[6R-[6α,7β(Z)]]-7-[2-furyl(methoxyimino)acetamido]-3-(hydroxymethyl)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

[6R-[6α,7β(Z)]]-7-[2-furyl(methoxyimino)acetamido]-3-(hydroxymethyl)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid

EC Number:

260-086-2

EC Name:

[6R-[6α,7β(Z)]]-7-[2-furyl(methoxyimino)acetamido]-3-(hydroxymethyl)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid

Cas Number:

56271-94-4

Molecular formula:

C15H15N3O7S

IUPAC Name:

[6R-[6α,7β(Z)]]-7-[2-furyl(methoxyimino)acetamido]-3-(hydroxymethyl)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

Identity: OK (640-2) (Furoxy Hydroxy)
CAS: 56271-94-4
Molecular weight: 381.35
Batch number: G316533
Expiry: 25 March 2018
Appearance: Cream coloured solid
Storage conditions: 2 to 8C, protected from light
Purity: 94.032% w/w
Weighing factor for formulation: 1.06g contains 1.000g, as OK (56271-94-4)
Date received: 23 October 2017

Method

Species / strain

Species / strain / cell type:

lymphocytes: Human

Details on mammalian cell type (if applicable):

Human blood was collected from two healthy, non-smoking, adult (between 18-35 years of age) 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 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 34 to 39°C, and the cells re-suspended (twice daily) by gentle inversion.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction, prepared from male Sprague-Dawley derived rats, dosed with phenobarbital and 5,6-benzoflavone to stimulate mixed-function oxidases in the liver,

Test concentrations with justification for top dose:

Preliminary toxicity test: 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 µg/mL
Main tests: -S9 mix (3 hours) 250, 500, 1000, and 2000 µg/mL
+S9 mix (3 hours) 250, 500, 1000, and 2000 µg/mL
-S9 mix (21 hours) 31.25, 62.5, 125, 250, 500, 1000, and 2000 µg/mL

The top dose tested was the limit dose for the test.

Vehicle / solvent:

DMSO to a final concentration of 1% in culture medium.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

1% v/v DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

3.4.1 Preliminary Toxicity Test Procedure
Cultures were treated approximately 48 hours after commencement of incubation of lymphocyte cultures. All cultures were uniquely identified. Cultures were prepared for each treatment (3-hour treatment in the absence and presence of S9 mix, and 21-hour treatment in the absence of S9 mix).
Duplicate cultures were used for treatment with the vehicle, and single cultures for treatment with the test item for each test condition. No positive control cultures were prepared. Cultures were incubated at 34 to 39C.
All cultures were centrifuged and re-suspended in fresh medium before treatment, and S9 fraction was present in appropriate cultures at a final concentration of 2% v/v.
OK (640-2) (Furoxy Hydroxy) was added to each culture in 50 µL aliquots. DMSO was used as the vehicle control.
At the end of the 3-hour treatment period, treated culture media was examined for the presence of precipitate. Cultures were then centrifuged at 500g for 5 minutes and the supernatant removed. Cultures were then washed in saline, re-suspended in fresh medium (final volume 5 mL) and incubated for 18 hours until the scheduled harvest time.
At the end of the 21-hour treatment period, treated culture media was examined for the presence of precipitate.
Harvesting and Fixation
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. After 2 hours incubation, each cell suspension was transferred to a centrifuge tube and centrifuged for 5 minutes at 500g. The cell pellets were treated with a hypotonic solution (0.075M KCl) for a 10 minute period at between 34 to 39°C. The suspensions were centrifuged at 500g for 5 minutes and the cell pellets fixed by addition of ice-cold fixative (methanol:glacial acetic acid (3:1 v/v)). Following further centrifugation the supernatant was removed and replaced with fixative; this was repeated until the fixative was clear.
Slide Preparation
The fixed pellets were re-suspended, then centrifuged at 500g for 5 minutes and re-suspended in a small volume of fixative. A few drops of the cell suspensions were dropped onto pre-cleaned microscope slides and allowed to air dry. One slide was prepared per culture. The
slides were then stained in 10% Giemsa, prepared in buffered water (pH 6.8). After rinsing in buffered water the slides were left to air-dry and mounted in DPX. The remainder of the cell suspensions in fixative were stored at 2 to 8C until slide analysis was completed.
Microscopic Examination
The prepared slides were examined by light microscopy and the incidence of mitotic cells per 1000 cells assessed. Slides were assessed for mitotic index (except when clear evidence of overt toxicity was observed, or in cultures where there were no signs of cytotoxicity).
3.4.2 Main Test Procedure
The procedure for the main tests was the same as that for the preliminary tests, with the following exceptions; positive control cultures were included for all tests, duplicate cultures were prepared for all cultures and two slides were prepared per culture.
3-Hour Treatment in the Absence of S9 Mix
OK (640-2) (Furoxy Hydroxy) was added to each culture in 50 µL aliquots. DMSO was used as the vehicle control and Mitomycin C was the positive control.
Following 3-hour treatment, cultures were centrifuged at 500g for 5 minutes and the supernatant removed. Cultures were then re-suspended in saline and centrifuged at 500g for 5 minutes. The saline was then removed and the cell pellets re-suspended in fresh medium (final volume of 5 mL). They were then incubated for 18 hours until the scheduled harvest time.
3-Hour Treatment in the Presence of S9 Mix
OK (640-2) (Furoxy Hydroxy) was added to each culture in 50 µL aliquots. DMSO was used as the vehicle control and Cyclophosphamide was the positive control.
Following 3-hour treatment, cultures were centrifuged at 500g for 5 minutes and the supernatant removed. Cultures were then re-suspended in saline and centrifuged at 500g for 5 minutes. The saline was then removed and the cell pellets re-suspended in fresh medium (final volume of 5 mL). They were then incubated for 18 hours until the scheduled harvest time.
21-Hour Treatment in the Absence of S9 Mix
OK (640-2) (Furoxy Hydroxy) was added to each culture in 50 µL aliquots. DMSO was used as the vehicle control and Mitomycin C was the positive control.
Following the end of the treatment period the cultures were harvested and slides prepared.
Microscopic Examination
The prepared slides were examined by light microscopy using a low power objective. 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).
Where no significant increase in toxicity was observed (i.e. no significant reduction in mitotic index), the maximum concentration tested (2000 µg/mL) was the highest concentration selected for metaphase analysis. Lower concentrations were also selected.
The selected slides were then coded. Metaphase cells were identified using a low power objective and examined at a magnification of x1000 using an oil immersion objective. From each culture 150 metaphase figures were examined, however, this number was reduced in cultures showing a high level of aberrant cells, where 15 cells with structural aberrations (excluding gaps) were observed. Chromosome aberrations were scored according to the classification of the ISCN (2009). Only cells with 44 - 48 chromosomes were analyzed. The vernier readings of all aberrant metaphase figures were recorded. A peer review of the metaphase analysis was performed by the analysis of 10 metaphases for the vehicle, highest concentration selected and positive control for each exposure condition.
Traditionally gaps have been excluded from the quantitation of chromosome aberrations. Some gaps, however, have been shown to be real discontinuities in DNA (Heddle and Bodycote, 1970, Satya-Prakash et al., 1981). In this study the total number of cells containing aberrations both with and without gaps has been calculated.
The incidence of polyploid and endoreduplicated cells (i.e. the ploidy status) were each recorded as a percentage of the 150 metaphases analyzed per slide, independently from the analysis for chromosome aberrations.

Rationale for test conditions:

The following criteria were applied for assessment of assay acceptability:
The concurrent vehicle control was considered acceptable for addition to the laboratories historical vehicle control database (lie below or close to the upper control limit). Where concurrent vehicle control data fell outside the 95% confidence limit it may be acceptable for inclusion in the historical control distribution as long as the data are not extreme outliers and there is evidence that the test system is ‘under control’ and there is evidence of no technical or human failure.
Concurrent positive controls induced a response that were compatible with the laboratories historical positive control database and produced statistically significant increases compared with the concurrent vehicle control.
The criteria for selection of the top dose concentration were consistent with those outlined previously.
Tests that did not fulfill the required criteria were rejected and therefore are not reported.

Evaluation criteria:

Providing that all of the acceptance criteria have been met, the test item was considered to be clearly positive if, in any of the experimental conditions examined:
At least one of the test concentrations exhibited a statistically significant increase compared with the concurrent vehicle control.
The increase was dose-related when evaluated with an appropriate trend test.
Any of the results are outside the distribution of the historical vehicle control data (above the upper 95% confidence limit).
If all of these criteria were met, the test item was considered able to induce chromosome breaks and/or gain or loss in the test system.
Providing that all of the acceptance criteria have been met, a negative response was claimed if, in all of the experimental conditions examined:
None of the test concentrations exhibited a statistically significant increase compared with the concurrent vehicle control.
There was no concentration-related increase when evaluated with an appropriate trend test.
All results are inside the distribution of the historical vehicle control data (within the 95% confidence limits).
If all of these criteria are met, the test item was considered unable to induce chromosome breaks and/or gain or loss in the test system.
The Study Director used scientific judgment to classify data that did not fall into either of the above categories.

Statistics:

The number of aberrant metaphase cells in each test item group was compared with the vehicle control value using the mid-p one-tailed Fisher exact test for an increase (Richardson et al 1989). Statistical significance was declared at 5%.
A Cochran-Armitage test for trend (Armitage, 1955) was applied to the control and all test item groups. If this was 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.
The data was analyzed using the SAFEStat Chromosome Aberrations application.

Results and discussion

Test results

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Any other information on results incl. tables

 Osmolality and pH Measurements

The osmolality and pH of OK (640-2) (Furoxy Hydroxy) in medium were measured by analysing samples of HML media, dosed at 1% (v/v), with either the vehicle (DMSO) or a OK (640-2) (Furoxy Hydroxy) formulation at 200 mg/mL (to give a final concentration of 2000 μg/mL). For medium dosed with OK (640-2) (Furoxy Hydroxy) at 2000 μg/mL; no fluctuations in osmolality of the medium of more than 50 mOsmol/kg and no fluctuations in pH of more than 1.0 unit were observed compared with the vehicle control. The maximum final concentration tested in the preliminary toxicity test was 2000 μg/mL as this is the standard limit concentration within this test system as recommended in the current OECD Guideline 473 (2016).

Preliminary Toxicity Test

Toxicity Data

In all exposure conditions the highest concentration tested was 2000 µg/mL and no precipitate was observed by eye at the end of treatment at 2000 µg/mL as assessed in concurrently treated HML media-only cultures.

In the absence of S9 mix following 3-hour treatment, OK (640-2) (Furoxy Hydroxy) caused no biologically significant reduction in the mitotic index at 2000 µg/mL, the highest tested concentration, when compared with the vehicle control.

In the presence of S9 mix following 3-hour treatment, OK (640-2) (Furoxy Hydroxy) caused no reduction in the mitotic index at 2000 µg/mL, the highest tested concentration, when compared with the vehicle control.

In the absence of S9 mix following 21-hour treatment, OK (640-2) (Furoxy Hydroxy) caused a reduction in the mitotic index to 74% of the vehicle control value at 2000 µg/mL, the highest tested concentration, when compared with the vehicle control.

The concentrations used in the main test were based upon these data.

 Main Test

In all treatment conditions the highest concentration tested was 2000 µg/mL since precipitation did not limit the top concentration tested.

3-Hour Treatment in the Absence of S9 Mix

Toxicity Data

OK (640-2) (Furoxy Hydroxy) caused no reduction in the mean mitotic index at 2000 µg/mL, compared with the mean vehicle control value. The concentrations selected for the metaphase analysis were 500, 1000 and 2000 µg/mL.

Metaphase Analysis

OK (640-2) (Furoxy Hydroxy) caused no statistically significant increases in the mean proportion of cells with chromosomal aberrations (excluding gaps) at any analyzed concentration, when compared with the vehicle control. There was no evidence of a linear dose-concentration relationship.

All mean values (excluding gaps) for the vehicle control (DMSO), and all OK (640-2) (Furoxy Hydroxy) treatment concentrations were within the laboratory historical 95% confidence limits.

The positive control compound, Mitomycin C, caused statistically significant increases (p<0.001) in the proportion of aberrant cells. This demonstrated the sensitivity of the test system.

Polyploidy and Endoreduplication Analysis

No statistically significant increases in polyploid or endoreduplicated metaphases were observed during metaphase analysis, when compared with the vehicle control.

3-Hour Treatment in the Presence of S9 Mix

Toxicity Data

OK (640-2) (Furoxy Hydroxy) caused no reduction in the mean mitotic index at 2000 µg/mL, compared with the mean vehicle control value. The concentrations selected for the metaphase analysis were 500, 1000 and 2000 µg/mL.

Metaphase Analysis

OK (640-2) (Furoxy Hydroxy) caused no statistically significant increases in the mean proportion of cells with chromosomal aberrations (excluding gaps) at any analyzed concentration, when compared with the vehicle control. There was no evidence of a linear dose-concentration relationship.

All mean values (excluding gaps) for the vehicle control (DMSO), and all OK (640-2) (Furoxy Hydroxy) treatment concentrations were within the laboratory historical 95% confidence limits.

The positive control compound, Cyclophosphamide, caused statistically significant increases (p<0.001) in the proportion of aberrant cells. This demonstrated the efficacy of the S9 mix and the sensitivity of the test system.

Polyploidy and Endoreduplication Analysis

No statistically significant increases in polyploid or endoreduplicated metaphases were observed during metaphase analysis, when compared with the vehicle control. 

21-Hour Treatment in the Absence of S9 Mix

Toxicity Data

OK (640-2) (Furoxy Hydroxy) caused no reduction in the mean mitotic index at 2000 µg/mL, compared with the mean vehicle control value. The concentrations selected for the metaphase analysis were 500, 1000 and 2000 µg/mL.

Metaphase Analysis

OK (640-2) (Furoxy Hydroxy) caused no statistically significant increases in the mean proportion of cells with chromosomal aberrations (excluding gaps) at any analyzed concentration, when compared with the vehicle control. There was no evidence of a linear dose-concentration relationship.

All mean values (excluding gaps) for the vehicle control (DMSO) and OK (640-2) (Furoxy Hydroxy) treatment concentrations (with the exception of the 2000 µg/mL concentration,

which lies marginally above the historical range), were within the laboratory historical 95% confidence limits.

The positive control compound, Mitomycin C, caused statistically significant increases (p<0.001) in the proportion of aberrant cells. This demonstrated the sensitivity of the test system.

Polyploidy and Endoreduplication Analysis

No statistically significant increases in polyploid or endoreduplicated metaphases were observed during metaphase analysis, when compared with the vehicle control.

Summary of results

Exposure period		S9 mix	Nominal concentration of OK (640-2) (Furoxy Hydroxy)	Cells with aberrations excluding gaps			Cells with aberrations including gaps			Relative Mitotic
(hours)			(µg/mL)	Individual values (%)		Mean (%)	Individual values (%)		Mean (%)	Index (%)
3		-	0 (DMSO)	1.3	2.0	1.7	1.3	2.7	2.0	100
			500	2.0	1.3	1.7	2.7	1.3	2.0	109
			1000	0.0	2.0	1.0	2.7	2.7	2.7	106
			2000	0.7	2.0	1.3	1.3	3.3	2.3	106
			0.2 (Mitomycin C)	25.9	16.5	20.1***	27.6	16.5	20.8***	95
3		+	0 (DMSO)	0.0	0.7	0.3	0.0	0.7	0.3	100
			500	0.7	0.0	0.3	0.7	0.7	0.7	107
			1000	1.3	0.0	0.7	2.7	1.3	2.0*	109
			2000	0.7	0.0	0.3	1.3	1.3	1.3	106
			7.5 (Cyclophosphamide)	46.9	41.7	44.1***	50.0	41.7	45.6***	100
	21	-	0 (DMSO)	2.0	0.7	1.3	3.3	1.3	2.3	100
			500	0.7	1.3	1.0	0.7	2.0	1.3	113
			1000	0.7	2.0	1.3	0.7	2.0	1.3	102
			2000	1.3	2.7	2.0	3.3	4.0	3.7	107
			0.1 (Mitomycin C)	32.6	27.8	30.0***	32.6	27.8	30.0***	105

One-tailed Fisher's exact test

***                    p<0.001

Otherwise         p>0.05

Applicant's summary and conclusion

Conclusions:

It is concluded that the test item OK (640-2) (Furoxy Hydroxy) has shown no evidence of causing an increase in the frequency of structural chromosome aberrations in this in vitro cytogenetic test system, under the experimental conditions described.