.1

±0.6

Organic solv.^2

67

0.0 – 2.5

1.1

±0.5

Total

52

0.0 – 3.0

1.2

±0.6

Total

48

0.0 – 2.5

1.1

±0.6

Total

86

0.0 – 3.5

1.1

±0.6

Positive control

Positive control

Positive control

EMS 550-825 µg/ml

51

7.5 – 14.0

9.6

±1.3

EMS 550-825 µg/ml

48

8.0 – 40.0

17.3

±5.3

CPA 2.5-20.0 µg/ml

84

7.5 – 37.0

13.6

±3.7

1 Aqueous solvents: deionised water (10 % v/v) and culture medium DMEM:F12

2 Organic solvents: dimethyl sulfoxide (0.5 or 1.0 % v/v), acetone, ethanol and tetrahydrofurane (0.5 % v/v)

Table 2. Structural Chromosome Aberrations Experiment IIC (Preparation Interval 22 hrs without S9 Mix: Exposure Period 22 hrs)

Slide no.	Cells scored	% Aberrantcells			Aberrations**
		incl.      excl.		carrying ex-	Gaps		Chromatidtype							Chromosome type						Other
		gaps*   gaps*		changes	g     ig		b	f		d		ex		ib	if		id		cx	macd
					Without S9 mix
Solvent control: DMSO 0.5 %
1	100				0	0	0		0		0		0	0	0	0		0		0	0
2	100				1	0	3		0		0		0	0	0	0		1		0	0
1 + 2	200	2.5	2.0	0.5	1	0	3		0		0		0	0	0	0		1		0	0
Positive control: EMS 660.0 µg / mL^#
1	50				6	0	21		2		0		11	1	1	0		0		1	0
2	50				3	0	27		1		0		5	2	3	0		0		2	0
1 + 2	100	52.0	47.0	13.0	9	0	48		3		0		16	3	4	0		0		3	0
Test item: 375.0 µg / mL
1	100				1	0	1		0		0		0	0	1	0		0		0	0
2	100				0	0	1		0		0		1	0	0	0		0		0	0
1 + 2	200	2.0	1.5	0.5	1	0	2		0		0		1	0	1	0		0		0	0
Test item: 400.0 µg / mL^##
1	200				1	0	9		0		0		0	0	0	0		0		0	0
2	200				0	0	6		0		0		1	0	0	0		0		1	0
1 + 2	400	4.5	4.3	0.3	1	0	15		0		0		1	0	0	0		0		1	0
Test item: 425.0 µg / mL^##
1	200				1	0	6		1		0		0	0	1	0		0		0	0
2	200				0	0	5		0		0		0	0	0	0		0		0	0
1 + 2	400	3.0	3.0	0.0	1	0	11		1		0		0	0	1	0		0		0	0
Test item: 450.0 µg / mL^##
1	200				0	0	12		5		0		0	0	1	0		0		0	0
2	200				0	0	10		4		0		0	1	1	0		0		0	0
1 + 2	400	6.8	6.8	0.0	0	0	22		9		0		0	1	2	0		0		0	0

* Including cells carrying exchanges

** Note: multiple aberrations may occur in a single cell, therefore, the numbers in these columns may not (nor are they intended to) correlate with the number in the columns of % Aberrant cells.

#50 metaphases per culture were evaluated due to strong clastogenic effects

##200 metaphases per culture were evaluated due to inhomogeneous data

 

Abbreviations

 g = gap, ig = iso-gap (gaps are achromatic lesions of chromatid or chromosome type where no or only a minimal misalignment of chromosomal material is visible), b = break, ib = iso-break, f = fragment, if = iso- fragment, d = deletion, id = iso-deletion, ma = multiple aberration (= more than 4 events in one cell [excluding gaps]), ex = chromatid type exchange, cx = chromosome type exchange, cd = chromosomal disintegration (= pulverization)

Conclusions:

In a GLP compliant OECD 473 study, under the experimental conditions reported, the test substance induced structural chromosomal aberrations in human lymphocytes in vitro. Therefore, the test substance is considered to be clastogenic in this chromosome aberration test, when tested up to precipitating or the highest evaluable concentrations.

Executive summary:

This GLP compliant OECD 473 in vitro assay was performed to assess the potential of the test substance to induce structural chromosomal aberrations in cultured human lymphocytes in the absence and presence of an exogenous metabolic activation system (liver S9 mix from phenobarbital/β-naphthoflavone treated male rats). In each experimental group two parallel cultures were analysed. Per culture at least 100 metaphases were evaluated for structural chromosomal aberrations, except for the positive control in Experiment IIC without S9 mix, where only 50 metaphases were evaluated. The highest applied concentration in this study (2760.0 µg/mL of the test substance, approx. 10 mM) was chosen with regard to the molecular weight and purity of the test substance and with respect to the current OECD Guideline 473. Concentration selection for the cytogenetic experiments was performed considering the toxicity data and test substance precipitation and in accordance with OECD Guideline 473.

In Experiment I in the absence and presence of S9 mix and in Experiment IIA in the presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration. In Experiment IIB and IIC in the absence of S9 mix concentrations showing clear cytotoxic effects were not evaluable for cytogenetic damage. However, the mitotic index was markedly reduced below 70 % of control in Experiment IIB. In Experiment I, IIA and IIB either with or without metabolic activation, no clastogenicity was observed at the concentrations evaluated. In Experiment IIC in the absence of S9 mix one statistically significant increase in chromosomal aberrations (6.8 % aberrant cells, excluding gaps) clearly exceeding the range of the laboratory solvent control data (0.0 – 2.5 % aberrant cells, excluding gaps) was observed after treatment with 450.0 µg/mL. In addition, at concentrations of 400.0 and 425.0 µg/mL, the percentage cells with aberrations (4.3 and 3.0 %) exceeded the range of the laboratory solvent control data, however these values did not reach statistical significance when compared to the concurrent control. A clear concentration-dependency was not observed. No evidence of an increase in polyploid metaphases was noticed after treatment with the test substance as compared to the control cultures. Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations.

In conclusion, it can be stated that under the experimental conditions reported, the test substance induced structural chromosomal aberrations in human lymphocytes in vitro. Therefore, the test substance is considered to be clastogenic in this chromosome aberration test, when tested up to precipitating or the highest evaluable concentrations. 

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Negative, micronucleus test - in vivo, Dony 2014

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:

29 Jan 2014 to 28 Feb 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

21 July 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

Version / remarks:

August 1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

30 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The rat is an animal that has been used for many years as a suitable experimental animal in cytogenetic investigations. There are many data available from such investigations, which may be helpful in the interpretation of results from the micronucleus test.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 8 to 10 weeks
- Weight at study initiation: 262.7 to 303.0 gram
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: Housed in groups, in Makrolon Type III / IV, with wire mesh top with granulated soft wood bedding.
- Diet: Pelleted standard diet, ad libitum
- Water: Tap water, ad libitum
- Acclimation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 23 – 65
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
29 Jan 2014 to 28 Feb 2014

Route of administration:

oral: unspecified

Vehicle:

- Vehicle used: 1% CMC (carboxymethyl cellulose) suspended in sterile water
- Justification for choice of vehicle: The vehicle was chosen by the Sponsor and due to its relative non-toxicity for the animals and ability to formulate a suitable dosing preparation.
- Concentration of test material in vehicle: 50, 100 and 200 mg/mL
- Amount of vehicle: 10 mL/kg bw
- Lot/batch no.: BCBD7651V (CMC), 134618061 (sterile water)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
On the day of the experiment, the test substance was suspended in 1% CMC. An Ultraturrax was used to formulate the test substance in the vehicle. All animals received a single standard volume once orally. The vehicle was chosen by the Sponsor and due to its relative non-toxicity for the animals and ability to formulate a suitable dosing preparation. The oral route was used as this is of relevance to human risk assessment. A correction factor of 1.06 was applied.

Duration of treatment / exposure:

Sampling of the bone marrow was done 24 and 48 hours after treatment.

Frequency of treatment:

The animals received one single oral dose.

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

Low dose group

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Mid dose group

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Remarks:

High dose group

No. of animals per sex per dose:

Pre-test: 2 males and 2 females for each pre-test
Main experiment: 7 males per group treated with test substance, 5 males per group for negative and positive control

Control animals:

yes, concurrent vehicle

Positive control(s):

- Positive control: cyclophosphamide
- Route of administration: oral
- Doses / concentrations: 20 mg/kg-bw

Tissues and cell types examined:

Bone marrow erythrocytes

Details of tissue and slide preparation:

PRE-EXPERIMENT:
A preliminary study of acute toxicity was performed in both male and female rats (two animals per sex and dose level) under identical conditions as in the mutagenicity study concerning: animal strain, vehicle, route, frequency, and volume of administration.
The animals were treated once orally with the test substance and examined for acute toxic symptoms at intervals of around 0-1 h, 2-4 h, 5-6 h, 24 h, 30 h, and 48 h after administration of the test substance.
The test dose levels were chosen using doses from the following scheme starting at 1250 mg/kg:
5 – 8 – 12.5 – 20 – 32 – 50 – 80 – 125 – 200 – 320 – 500 – 800 – 1250 – 2000 mg/kg bw.

CRITERIA FOR DOSE SELECTION:
It is generally recommended to use the maximum tolerated dose or the highest dose that can be dissolved and administered reproducibly or 2000 mg/kg as the upper limit for non-toxic test substances.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Three adequately spaced dose levels spaced by a factor of 2 were applied (500, 1000 and 2000 mg/kg bw), and bone marrow samples were collected at the central sampling interval of 24 h after treatment. For the highest dose level an additional bone marrow sample was taken at 48 h after treatment.

DETAILS OF SLIDE PREPARATION:
The animals were sacrificed using CO2 followed by bleeding. The femora were removed, the epiphyses were cut off and the marrow was flushed out with fetal calf serum using a syringe. The nucleated cells were separated from the erythrocytes using the method of Romagna. The cell suspensions were passed through a column consisting of α-Cellulose and Cellulose. The columns will then be washed with Hank´s buffered saline. The cell suspension was centrifuged at 1500 rpm (390 × g) for 10 minutes and the supernatant was discarded. A small drop of the re-suspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald /Giemsa. Cover slips were mounted with EUKITT. At least one slide was made from each bone marrow sample.

METHOD OF ANALYSIS:
Evaluation of the slides was performed using NIKON microscopes with 100× oil immersion objectives. Per animal 2000 polychromatic erythrocytes (PCE) were analysed for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined from the same slide and expressed in polychromatic erythrocytes per 2000 erythrocytes. The analysis was performed with coded slides. Immature and mature erythrocytes were identified by their pale and blue to green colour, respectively. Micronuclei are distinguished by being small nuclei separate from and additional to the main nuclei of the cells.

OTHER:
The animals of all dose groups, except the positive control were examined for acute toxic symptoms at intervals of around 0-1 h, 2-4 h, 5-6 h, 24 h, and 48 h after administration of the test substance or the vehicle controls.

Evaluation criteria:

ACCEPTANCE CRITERIA
The study was considered valid as the following criteria were met:
- at least 5 animals per group could be evaluated.
- PCE to erythrocyte ratio was not less than 20 % of the negative control.
- The positive control showed a statistically significant and biologically relevant increase of micronucleated PCEs compared to the negative control.

EVALUATION OF RESULTS
A test substance is classified as mutagenic if it induces either a dose-related increase or a clear increase in the number of micronucleated polychromatic erythrocytes in a single dose group. Statistical methods (nonparametric Mann-Whitney test) were used as an aid in evaluating the results. However, the primary point of consideration was the biological relevance of the results.

A test substance that fails to produce a biologically relevant increase in the number of micronucleated polychromatic erythrocytes is considered non-mutagenic in this system.

A test substance failing to meet the criteria for a positive or negative response may be judged equivocal in this assay and may be considered for further investigation.

Statistics:

Statistical methods (nonparametric Mann-Whitney test) were used as an aid in evaluating the results.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF PRE-EXPERIMENT
In the first pre-experiment 2 male and 2 female animals received a single oral dose of the test substance (1250 mg/kg bw) suspended in 1% CMC (10 mL/kg bw). Two females treated with 1250 mg/kg bw displayed ruffled fur 24 hours post-treatment.
In the second pre-experiment 2 male and 2 female animals received a single oral dose of the test substance (2000 mg/kg bw) suspended in 1% CMC (10 mL/kg bw). The animals treated with 2000 mg/kg bw did not show any clinical symptoms.
On the basis of these data 2000 mg/kg bw, the maximum OECD Guideline recommended dose for this assay was considered suitable. No substantial gender specific differences in toxicity were observed, thus, the main study was performed using male animals only, as permitted by the Guideline.

RESULTS OF DEFINITIVE STUDY
In the main experiment for the different dose groups 7, 7 and 14 males (2 × 7 males per group) received orally a single dose of 500, 1000 and 2000 mg/kg bw test substance suspended in 1% CMC, respectively. The volume administered was 10 mL/kg bw. The animals treated with the test substance or the vehicle (1% CMC) alone did not show any clinical symptoms.

The mean number of polychromatic erythrocytes was not substantially decreased after treatment with the test substance as compared to the mean value of PCEs of the vehicle control, indicating that the test substance did not have any significant cytotoxic properties on the bone marrow (Table 1 in ‘Any other information on results incl. tables’).
In comparison to the corresponding vehicle controls there was no biologically relevant enhancement or statistically significant increase in the frequency of the detected micronuclei at any preparation interval and dose level after administration of the test substance (Table 1 and 2 in ‘Any other information on results incl. tables’).
For all treatment groups the mean values of micronuclei observed after treatment with the test substance were well within the historical vehicle control range (Table 3 in 'Any other information on results incl. tables'). Additionally no dose dependence was observed.
A dose of 20 mg/kg bw cyclophosphamide administered orally was used as positive control which showed a statistically significant increase of induced micronucleus frequency. The volume of the positive control administered was 10 mL/kg bw.

Table 1. Summary of Micronucleus Test Results

test group	dose mg/kg b.w.	sampling time (h)	PCEs with micronuclei (%)	range	PCE per 2000 erythrocytes
negativecontrol	0	24	0.210	2 - 6	1158
test substance	500	24	0.329	2 -11	1163
test substance	1000	24	0.186	2 - 8	1129
test substance	2000	24	0.329	2 -12	1152
positive control	20	24	1.540	17 -49	1076
negativecontrol	0	48	0.190	2 - 9	1136
test substance	2000	48	0.186	1 - 8	1120

 

 

Table 2. Biometry

Statistical significance at the five per cent level (p < 0.05) for the incidence of micronuclei was evaluated by means of the non-parametric Mann-Whitney test.

Negative control versus test group	Significance	p
500 mg test substance/kg bw; 24 h	-	0.1098
1000 mg test substance/kg bw; 24 h	n.t.	-
2000 mg test substance/kg bw; 24 h	-	0.2058
20 mg CPA/kg bw; 24 h	+	0.0040
2000 mg test substance/kg bw; 48 h	n.t.	-

+ = significant;

- = not significant;

n.t.= not tested, as the mean micronucleus frequency was not above the vehicle control value

 

Table 3. Historical Control Data (2009 – 2013)

Micronucleated cells	Negative Controls	Positive Controls (CPA)
	Males	Males
Mean ± SD (%)	0.196 ± 0.123	2.276 ± 1.061
Range of mean group value (%)	0.000 - 0.550	0.300 - 5.700
Range (individual animal data)	0 - 11	6 - 114
No. of Experiments	36	36

Conclusions:

In a GLP compliant OECD 474 study under the experimental conditions reported, the test substance did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the rat. Therefore, the test substance is considered to be non-mutagenic in this bone marrow micronucleus assay.

Executive summary:

This GLP compliant OECD 474 study was performed in order to investigate the potential of the test substance to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the rat. The test substance was suspended in 1% carboxymethylcellulose (CMC), which was also used as the vehicle control. The volume administered orally was 10 mL/kg bw. At 24 and 48 hours after a single administration of the test substance, the bone marrow cells were collected for micronuclei analysis. Seven males per test group (except the negative and positive control groups with five males only) were evaluated for the occurrence of micronuclei. Per animal 2000 polychromatic erythrocytes (PCEs) were scored for micronuclei. To describe a cytotoxic effect due to the treatment with the test substance the ratio between polychromatic and normochromatic erythrocytes was determined per slide and reported as the number of PCEs per 2000 erythrocytes. The following dose levels of the test substance were investigated: 24 h preparation interval: 500, 1000, and 2000 mg/kg bw; 48 h preparation interval: 2000 mg/kg bw. The highest dose was estimated to be a suitable maximum tolerated dose based on a pre-experiment.

After treatment with the test substance the number of PCEs per 2000 erythrocytes was not substantially decreased as compared to the mean value of PCEs per 2000 erythrocytes of the vehicle control, thus indicating that the test substance did not exert any significant cytotoxic effects in the bone marrow. In comparison to the corresponding vehicle controls, there was no biologically relevant or statistically significant enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test substance with any dose level used. For all treatment groups the mean values of micronuclei observed after treatment with the test substance were well within the historical vehicle control range. Additionally, no dose dependency was observed. A dose of 20 mg/kg bw cyclophosphamide administered orally was used as the positive control, which showed a substantial increase of induced micronucleus frequency. The volume of the positive control administered was 10 mL/kg bw.

In conclusion, it can be stated that under the experimental conditions reported, the test substance did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the rat. Therefore, the test substance is considered to be non-mutagenic in this bone marrow micronucleus assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Ames test (bacterial reverse mutation test in vitro): Negative

This GLP-compliant OECD 471 study was performed to investigate the potential of the test substance to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA1535, TA1537, TA98, and TA100, and the Escherichia coli strains WP2uvrApKM101 and WP2 pKM101.The assay was performed with and without Phenobarbital/ß-naphthoflavone induced rat liver S9 metabolic activation. Each concentration, including the controls, was tested in triplicate. The test substance was tested at the following concentrations: Experiment I and II: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate.

The plates incubated with the test substance showed reduced background growth at higher concentrations with all strains used in the first experiment and some of the strains in the second experiment. Cytotoxic effects, evident as a reduction in the number of revertants (below an induction factor of 0.5), were observed at high concentrations in nearly all strains used. No increase in revertant colony numbers of any of the six tester strains was observed following treatment with the test substance at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies.

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

Chromosome aberration test in vitro: Positive

This GLP compliant OECD 473 in vitro assay was performed to assess the potential of the test substance to induce structural chromosomal aberrations in cultured human lymphocytes in the absence and presence of an exogenous metabolic activation system (liver S9 mix from phenobarbital/β-naphthoflavone treated male rats).In each experimental group two parallel cultures were analysed. Per culture at least 100 metaphases were evaluated for structural chromosomal aberrations, except for the positive control in Experiment IIC without S9 mix, where only 50 metaphases were evaluated.The highest applied concentration in this study (2760.0 µg/mL of the test substance, approx. 10 mM) was chosen with regard to the molecular weight and purity of the test substance and with respect to the current OECD Guideline 473. Concentration selection for the cytogenetic experiments was performed considering the toxicity data and test substance precipitation and in accordance with OECD Guideline 473.

In Experiment I in the absence and presence of S9 mix and in Experiment IIA in the presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration. In Experiment IIB and IIC in the absence of S9 mix concentrations showing clear cytotoxic effects were not evaluable for cytogenetic damage. However, the mitotic index was markedly reduced below 70 % of control in Experiment IIB. In Experiment I, IIA and IIB either with or without metabolic activation, no clastogenicity was observed at the concentrations evaluated. In Experiment IIC in the absence of S9 mix one statistically significant increase in chromosomal aberrations (6.8 % aberrant cells, excluding gaps) clearly exceeding the range of the laboratory solvent control data (0.0 – 2.5 % aberrant cells, excluding gaps) was observed after treatment with 450.0 µg/mL. In addition, at concentrations of 400.0 and 425.0 µg/mL, the percentage cells with aberrations (4.3 and 3.0 %) exceeded the range of the laboratory solvent control data, however these values did not reach statistical significance when compared to the concurrent control. A clear concentration-dependency was not observed. No evidence of an increase in polyploid metaphases was noticed after treatment with the test substance as compared to the control cultures. Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations.

In conclusion, it can be stated that under the experimental conditions reported, the test substance induced structural chromosomal aberrations in human lymphocytes in vitro. Therefore, the test substance is considered to be clastogenic in this chromosome aberration test, when tested up to precipitating or the highest evaluable concentrations. 

Bone marrow micronucleus test in vivo: Negative

This GLP compliant OECD 474 study was performed in order to investigate the potential of the test substance to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the rat. The test substance was suspended in 1% carboxymethylcellulose (CMC), which was also used as the vehicle control. The volume administered orally was 10 mL/kg bw. At 24 and 48 hours after a single administration of the test substance, the bone marrow cells were collected for micronuclei analysis. Seven males per test group (except the negative and positive control groups with five males only) were evaluated for the occurrence of micronuclei. Per animal 2000 polychromatic erythrocytes (PCEs) were scored for micronuclei. To describe a cytotoxic effect due to the treatment with the test substance the ratio between polychromatic and normochromatic erythrocytes was determined per slide and reported as the number of PCEs per 2000 erythrocytes. The following dose levels of the test substance were investigated: 24 h preparation interval: 500, 1000, and 2000 mg/kg bw; 48 h preparation interval: 2000 mg/kg bw. The highest dose was estimated to be a suitable maximum tolerated dose based on a pre-experiment.

After treatment with the test substance the number of PCEs per 2000 erythrocytes was not substantially decreased as compared to the mean value of PCEs per 2000 erythrocytes of the vehicle control, thus indicating that the test substance did not exert any significant cytotoxic effects in the bone marrow. In comparison to the corresponding vehicle controls, there was no biologically relevant or statistically significant enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test substance with any dose level used. For all treatment groups the mean values of micronuclei observed after treatment with the test substance were well within the historical vehicle control range. Additionally, no dose dependency was observed. A dose of 20 mg/kg bw cyclophosphamide administered orally was used as the positive control, which showed a substantial increase of induced micronucleus frequency. The volume of the positive control administered was 10 mL/kg bw.

In conclusion, it can be stated that under the experimental conditions reported, the test substance did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the rat. Therefore, the test substance is considered to be non-mutagenic in this bone marrow micronucleus assay.

Justification for classification or non-classification

Based on the results of GLP compliant guideline studies on genetic toxicity, classification is not warranted in accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation No. 1272/2008.

The positive effects observed in the in vitro chromosome aberration test were not confirmed in the in vivo micronucleus test, both investigating the endpoint clastogenicity. Therefore, the data are considered sufficient to demonstrate that the test substance is not genotoxic in vivo.