Polyamide wax

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From 2000-01-19 to 2000-07-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Method

Target gene:

n/a

Species / strain

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

Cultures of human lymphocytes are primary cell cultures recommended by international regulations for the mammalian chromosome aberration test; they have a stable karyotype with 46 chromosomes and an average cell cycle time of 13-14 hours.
Cultures of human lymphocytes were prepared from whole blood pooled from healthy males donors.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Liver homogenate derived from Aroclor 1254 induced rats (S-9 mix)

Test concentrations with justification for top dose:

Mitotic index analysis:
Test 1 (3-hour treatment without activation): 0, 500, 1000, 1500, 2000, 2500, 2750, 3000, 3250, 3500, 3750 and 4000 µg/mL
Test 1 (3-hour treatment with activation): 0, 625, 1250, 2500, 3500, 4000, 4500 and 5000 µg/mL
Test 2 (20-hr without activation ): 0, 500, 1000, 1250, 1500, 1750, 2000, 2500, 3000, 3500, 4000, 4500 and 5000 µg/mL
Test 2 (3-hour treatment with activation):0, 625, 1250, 2500, 3500, 4000, 4500 and 5000 µg/mL

Metaphase analysis
Test 1 (3-hour treatment without activation): 0, 1000, 2000, 3250 and 3500 µg/mL,
Test 1 (3-hour treatment with activation): 0, 1250, 2500 and 5000 µg/mL,
Test 2 (20-hour without activation ): 0, 1500, 2000 and 2500 µg/mL,
Test 2 (3-hour treatment with activation):0, 1250, 2500 and 4500 µg/mL,

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Culture medium
- Justification for choice of solvent/vehicle: Information supplied by the sponsor stated that the test item was insoluble in water. it was found to form a doseable suspension in culture medium at 10 mg/ml.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
In the first experiment, lymphocyte cultures were exposed to the test or control items (with or without S9 mix) for 3 hours then rinsed. Cells were harvested 20 hours after the beginning of treatment, corresponding to approximately 1.5 normal cell cycles.

The second experiment was performed as follows:
- without S9 mix, cells were exposed continuously to the test or control items until harvest,
- with S9 mix, cells were exposed to the test or control items for 3 hours and then rinsed. Cells were harvested 20 hours after the beginning of treatment.

SPINDLE INHIBITOR (cytogenetic assays): Colcemid at a final concentration of 0.1 µg/ml

STAIN (for cytogenetic assays): Giemsa 10%

NUMBER OF CELLS EVALUATED: 200 metaphases/dose-level

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes, noted when seen
- Determination of endoreplication: yes, noted when seen

Evaluation criteria:

The test substance is considered to cause a positive response if the following conditions are met:
Statistically significant increases (P<0.01) in the frequency of metaphases with aberrant chromosomes (excluding gaps) are observed at one or more test concentration.
The increases exceed the negative control range of the laboratory, taken at the 99% confidence limit.
The increases are reproducible between replicate cultures.
The increases are not associated with large changes in osmolality of the treatment medium or extreme toxicity.
Evidence of a dose-relationship is considered to support the conclusion.

A negative response is claimed if no statistically significant increases in the number of aberrant cells above concurrent control frequencies are
observed, at any dose level.

Statistics:

For each experiment and for each harvest time, the frequency of cells with structural chromosome aberration in treated cultures was compared to that of the vehicle control cultures using Fisher's test.

Results and discussion

Test results

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Test 1:
In the absence of S9 mix, the test item caused a reduction of 5% of the vehicle control value at 5000 µg/ml. Due to steep toxic response at 5000 µg/ml, this test was repeated in order to achieve a concentration causing a decrease in mitotic index closer to 50% of the vehicle control value. In the repeat test, the test item again caused a steep decrease, causing a reduction in the mitotic index to 16% of the vehicle control value at 3000 µg/ml. This test was repeated again and at 3500 µg/ml the test item caused a reduction of 25% to the vehicle control value. One culture at this concentration was considered to have less than 100 metaphases available for chromosome analysis and most of the metaphases were of poor quality. At 3250 µg/ml a reduction of the index mitotic to 91% of the vehicle ontrol value was recorded.
The mitotic indices data obtained in all the three tests showed changes in the toxicity profile which may have been due to the treatment of the test
substance as a suspension. It was decided that as the dose levels at 3250 and 3500 µg/ml were relatively close, slides prepared from these
cultures treated at 3250 µg/ml would be scored as the highest treatment level. The duplicate scoreable culture at 3500 µg/ml was also analysed to
give an indication of chromosome aberrations at this level.
The dose levels selected for the metaphase analysis were 1000, 2000, 3250 and 3500 µg/ml.

In the presence of S9 mix, the test item caused a reduction of the mitotic index to 30% of the vehicle control value at 5000 µg/ml. Due to steep toxic response this test was repeated in order to achieve a concentration causing a decrease in mitotic index closer to 50% of the vehicle control value. In the repeat test, the test item caused a reduction of the mitotic index to 34% of the vehicle control value at 5000 µg/ml. A second repeat test was performed and a reduction to 70% of the control vehicle value at 5000 µg/ml was recorded.
The dose levels selected for metaphase analysis were 1250, 2500 and 5000 µg/ml.

Test 2
In the absence of S9 mix, the test item caused a reduction of the mitotic index to 18% of the vehicle control value at 3000 µg/ml. Due to steep toxic response this test was repeated in order to achieve a concentration causing a decrease in mitotic index closer to 50% of the vehicle control value. The test item failed to cause a significant reduction in the mitotic index and slight bacterial contamination was observed in all cultures, therefore the test was repeated again. In the second repeat test the test item caused a reduction of the mitotic index to 45% of the vehicle control value at 2500 µg/ml.
The dose levels selected for the metaphase analysis were 1500, 2000 and 2500 µg/ml.

In the presence of S9 mix, the test item caused a reduction of the mitotic index to 58% of the vehicle control value at 4500 µg/ml.
The dose levels selected for the metaphase analysis were 1250, 2500 and 4500 µg/ml.

Any other information on results incl. tables

Table 7.6.1.1 Metaphase analysis in Test 1

Substance concentration (µg/mL)	Cells with aberrations excluding gaps			Cells with aberrations including gaps			Relative cell count
	Individual values per 100 cells observed (%)		Mean (%)	Individual values per 100 cells observed (%)		Mean (%)
Method without S9 mix / 3 h. time exposure
0 (culture medium)	0	0	0.0	0	0	0.0	100
1000	0	0	0.0	1	0	0.5	115
2000	0	1	0.5	0	1	0.5	109
3250	1	1	1.0	1	3	2.0	91
3500	2		2.0	2		2.0	25
0.1 (Mitomycin C)	10	12	11.0***	11	13	12.0***	-
Method with S9 mix / 3 h. time exposure
0 (Culture medium)	0	0	0.0	1	0	0.5	100
1250	0	1	0.5	1	2	1.5	110
2500	1	2	1.5	4	3	3.5	100
5000	0	0	0.0	1	3	2.0	70
6 (Cyclophosphamide))	15	16	15.5***	23	20	21.5***	-

***P<0.001; **P<0.01; otherwise P>0.01

Table7.6.1.2 Metaphase analysis in Test 2

Substance concentration (µg/mL)	Cells with aberrations excluding gaps			Cells with aberrations including gaps			Relative cell count
	Individual values per 100 cells observed (%)		Mean (%)	Individual values per 100 cells observed (%)		Mean (%)
Method without S9 mix / 3 h. time exposure
0 (Culture medium)	2	2	2.0	5	5	5.0	100
1500	2	2	2.0	3	2	2.5	79
2000	2	1	1.5	4	2	3.0	56
2500	1	2	1.5	2	4	3.0	45
0.1 (Mitomycin C)	19	21	20.0***	28	24	26.0***	-
Method with S9 mix / 20 h. time exposure
0 (Culture medium)	1	0	0.5	1	1	1.0	100
1250	2	0	1.0	3	1	2.0	100
2500	2	0	1.0	4	1	2.5	95
4500	2	1	1.5	2	2	2.0	58
6 (Cyclophosphamide))	13	11	12.0***	19	14	16.5***	-

***P<0.001; **P<0.01; otherwise P>0.01

Applicant's summary and conclusion

Conclusions:

The substance was not considered to be clastogenic to the human lymphocytes in the in vitro chromosomal aberration assay either in the presence or in the absence of a rat liver metabolizing system.

Executive summary:

An in vitro chromosomal aberration assay was conducted to evaluate the clastogenic potential of the test substance in human lymphocytes according to the OECD Guideline 473 and in compliance withGood Laboratory Practices.

Human lymphocytes, in whole blood culture, were exposed to the test substance both in the absence and presence of S9 mix derived from rat livers. Three hours before the end of the incubation period, cell division was arrested using Colcemid®. The cells were then harvested and slides prepared, so that metaphase cells could be examined for chromosomal damage. On the basis of the mitotic index data obtained from a toxicity test, the following concentrations were selected for two independent tests evaluating the metaphase analysis: 

-        Test 1: 3-h treatment without S9 mix at dose levels of 0, 1000, 2000, 3250 and 3500 µg/mL and 3-h treatment with S9 mix at dose levels of 0, 1250, 2500 and 5000 µg/ml

-        Test 2: 20 -hr treatment without S9 mix at dose levels of: 0, 1500, 2000 and 2500 µg/mL and 3-h treatment with S9 mix at 0, 1250, 2500 and 4500 µg/mL .

Concurrent solvent and positive controls (mitomycin-C (in the absence of S9 mix) and cyclophosphamide (in the presence of S9 mix)) were also included. In both tests, the substance caused no statistically significant increases in the proportion of metaphase figures containing chromosomal aberrations at any concentration in the absence and presence of S9 mix when compared with the vehicle control. Also, no statistically significant increases in the proportion of polyploid or endoreduplicated metaphase cells were observed during metaphase analysis when compared with the vehicle control. All positive control compounds caused statistically significant increases in the proportion of aberrant cells, demonstrating the sensitivity of the test system and the efficacy of the S9 mix.Under the study conditions, the test substance was therefore not considered to be clastogenic to human lymphocytes in the in vitro chromosomal aberration assay.