Bis(5-oxo-L-prolinato-N1,O2)zinc

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

Study period:

29 August 2012 to 16 December 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

other: read-across target

Reference

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted on read-across substance.

Justification for type of information:

See the attached justification.

Reason / purpose for cross-reference:

read-across source

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Deviations:

no

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:

sodium (2S)-5-oxopyrrolidine-2-carboxylate

Cas Number:

28874-51-3

Molecular formula:

C5H6NO3. Na

IUPAC Name:

sodium (2S)-5-oxopyrrolidine-2-carboxylate

Test material form:

solid: particulate/powder

Details on test material:

- Physical state: White to pale yellow powder.
- Storage condition of test material: Controlled room temperature (15-25 °C, below 70 RH%), protected from humidity.

Method

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: Dulbecco’s Modified Eagle’s Medium.
- Properly maintained: Yes, stocks were kept in a freezer at -80 ± 10 °C. The laboratory cultures were maintained in 150 cm² plastic flasks at 37 ± 0.5 °C in a humidified atmosphere containing approximately 5 % CO2 in air.
- Periodically checked for Mycoplasma contamination: Yes, no infection was noted.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

Assay 1 and 2 test concentrations: 5000, 2500, 1250, 625 and 312.5 μg/mL (with and without metabolic activation).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: distilled water.
- Justification for choice of solvent/vehicle: Based on the results of a short preliminary solubility test, the test material was soluble in distilled water at 500 mg/mL. Therefore, distilled water was selected as solvent for the study.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Remarks:

Positive control solutions were prepared immediately before the treatment of the cells and filtered sterile using a 0.22 μm syringe filter before use.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium.
Cells were treated with test material solutions, negative (solvent) or positive control solution (treatment volume: 100 μL/dish in all cases) for the given period of time at 37°C in the absence or presence of S9-mix. After the exposure period, the cultures were washed with Dulbecco’s Modified Eagle’s Medium (supplemented with 2 mM L-glutamine and 1 v/v% Antibiotic-antimycotic solution). Then, 10 mL of fresh culture medium were added into the dishes and cells were incubated further until the scheduled harvesting time.

DURATION
- Exposure duration:
> Assay 1: 3 hours with and without S9-mix.
> Assay 2: 20 hours without S9-mix and 3 hours with S9-mix.
- Expression time:
> Assay 1: Cells were harvested 20 hours after the beginning of treatment (approximately 1.5 normal cell cycles).
> Assay 2: Cells were harvested 28 hours after the beginning of treatment (approximately 2 normal cell cycles).

SPINDLE INHIBITOR: Colchicine (0.2 μg/mL) was added to cultures 2 to 2.5 hours prior to harvesting.
The cells were swollen with 0.075 M KCl hypotonic solution, then were washed in fixative (Methanol:Acetic acid 3:1 (v:v) mixture) until the preparation became plasma free (4 washes). Then, a suspension of the fixed cells was dropped onto clean microscope slides and air-dried.
STAIN: The slides were stained with 5 % Giemsa solution, air-dried and coverslips were mounted.

NUMBER OF REPLICATIONS: Two replications were performed.

NUMBER OF CELLS EVALUATED: At least one hundred metaphases with 22 ± 2 chromosomes (dicentric chromosomes were counted as two chromosomes) from each culture were examined for the presence or absence of chromosomal aberrations (approximately 1000x magnification), where possible. Examination of slides from a culture was halted when 15 or more metaphases with aberrations (excluding gaps) had been recorded for that culture. Chromatid and chromosome type aberrations (gaps, deletions and exchanges) were recorded separately.

DETERMINATION OF CYTOTOXICITY
- Method: For concurrent measurement of cytotoxicity an extra dish was plated for each sample and treated in the same manner. At the scheduled harvesting time, the number of surviving cells was determined using a haemocytometer. Results are expressed compared to the negative (solvent) control as % relative survival.

OTHER EXAMINATIONS:
The occurrence of polyploid and endoreduplicated metaphases was recorded in the main tests.

Evaluation criteria:

The assay is considered valid, if the following criteria are met:
- The solvent control data are within the laboratory’s normal range for the spontaneous aberration frequency.
- The positive controls induce increases in the aberration frequency, which are significant.

A test material is considered to have shown clastogenic activity in this study if all of the following criteria are met:
- Increases in the frequency of metaphases with aberrant chromosomes are observed at one or more test concentrations (only data without gaps will be considered).
- The increases are reproducible between replicate cultures and between tests (when treatment conditions were the same).
- The increases are statistically significant.
- The increases are not associated with large changes in pH or osmolarity of the treated cultures.
The historical control data for this laboratory were also considered in the evaluation. Evidence of a dose-response relationship (if any) was considered to support the conclusion.

A test material is concluded to have given a negative response if no reproducible, statistically significant increases are observed.

Statistics:

For statistical analysis, Fisher’s exact test was used. The parameter evaluated for statistical analysis was the number of cells with one or more chromosomal aberrations excluding gaps.

Results and discussion

Test results

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No large changes in the pH were observed.
- Effects of osmolality: A slightly higher than usual difference was observed in osmolality was observed at the highest concentration (5000 μg/mL) in both experiments of Assays 1 and 2.
- Water solubility: No insolubility was detected at the end of the treatment period in the final treatment medium for either Assay 1 or 2.

RANGE-FINDING/SCREENING STUDIES: A total of eight test concentrations between 5000 and 2.29 μg/mL were used to evaluate toxicity in the presence and absence of metabolic activation in each cytotoxicity assay.
Cells survival relative to the negative solvent control ranged from (exposure time/harvest time):
> Assay A: 88 - 114% (3/20 without S9-mix) and 84 – 123 % (3/20 with S9-mix).
> Assay B: 77 - 127% (20/28 without S9-mix) and 88 – 116 % (3/28 with S9-mix).
All observations were normal throughout the study, pH measured 7.4 in all samples and osmolarity ranged from 321 to 402 mmol/kg.

COMPARISON WITH HISTORICAL CONTROL DATA: The negative (solvent) control data were within the laboratory’s normal range for the spontaneous aberration frequency, the positive control substances caused a statistically significant increase in the number of structural aberrations excluding gaps in the experiments with or without metabolic activation demonstrating the sensitivity of the test system.

ADDITIONAL INFORMATION ON CYTOTOXICITY: No cytotoxicity was observed in both experiment in Assays 1 and 2.

CHROMOSOME ADERRATION EVALUATION
Concentrations of 5000, 2500 and 1250 μg/mL (a total of three) were chosen for evaluation in both cases in Assays 1 and 2. Based on the observed results, an additional concentration (625 μg/mL) was evaluated in Assay 1 in the experiment with metabolic activation.
None of the treatment concentrations caused a significant increase in the number of cells with structural chromosome aberrations in either assay without metabolic activation or in Assay 2 with metabolic activation. Following the initial analysis of three concentrations, a significant increase in chromosome aberrations at 1250 μg/mL in Assay 1 with metabolic activation was observed. No increase was found at two higher concentrations (2500 and 5000 μg/mL). A further concentration, 625 μg/mL, was then analysed and no increase in aberrations was observed. Therefore the increase was not repeatable either at other concentrations within the assay or between assays.

POLYPLOID AND ENDOREDUPLICATED METAPHASES
The occurrence of polyploid and endoreduplicated metaphases was recorded in the main tests. Polyploid metaphases (1-3) were found in some cases in the negative (solvent) control or test material treated samples in the performed experiments. No endoreduplicated metaphases were found in the main tests.

Remarks on result:

other: all strains/cell types tested

Any other information on results incl. tables

Table 1: Summary Table of Chromosome Aberration Assay 1

S9-mix	Concentration (µg/mL)	Exposure/Harvest Time	Relative Survival (%) #	Mean % Aberrant Cells ##
-	Negative (solvent) Control	3h/20h	100	0.5
	5000	3h/20h	95	0.0
	2500	3h/20h	115	0.5
	1250	3h/20h	109	2.0
	625	3h/20h	83	NE
	312.5	3h/20h	101	NE
	Positive Control	3h/20h	80	10.5***
+	Negative (solvent) Control	3h/20h	100	1.0
	5000	3h/20h	90	0.0
	2500	3h/20h	96	3.5
	1250	3h/20h	92	6.5**
	625	3h/20h	102	3.0
	312.5	3h/20h	100	NE
	Positive Control	3h/20h	59	100.0***

Negative (solvent) control: 1% (v/v) Distilled water

Positive control (-S9): Ethyl methanesulfonate, 1 μL/mL

Positive control (+S9): Cyclophosphamide, 6 μg/mL

NE: not evaluated

# compared to the negative (solvent) control

## excluding gaps

** p < 0.01 comparing numbers of aberrant cells excluding gaps with corresponding negative control.

*** p < 0.001 comparing numbers of aberrant cells excluding gaps with corresponding negative control.

Table 2: Summary Table of Chromosome Aberration Assay 2

S9-mix	Concentration (µg/mL)	Exposure/Harvest Time	Relative Survival (%) #	Mean % Aberrant Cells ##
-	Negative (solvent) Control	20h/28h	100	2.0
	5000	20h/28h	81	2.0
	2500	20h/28h	87	0.5
	1250	20h/28h	85	0.5
	625	20h/28h	95	NE
	312.5	20h/28h	83	NE
	Positive Control	20h/28h	62	30.4***
+	Negative (solvent) Control	3h/28h	100	1.5
	5000	3h/28h	85	2.5
	2500	3h/28h	85	1.5
	1250	3h/28h	83	2.0
	625	3h/28h	93	NE
	312.5	3h/28h	89	NE
	Positive Control	3h/28h	35	100.0***

Negative (solvent) control: 1% (v/v) Distilled water

Positive control (-S9): Ethyl methanesulfonate, 0.4 μL/mL

Positive control (+S9): Cyclophosphamide, 6 μg/mL

NE: not evaluated

# compared to the negative (solvent) control

## excluding gaps

*** p < 0.001 comparing numbers of aberrant cells excluding gaps with corresponding negative control.

 

Table 3: Summary Table of Polyploidy and Endoreduplicated Cells Assay 1

S9-mix	Concentration (µg/mL)	Exposure/Harvest Time	No. of Cells Observed
			Polyploid	Endoreduplicated
-	Negative (solvent) Control	3h/20h	0	0
	1250	3h/20h	2	0
	2500	3h/20h	0	0
	5000	3h/20h	3	0
	Positive Control	3h/20h	0	0
+	Negative (solvent) Control	3h/20h	0	0
	625	3h/20h	0	0
	1250	3h/20h	1	0
	2500	3h/20h	2	0
	5000	3h/20h	1	0
	Positive Control	3h/20h	0	0

 

Table 4: Summary Table of Polyploidy and Endoreduplicated Cells Assay 2

S9-mix	Concentration (µg/mL)	Exposure/Harvest Time	No. of Cells Observed
			Polyploid	Endoreduplicated
-	Negative (solvent) Control	20h/28h	1	0
	1250	20h/28h	0	0
	2500	20h/28h	2	0
	5000	20h/28h	0	0
	Positive Control	20h/28h	0	0
+	Negative (solvent) Control	3h/28h	0	0
	1250	3h/28h	0	0
	2500	3h/28h	0	0
	5000	3h/28h	2	0
	Positive Control	3h/28h	0	0

Applicant's summary and conclusion

Conclusions:

Under the conditions of the study, the test material did not induce reproducible, statistically significant increases in chromosome aberrations in either assay, with or without metabolic activation. Therefore, the test material is considered not clastogenic in this test system.

Executive summary:

The clastogenic potential of the test material was determined in an in vitro mammalian cell chromosome aberration assay performed under GLP conditions and in line with the standardised guideline OECD 473, EU Method B.10 and EPA OPPTS 870.5375.

Cultures of Chinese hamster lung fibroblasts (V79) cells were exposed to the test material at concentrations of 5000, 2500, 1250, 625 and 312.5 μg/mL, in two assays, with and without metabolic activation. Assay 1 was performed with two experiments both with a three hour exposure period and 20 hours harvesting time. Both experiments were performed in the presence or absence of a metabolic activation system, which was a cofactor-supplemented post-mitochondrial S9 fraction prepared from the livers of phenobarbital/β-naphthoflavone induced rats. Assay 2 was performed either in the presence of S9-mix with a three hour exposure period, or in the absence of S9-mix with a 20 hour exposure period. Both experiments were conducted with a harvesting time of 28 hours.

Treatment with the test material did not result in a statistically and biologically significant, repeatable, dose-dependent increase in the frequency of the cells with structural chromosome aberrations either in the presence or absence of a metabolic activation system. Additionally there was no indication of polyploidy or endoreplication.

The test material is therefore considered not to be clastogenic in this test system.