Zinc, [1,2,3-propanetriolato(2-)-.kappa.O1,.kappa.O2]-, homopolymer, stereoisomer

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:

1994

Report date:

1994

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

(1983)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

zinc(2+) 3-hydroxypropane-1,2-bis(olate)

EC Number:

700-107-9

Cas Number:

87189-25-1

Molecular formula:

(C3H6O3Zn)n

IUPAC Name:

zinc(2+) 3-hydroxypropane-1,2-bis(olate)

Details on test material:

White Powder

Method

Target gene:

not applicable

Species / strain

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

- Source: human blood was collected aseptically from healthy male donors.
- Type and identity of media: RPMI 1640 tissue culture medium (Sigma Chemical Company Ltd.)
- Preparation: Lymphocytes were separated by centrifugation
- Media after preparation: RPMI 1640 + 20% foetal calf serum (Imperial Laboratories) + phytohaemagglutinin (Wellcome) (approx. 175 µg/ml) to stimulate cell division
- Cell density: 1 x E+6 cells per ml.
- Incubation time: approx. 48 hours (37°C, 5% CO2)

Metabolic activation:

with and without

Metabolic activation system:

S9-mix prepared from the livers of male 7-8 weeks old Sprague-Dawley derived rats, stimulated with Aroclor 1245 administered as a single intraperitoneal injection in Arachis oil at a dosage of 500 mg/kg bw.

Test concentrations with justification for top dose:

Test article (first experiment): 1.0, 2.0, 3.9, 7.8, 15.6, 31.1, 62.5, 125, 250 and 500 µg/ml
Test article (second experiment): 2.5, 5, 7.5, 10, 15, 20, 30, 40, 50, 62.5 and 100 µg/ml (without S9); 5, 7.5, 10, 15, 20, 30, 40, 50, 62.5 and 100 µg/ml (with S9)
Solvent control: 0.1 M HCl
Positive control: 500 and 750 µg/ml Ethyl methanesulphonate (without S9); 10 and 15 µg/ml Cyclophosphamide (with S9)

Vehicle / solvent:

- Solvent used: 0.1 M hydrochloric acid (HCl)
- Justification for choice of solvent: The test substance formed a dosable suspension in 0.1 M HCl on sonication at 50 mg/ml. This formed a fine dispersion in the culture medium when dosed at 1% v/v to give a final concentration of 500 µg/ml. This was used as the highest concentration for subsequent testing.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

(0.1 M HCl)

Positive controls:

yes

Positive control substance:

other: Ethyl methanoesulphonate (without S9) and Cyclophosphamide (with S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period (+ phytohaemagglutinin): 48 h
- Expression time (cells in growth medium): 18 h (without S9-mix), 15 h (with S9-mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 18 h
- Treatment time: 18 h, no recovery period (without S9-mix); 3 h and 15 h recovery (with S9-mix)

SPINDLE INHIBITOR (cytogenetic assays): Colchicin (0.25 µg/ml), added 2 hours before harvest
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2 experiments à 2 plates per dose

NUMBER OF CELLS EVALUATED: 100 per dose per experiment

CONCENTRATIONS OF THE TEST SUBSTANCE SELECTED FOR ANALYSIS:
- First test: 15.6, 7.8 and 3.9 µg/ml (without S9); 31.1, 15.6 and 7.8 µg/ml (with S9); posive cotrols: ehtyl methanesulphonate and cyclophosphamide: 500 and 15 µg/ml, respectively
- Second test: 20, 10 and 5 µg/ml (without S9); 40, 30, 20 and 10 µg/ml (with S9); posive cotrols: ehtyl methanesulphonate and cyclophosphamide: 500 and 10 µg/ml, respectively

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

SCORING OF SLIDE PREPARATION:
Metaphase cells were examined under a microscope for structural and numerical abnormalities. Structural abnormalities of chromosome were classified as gap (included chromosome type and chromatid type), chromatid break (ctb), chromosome break (csb), chromatid exchange (cte), chromosome exchange (cse), and other (fragmentation and so on). Observations of slides were carried out by the plural number of observers. The cell having at least one aberration was recorded as an aberrant cell.

Evaluation criteria:

Numbers of cells with chromosome aberration and polyploidy cells in the groups treated by the test substance are compared with those of the control groups for each dose. In principle, the test substance is judged to be clastogenic or aneugenic, if significant increase in aberration frequency, dose response and reproducibility of the result could be confirmed. For evaluation of the number of aberrant cells gaps were not included.

Statistics:

no data

Results and discussion

Test results

Test results 1

Species / strain:

lymphocytes: human

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(>15.6 µg/ml)

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

lymphocytes: human

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

(30, 31.3 and 40 µg/ml)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(> 10 µg/ml)

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of osmolality: Following the exposure of the cells to the test substance, a sample of the supenatant medium from cultures treated with the solvent control and the two highest concentrations of test substance was removed and stored at -20°C. This was retained for osmolality measurement in the case of a positive response which is only detected at high dose levels and may be attributed to a hyperosmotic effect

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Toxicity data:

First experiment: In the absence of S9 -mix, there was a decrease in mitotic index to 47% of the solvent control value at 15.6 µg/ml. All higher concentrations were too toxic for analysis. In the presence of S9 -mix, the test substance caused a dose-related reduction in mitotic index leading to complete cell death at 125 µg/ml. The highest concentration suitable for analysis, 31.3 µg/ml, caused a decline in mitotic index to 45% of the control level.

Second experiment: In the absence of S9 -mix, there was a decrease in mitotic index to 53% of the solvent control value at 20 µg/ml. All higher concentrations were too toxic for analysis. In the presence of S9 -mix, the test substance caused a decrease in mitotic index to 51% of control levels at 40 µg/ml. The additional concentration of 30 µg/ml was included to assess the reproducibility of the response seen in the first test.

Metaphase data:

First experiment: In the absence of S9 -mix, the test substance caused no statistically significant increases in the proportion of metaphase figures with chromosome damage at any concentration. In the presence of S9 -mix, a statistically significant increase in the number of aberrant cells was observed at 31.3 µg/ml. This increase, to 10.5%, lies outside the historical control range.

Second experiment: In the absence of S9 -mix, the test substance caused no statistically significant increases in the number of aberrant cells at any concentration. In the presence of S9 -mix, a statistically significant increase in the number of aberrant cells was observed at 30 and 40 µg/ml. The increases, to 5.5 and 16%,respectively, lie outside the historical control range (0 -5.25%).

The test substance has shown evidence of clastogenic activity in human lymphocytes in the presence of S9 -mix, but not in its absence, in this in vitro cytogenetic test system.

First experiment, without metabolic activation, 18 hour harvest:

Concentration test agent (µg/ml)	Relative mitotic index (%)	No. of aberrant cells (exc. gaps) (%)
Solvent control	100	4.0
1.0	98	-
2.0	105	-
3.9	88	4.5
7.8	58	3.0
15.6	47	4.0
31.3	28	-
62.5	12	-
125	-	-
250	-	-
500	-	-
Etylmethanesulphonate	-	15.0

First experiment, with metabolic activation, 18 hour harvest:

Concentration test agent (µg/ml)	Relative mitotic index (%)	No. of aberrant cells (exc. gaps) (%)
Solvent control	100	2.25
1.0	96	-
2.0	88	-
3.9	96	-
7.8	63	3.0
15.6	73	3.0
31.3	45	10.5
62.5	6	-
125	-	-
250	-	-
500		-
Cyclophosphamide	-	21.5

Second experiment, without metabolic activation, 18 hour harvest:

Concentration test agent (µg/ml)	Relative mitotic index (%)	No. of aberrant cells (exc. gaps) (%)
Solvent control	100	1.5
2.5	89	-
5.0	83	1.5
7.5	69	-
10.0	61	1.5
15.0	56	-
20.0	53	2.5
30.0	8	-
40.0	11	-
50.0	11	-
62.5	28	-
100	-	-
Ethylmethanesulfonat	-	21.0

Second experiment, with metabolic activation, 18 hour harvest:

Concentration test agent (µg/ml)	Relative mitotic index (%)	No. of aberrant cells (exc. gaps) (%)
Solvent control	100	1.25
2.5	-	-
5.0	77	-
7.5	77	-
10.0	74	2.5
15.0	54	-
20.0	51	3.5
30.0	80	7.0
40.0	51	16.5
50.0	29	-
62.5	26	-
100	3	-
Cyclophosphamide	-	24.0

Applicant's summary and conclusion

Conclusions:

The test item has shown evidence of clastogenic activity in the presence of S-9 mix, but not in its absence, in this in vitro cytogenetic test system.