1,4-naphthoquinone

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Three in vitro studies are available assessing the genotoxic potential of the substance. This includes a reverse mutation assay (Ames test) a chromosome aberration assay and a HPRT assay.

The results of the Ames test indicated a positive response in the Salmonella strain TA1537 with metabolic activation only.

In the in vitro chromosome aberration assay, the incidences of chromosomal structural aberrant cells were higher than the historical negative control range at the test facility and increased significantly compared with the negative controls in the assay without S9 mix and in the 24 hour assay. A dose dependant increase was also observed in these assays. Therefore the substance was considered to have the potential to induce chromosomal aberration in CHL/IU cells under the conditions employed in the test.  

In the in vitro HPRT assay, no mutagenicity was observed both with and without metabolic activation (no enhancement of mutation rate over the range of controls was observed).

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

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

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 Mix

Test concentrations with justification for top dose:

Preliminary test: 1.22, 4.88, 19.5, 78.1, 313, 1250 and 5000 µg/plate.

Main test 1 and 2:
Without S9 mix: 0.153, 0.305, 0.610, 1.22, 2.44 and 4.88 µg/plate.
With S9 mix: 2.44, 4.88, 9.77, 19.5, 39.1 and 78.1 µg/plate.

The main tests were conducted based on the results of the preliminary test using doses at which the maximum dose that microbial growth inhibition was clearly observed.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Standard vehicle for tests of this type

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide, sodium azide, 9-aminoacridine hydrochloride monohydrate and 2-aminoanthracene.

Remarks:

Varying positive control substances depending on the strain/metabolic activation used.

Details on test system and experimental conditions:

METHOD OF APPLICATION: Preincubation method (standard as per OECD guidance)

DURATION
- Preincubation period: 20 minutes at 37°C
- Exposure duration: 48 hours at 37°C

NUMBER OF REPLICATIONS: In the main test 3 plates/dose were used (triplicate). In the preliminary test 1 plate per dose was used (2 for controls).

MEASUREMENT OF COLONY NUMBER: Revertant colonies were measured using an automatic colony counter or manual counting.

DETERMINATION OF CYTOTOXICITY
- Method: Background lawn of the bacterial cells that have amino-acid requirements were observed by stereoscopic microscope and microbial inhibition (cytotoxicity) was judged by the relationship between the test substance treated plates and the solvent control plates.

Rationale for test conditions:

Pre-incubation method standard as per OECD guidance. Main experiment was repeated to confirm the positive result.

Evaluation criteria:

The substance was judged to be positive when it induced a dose dependant increase in the number of revertant colonies (mean) to a level equal to or greater than 2 fold of the solvent control value (mean) in any one of the tester strains with or without S9 mix, and when a dose dependant increase was reproducible. Other results were judged to be negative.

Statistics:

No statistical analysis was performed with the results in the study.

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Observed at >/= 39.1 µg/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Observed at >/= 2.44 µg/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Observed at >/= 39.1 µg/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Observed at >/= 2.44 µg/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Observed at >/= 39.1 µg/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Observed at >/= 2.44 µg/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Observed at >/= 39.1 µg/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Observed at >/= 2.44 µg/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Observed at >/= 78.1 µg/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Observed at >/= 2.44 µg/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Preliminary test:
Cytotoxicity without S9 mix: 4.88 µg/plate or greater (all strains).
Cytotoxicity with S9 mix: 78.1 µg/plate or greater (all strains).

Main test 1:
Cytotoxicity without S9 mix: 2.44 µg/plate (all strains)
Cytotoxicity with S9 mix: 39.1 µg/plate or greater (TA100, TA1535, TA98, TA1537). 78.1 µg/plate (WP2urvA).
Precipitation: precipitation of the test substance was not observed in all tested strains both with and without S9 mix.

Main test 2:
Cytotoxicity without S9 mix: 2.44 µg/plate (all strains)
Cytotoxicity with S9 mix: 39.1 µg/plate or greater (TA100, TA1535, TA98, TA1537). 78.1 µg/plate (WP2urvA).
Precipitation: precipitation of the test substance was not observed in all tested strains both with and without S9 mix.

Sterility Test: Bacteria or fungus contamination was not observed in plates of the highest dose of the test substance solution, S9 mix or the 0.1 mol/L sodium phosphate buffer in the preliminary test and two main tests.

Conclusions:

Under the conditions of the study, the substance was deemed to be potentially mutagenic as greater than a 2-fold number of revertant colonies was induced by the test substance compared with controls when considering the strain TA1537 with metabolic activation. This result was found in two experiments and the acceptability of the study was observed.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

other: In vitro chromosome aberration assay

Species / strain / cell type:

other: CHL/IU (derived from the lungs of female Chinese Hamsters)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: DS Pharma Biomedical Co., Ltd.
- Suitability of cells: Standard cells as detailed in the OECD guideline
- Cell doubling time: 15.2 hours
- Modal number of chromosomes: 25 (2n)

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Culture medium containing DMSO
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Cell growth inhibition test:
Short term and continuous assays: 0, 0.0781, 0.156, 0.313, 0.625, 1.25, 2.5 5, 10 and 20 µg/mL
A preliminary test was conducted at 16, 160 and 1600 µg/mL in each treatment condition before conducting the cell growth inhibition test. The tested plates were observed by an inverted phase-contrast microscope to determine cell densities after the treatment (expressed as the relative percentage (%) to the negative control plate). Based on these results, 20 µg/mL at which the cell growth inhibition was predicted to be more than 50% was selected as the highest concentration in the cell growth inhibition test.

Chromosomal aberration test (short term assay):
Without S9 mix: 0, 0.4, 0.6, 0.7, 0.8, 0.9, 1.0, 1.25, 1.5 and 2 µg/mL.
With S9 mix: 0, 1, 2, 4, 5, 6, 7, 8 and 10 µg/mL.

Chromosomal aberration test (continuous assay):
0, 0.4, 0.6, 0.7, 0.8, 0.9, 1, 1.25, 1.5 and 2 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: In the results of a solvent selection test the substance was dissolved at 160 mg/mL in DMSO and in acetone, but was not dissolved or suspended in physiological saline. Exothermic reactions, discolouration or foaming were not observed in the preparation and therefore DMSO was selected as the vehicle of choice and was used as the solvent control substance (as is standard in the OECD guideline).

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

mitomycin C

Details on test system and experimental conditions:

CELL GROWTH INHIBITION TEST:
With and without S9 mix: short-term treatment assay (6 hour treatment and 8 hour recovery).
24 hour assay: 24 hours continuous treatment (no S9 mix).
4000 cells/mL cell suspension was initially used and the population doubling and relative population doubling (%) were calculated. The concentration giving 50% inhibition of cell growth (IC50) were calculated for all assays from a linear equation derived from two data points showing cell growth indices higher and lower than 50%, but closest to 50%.

CHROMOSOME ABERRATION ASSAY:
2 hours before the end of culture, colcemid was added to the medium in each plate to accumulate the metaphase cells. The cells were processed in accordance with standard procedures and the RPD measured. The concentrations were then selected for metaphase analysis at which the cell growth index was around 50% (1.0 and 0.8 µg/mL without S9 mix and in the 24 hour assay, respectively). With S9, the cell growth indices were 66.2% and 19.2% at 5 and 6 µg/mL, respectively. Based on these results the following were used for metaphase analysis:
Without S9 mix: 0.8, 0.9 and 1.0 µg/mL
With S9 mix: 2, 4 and 5 µg/mL
24-hour assay: 0.6, 0.7 and 0.8 µg/mL

Test specimens were arranged ramdomly. The criteria for selection of observable metaphase cells was that the chromosomes should be well spread, for structural aberrant cells the number of centromeres should be 25 +/- 2 and for numerical aberrant cells the number of centromeres should be 25 +/- 2 or 38 or more.

The number of cells analysed was 150 cells per plate (300 cells per concentration). These were assessed for chromatid break, chromatid exchange, chromosome break, chromosome exchange, fragmentation (all structural aberrations) and polyploidy cells with 38 or more centromers and endoreduplicated cells (all numerical aberrations). The number of 'gaps' was also noted.
A structural aberrant cell was assessed as a cell with at least one structural chromosome aberration. A numerical aberrant cell was assessed as a cell with at least one numerical chromosome aberration.

Rationale for test conditions:

Standard test approach as per the OECD guidelines

Evaluation criteria:

Negative: Either the incidence of aberrant cells in the test substance treated groups was within the historical negative control range or no statistically significant difference from the solvent controls were detected.
Inconclusive: A significant difference from a negative control value in a least one of the test substance treated groups was detected, but a dose dependant increase was not detected in the statistical analysis.
Positive: A significant difference from the negative control value in at least one of the test substance treated groups and a dose dependant increase were detected in the statistical analysis.

Statistics:

Statistical analysis was performed to detect the differences in the incidents of aberrant cells between the solvent control and the test substance groups in which the incidence of aberrant cells was more than the historical control range. It was also performed to detect the differences in the incidence of aberrant cells between the negative and positive control groups. Fischer's exact test was used for statistical analysis with a level of significance of 5%. When a significant difference from the negative control was detected the dose response relationship was examined by Cochran-Armitage test (also with the level of significance of 5%).

Species / strain:

other: CHL/IU (derived from the lungs of female Chinese Hamsters)

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

At the concentrations where metaphase analysis was determined

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Cell growth inhibition test:
No precipitation was observed within the test. The IC50 values were 1.0, 5.0 and 0.9 µg/L without and with S9 mix and in the 24 hour assay respectively.

Chromosome aberration test:
No precipitation was observed within the test. The concentrations at which the cell growth index was around 50% were 1.0 and 0.8 µg/mL without S9 mix and in the 24 hour assay, respectively. With S9 mix, the cell growth indices were 66.2% and 19.2% at 5 and 6 µg/mL, respectively. Based on the results of the cell growth index, the concentration for the metaphase analysis were selected as 0.8, 0.9 and 1.0 µg/mL without S9 mix, 2, 4 and 5 µg/mL with S9 mix and 0.6, 0.7 and 0.8 µg/mL in the 24 hour assay. In the preliminary observation, all selected specimens were acceptable for the metaphase analysis.

As a result of the metaphase analysis, the incidences of both structural and numerical aberrant cells with S9 mix and the incidence of numerical aberrant cells without S9 mix and the 24 hour assay in the test treatment groups were within the negative historical control range of the test facility.

However, the incidences of structural aberrant cells were 4.0 and 4.3% at 0.9 and 1.0 µg/mL without S9 mix and 8.7 and 7.7% at 0.7 and 0.8 µg/mL in the 24 hour assay, respectively and outside the historical negative control range. Also, statistically significant increases of the incidence of chromosomal structural aberrant cells compared to the solvent control values were detected at the concentrations employed without S9 mix and in the 24 hour assay. A dose dependant increase was also detected in these assays.

In the negative control group, the incidences of both structural and numerical aberrant cells were less than 5%. In the positive controls this was > 10% under the treatment conditions.

Conclusions:

A reliable guideline/GLP study is available to assess the potential for clastogenicity of the test substance. The incidences of chromosomal structural aberrant cells were higher than the historical negative control range at the test facility and increased significantly compared with the negative controls in the assay without S9 mix and in the 24 hour assay. A dose dependant increase was also observed in these assays. Therefore the substance was considered to have the potential to induce chromosomal aberration in CHL/IU cells under the conditions employed in the test.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

other: Noted as being performed according to OECD recommendations of 1983 and U.S. EPA guidelines of 1982.

Deviations:

not specified

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

not specified

Principles of method if other than guideline:

The study predates the adoption of the formal OECD 476 guideline but is stated as being conducted in accordance with OECD and EPA recommendations at the time.

GLP compliance:

yes

Type of assay:

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

Target gene:

The gene for HGPRT is located on the x-chromosome of the V79 cells. HGPRT catalyzes the salvage of hypoxanthine and guanine by conversion of these purines with phosphoribosyl pyrophosphate.

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Taken from stocks of V79 cell lines stored at the laboratory.
- Suitability of cells: Cells possessed a stable karyotype.
- Cell cycle length, doubling time or proliferation index: Doubling time 12 to 16 hours.
- Modal number of chromosomes: 22
- Normal (negative control) cell cycle time:

MEDIA USED
- A version of Eagle's minimal essential medium enriched with amino acids and vitamins but free of added thymidine and hypoxanthine (Dulbecco's modified medium).

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

Not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix prepared from the livers of rats treated with aroclor 1254.

Test concentrations with justification for top dose:

Experiment 1 and 2:

0.005, 0.02, 0.04 and 0.05 µg/mL without S9 mix.
0.8, 4.0, 5.0 and 6.0 µg/mL with S9 mix.

The concentrations were chosen from the plotted concentration-response curve obtained in the pre-experiment. The highest concentration selected was in the range of about 40% survival in the presence and absence of S9 mix.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen because it proved to be relatively non-toxic. The test substance could also be dissolved up to toxic concentrations.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

9,10-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

Experimental procedures:

Two day old logarithmically growing stock cultures more than 50% confluent were trypsinised and a single cell suspension prepared. After 24 hours, the medium was replaced with that containing the test substance where appropriate (with and without S9 mix). This was maintained for 4 hours before the medium replaced. All incubations were performed at 37°C with staining performed using methylene blue solutions. Stained colonies with more than 50 cells were counted with a preparation microscope.

Rationale for test conditions:

Standard as per requirements at the time the study was conducted.

Evaluation criteria:

A test substance is considered mutagenic if it induces with at least one of the test substance concentrations reproducibly a mutation frequency that is three times higher than the spontaneous mutant frequency in the experiment.

It is also considered mutagenic if there is a reproducible concentration dependant increase in the mutation frequency, and such evaluation may be considered independantly of the enhancement factor for the induced mutants.

Statistics:

Not required as there was no reproducible enhancement in the groups treated with the test substance and compared to the negative controls, both with and without S9 mix.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Toxicity data (experiment 1)

Treatment	Conc. µg/mL	S9 Mix	No. cells per flask (mean)^a	PE% absolute^b	PE% relative^c
Negative control	0	-	498	100.4	100
Negative control	0	+	345	69.6	100
Negative control and solvent		-	419.5	84.6	100
Negative control and solvent	0	+	401	80.8	100
Positive control and EMS	1000	-	337.5	68	67.8
Positive control and DMBA	15.4	+	54.5	11	13.6
Test substance	0.005	-	477	96.2	113.7
Test substance	0.8	+	326	65.7	81.3
Test substance	0.02	-	327	65.9	77.9
Test substance	4	+	258.5	52.1	64.5
Test substance	0.04	-	171	34.5	40.8
Test substance	5	+	151	30.4	37.7
Test substance	0.05	-	96.5	19.5	23
Test substance	6	+	49.5	10	12.3

^a– only colonies with more than 50 cells 7 days after seeding in medium were scored.

^b– PE absolute = (mean number of cells per flask/number of cells seeded)*100.

^c– PE relative = (mean number of cells per flask/mean number of cells per corresponding control)*100

 

Mutagenicity data (experiment 1)

Treatment	Conc. µg/mL	S9 Mix	No. mutant colonies (mean)^a	SD	Mutant colonies per 10^6cells
Negative control	0	-	5.6	1.1	24.8
Negative control	0	+	3.6	1.1	15.9
Negative control and solvent		-	4.2	2.5	9.8
Negative control and solvent	0	+	5.2	2.4	17.3
Positive control and EMS	1000	-	181.2	24.1	556.7
Positive control and DMBA	15.4	+	45.2	5.2	220.6
Test substance	0.005	-	11.0	4.1	26.8
Test substance	0.8	+	5.4	2.2	17.9
Test substance	0.02	-	15.2	3.7	35.6
Test substance	4	+	6.6	1.1	27.9
Test substance	0.04	-	7.4	2.1	16.6
Test substance	5	+	4.6	1.7	10.0
Test substance	0.05	-	15.6	1.9	45.8
Test substance	6	+	3.0	2.5	14.7

^a– only colonies with more than 50 cells 7 days after seeding in medium were scored.

Toxicity data (experiment 2)

Treatment	Conc. µg/mL	S9 Mix	No. cells per flask (mean)^a	PE% absolute^b	PE% relative^c
Negative control	0	-	329.5	66.8	100
Negative control	0	+	294.5	59.7	100
Negative control and solvent		-	316	64.1	100
Negative control and solvent	0	+	315.5	64	100
Positive control and EMS	1000	-	291.5	59.1	88.5
Positive control and DMBA	15.4	+	71	14.4	22.5
Test substance	0.005	-	302.5	61.4	95.7
Test substance	0.8	+	316.5	64.2	100.3
Test substance	0.02	-	287	58.2	90.8
Test substance	4	+	301.5	61.2	95.6
Test substance	0.04	-	168	34.1	53.2
Test substance	5	+	212.5	43.1	67.4
Test substance	0.05	-	99.5	20.2	31.5
Test substance	6	+	83.5	16.9	26.5

^a– only colonies with more than 50 cells 7 days after seeding in medium were scored.

^b– PE absolute = (mean number of cells per flask/number of cells seeded)*100.

^c– PE relative = (mean number of cells per flask/mean number of cells per corresponding control)*100

Mutagenicity data (experiment 2)

Treatment	Conc. µg/mL	S9 Mix	No. mutant colonies (mean)^a	SD	Mutant colonies per 10^6cells
Negative control	0	-	9.2	4.0	21.2
Negative control	0	+	9.2	3.5	21.6
Negative control and solvent		-	8.8	2.7	23.7
Negative control and solvent	0	+	1.0	1.4	3.1
Positive control and EMS	1000	-	171.6	14.0	545.2
Positive control and DMBA	15.4	+	55.8	5.8	201.9
Test substance	0.005	-	4.4	1.7	13.5
Test substance	0.8	+	5.2	1.9	26.1
Test substance	0.02	-	7.2	4.6	21.6
Test substance	4	+	4.6	2.3	14.0
Test substance	0.04	-	3.6	0.5	10.3
Test substance	5	+	2.6	1.7	8.4
Test substance	0.05	-	8.0	3.1	23.6
Test substance	6	+	0.6	0.5	1.8

^a– only colonies with more than 50 cells 7 days after seeding in medium were scored.

Conclusions:

The test substance was assessed for potential mutagenic properties in the HGPRT test in two independant experiments. Under the conditions of the test no mutagenicity was observed both with and without metabolic activation. No enhancement of mutation rate over the range of controls was observed and the sensitivity of the test system was demonstrated by the enhanced mutation rates found in the groups treated with positive control substances.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

One in vivo study is available which assess the genotoxic potential of the substance. This was a chromosome aberration assay conducted with Chinese Hamsters.

Under the conditions of this study, no chromosome damaging effects were observed in the bone marrow metaphases of male and female Chinese hamsters

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

Version / remarks:

1983 version

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EEC Directive 84/449

Version / remarks:

1984 version

Deviations:

not specified

GLP compliance:

yes

Type of assay:

mammalian bone marrow chromosome aberration test

Species:

hamster, Chinese

Strain:

not specified

Details on species / strain selection:

Detailed as EMD (brother-sister inbreeding)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: E. Merck, Darmstadt
- Age at study initiation: 7 to 9 weeks
- Weight at study initiation: 44.2 g (males) and 37.6 g (females)
- Assigned to test groups randomly: yes
- Fasting period before study: No
- Housing: Housed individually in Makrolon cages type 1 on softwood chippings under conventional conditions.
- Diet (e.g. ad libitum): Ad libitum (free access to ssniff hamster mixed food.
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: At least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 to 29°C
- Humidity (%): 33 to 71%
- Air changes (per hr): Not specified
- Photoperiod (hrs dark / hrs light): 12 hours light/dark cycle

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: 0.25% aqueous Methocel K4M Premium
- Justification for choice of solvent/vehicle: Standard vehicle
- Concentration of test material in vehicle: Not specified
- Amount of vehicle (if gavage or dermal): Not specified

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

Dosed via oral gavage

Duration of treatment / exposure:

Single dose for all relevant animals (1 day)

Frequency of treatment:

Once on day of treatment

Post exposure period:

6 to 48 hours

Dose / conc.:

15 mg/kg bw/day

Dose / conc.:

40 mg/kg bw/day

Dose / conc.:

120 mg/kg bw/day

No. of animals per sex per dose:

5 males and 5 females per dose group (70 in total). Any animals that died during the study were replaced with those from the same stock. At 3 hours before sacrifice and bone marrow sampling, the animals were treated with colcemide to arrest dividing bone marrow cells in the metaphase.

Control animals:

yes, concurrent vehicle

Positive control(s):

- Name: Endoxan (Cyclophosphamide).
- Justification for choice of positive control(s): Standard positive control
- Route of administration: Oral gavage
- Doses / concentrations: 20 mg dissolved in 10 mL Aqua pro injectione directly before use. A dose of 20 mg/kg was used on study.

Tissues and cell types examined:

Bone marrow

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Based on results of a preliminary study.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Bone marrow sampled at 24 hours post dose in animals receiving 15 or 40 mg/kg and 6, 24 and 48 hours post dose in animals receiving 120 mg/kg. Positive control animals were sampled at 24 hours post dose.

DETAILS OF SLIDE PREPARATION: Immediately after the hamsters were euthanized by CO2, both femora of each animal were dissected, cleaned from the rest of teh muscles and the epiphyses removed. bone marrow cells from both the femora per animal were flushed out withs aline and collected in a centrifuge tube and suspended. After centrifugation the supernatant was drawn off and the sediment suspended in KCl solution at 37°C. After hypotonic treatment for 10 minutes, the cells were futher centrifuged and the supernatant discarded. The cells were fixed twice in a mixture of acetic acid and methanol, dropped onto the slides in fresh fixative and flame dried. After over-night air drying at room temperature the slides were stained with aceto-orcein solution, differentiated in ethanol dipped twice in xylene and mounted in Eukitt.

METHOD OF ANALYSIS: All slides were coded before microscopic examination to ensure a lack of bias. 100 metaphases per animal were socred for structural chromosome aberrations using zeiss light microscopes with phase optics.

Morphological observations were scored as follows:
Gap - achromatic region in chromatid(s) not greater than the width of teh chromatid.
Break - achromatic region in chromatid(s) greater than the width of a chromatid or a discontinuity with displacement.
Exchange - aberrations arising from an exchange between one or two chromatids. These may be chromosome or chromatid interchanges. Only asymmetrical or chromatid exchanges will normally be recognised.
Multiple aberration - cells with more than 5 aberrations, gaps excluded.
Specific aberration - atypic chromosomes and pulverised metaphases were scored as specific aberrations.

The position of each aberrant metaphase was recorded by the Vernier reading and photographs taken of each aberrant metaphase. The frequency of polyploid cells were based on scoring of 1000 mitoses per animal. The estimation of the mitotic index was based on scording 1000 cells per animal.

Evaluation criteria:

Statistical significance compared with control groups would indicate a positive response.

Statistics:

The percentage of structural aberrant metaphases per animal, number of mitotic cells per 1000 cells per animal and the number of polyploid cells per 1000 mitoses per animal were used as parameters for statistical analysis.

Regarding structural aberrations two evaluations were conducted; one in which cells carrying gaps and isogaps as the only aberration are included and one where such cells are excluded.

The evaluation was performed applying a non parametric test (i.e. a modification of the Mann-Whitney-Wilcoxon test for tied values. Testing was performed against the one-sided alternative.

As more than one group was compared with controls, the level of significance was modified by the Bonferroni correction according to the number of groups. The values of positive controls were treated in the same way.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
Chinese Hamsters were dosed at 100, 120, 150, 200 or 250 mg/kg. Mortality was observed at the three highest doses. The dose of 120 mg/kg showed clear toxic effects (dyspnea and diarrhoea). Consequently this dose was chosen as the highest dose in the main study.

RESULTS OF DEFINITIVE STUDY

Structural aberrations: No chromosamal damaging affect was observed.
Polyploid cells: No statistically signifcant increase of polyploid cells were observed.
Mitotic index: No treatment-related variation was observed.
Bodyweight: No treatment-related variation was observed.
Clinica signs: 15 mg/kg (one animal observed to be prone), 120 mg/kg (three animals showed dyspnea, two showed ptosis, one had diarrhoea and one animal showed titubation. Most of the hamsters showed bluish urine.
Mortality: One animal died in each dose group.
Positive control: For the positive control, a statistically significant increase of aberrant metaphases for both evaluations (gaps included and excluded) was found.
Evidence of exposure: Clear evidence of systemic exposure and absorption of the test substance was apparent based on the observed clinical signs and mortality. Based on this obvious exposure it is very likely that the bone marrow was exposed to the test substance allowing for a full assessment of the clastogenic potential of the test substance.

The mean percentage of aberrant metaphases found 24 hours after treatment (gaps included) was 1.6% (negative control), 1.4% (15 mg/kg), 1.2% (40 mg/kg) and 1.3% (120 mg/kg). For the 120 mg/kg group the values were 2.0 and 1.3% at 6 and 48 hours respectively.
The corresponding values (gaps exlcuded) were 1.2, 1.0, 0.9 and 1.2% for the 24 hour groups and 1.7 and 1.1% for the 6 and 48 hour groups.

In the positive control the values were 6.1% (gaps included) and 5.9% (gaps excluded).

Aberrations per 100 cells (mean values)

Dose (mg/kg)	Time (hours)	Gaps	Iso-gaps	Breaks	Iso-breaks	Exchanges	Multiple aberr.	Specific aberr.	Total aberr. Incl.	Total aberr. Excl.
0	24	0.4	0	0.7	0.	0	0	0	1.8	1.4
15	24	0.4	0	0.6	0.4	0	0	0	1.4	1.0
40	24	0.3	0	0.6	0.3	0	0	0	1.2	0.9
120	6	0.3	0	0.8	1.0	0	0	0	2.1	1.8
120	24	0.1	0	0.6	0.6	0	0	0	1.3	1.2
120	48	0.2	0	0.6	0.7	0	0	0	1.5	1.3
20 (EMD)	24	0.2	0	3.5	2.1	1	0.5	0	7.3	7.1

Conclusions:

Under the conditions of the study, no chromosome damaging effects were observed in the bone marrow metaphases of male and female Chinese hamsters after a single oral dose at 15, 40 or 120 mg/kg of the test substance.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

The substance was shown to be mutagenic in the in vitro bacterial reverse mutation assay. However, a positive response for mutagenicity was observed only in one Salmonella strain with metabolic activation. A second in vitro study is available (HPRT assay) which provides a negative result for mutagenicity both with and without metabolic activation. As a higher tier in vitro mutagenicity assay (HPRT) is available providing a negative result, and as the initial positive response in the reverse mutation assay was only observed in one strain with metabolic activation, the substance is considered to not possess mutagenic potential and no further testing is considered.

The substance was also shown to be clastogenic in an in vitro chromosome aberration assay. However, the in vivo chromosome aberration assay conducted showed that this response was not replicated under appropriate conditions and therefore the substance is not considered to possess clastogenic activity. Evidence of absorption and subsequent exposure was also observed in the in vivo study indicated by clinical signs and mortality.

Overall the positive responses observed in the initial lower tier genotoxicity tests were not replicated in higher tier in vitro/vivo tests and accordingly the test substance is not considered to be classified as mutagenic in accordance with the CLP Regulation (1272/2008, as amended). No further testing is proposed.