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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
Not reported
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and guideline compliant proprietary study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Sodium terephthalate: crystalline powder of 99% w/w purity
Batch no. E24L42 Ex Avocado Research Chemicals.

Method

Target gene:
Not relevant
Species / strain
Species / strain / cell type:
lymphocytes: human, peripheral
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
rat liver derived metabolic system (S9-mix)
Test concentrations with justification for top dose:
1000 µg/mL to 2100 µg/mL.
Vehicle / solvent:
Double deionised water.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Mitomycin C, cyclophosphamide
Details on test system and experimental conditions:
Duplicate human peripheral blood cultures were exposed to the solvent, test substance or positive control substances at appropriate concentrations in the following experiments:

1. A cytogenetic experiment conducted using a sample of pooled blood. Cells were exposed to the test substance and control substances for 3 hours, both in the presence and absence of S9 mix. Solvent, untreated and positive control cultures were included.
2. A second independent cytogenetic experiment was conducted using a sample of pooled blood. Cells were exposed to the test substance and control substances for a period of 3 hours in the presence of S9 mix and 20 hours in the absence of S9 mix. Solvent, untreated and positive control cultures were included.

Human blood cells were obtained from 4 female healthy, non smoking donors (2 samples of blood for experiment 1 and 2 samples of blood for experiment 2). At 0 hours, cultures (10 mL) were established by the addition of 0.5 mL of whole blood to RPMI-1640 (Dutch modifcation) tissue culture medium supplemented with approximately 10% foetal bovine serum (FBS), 1.0 IU/mL heparin, L-glutamine (2mM), 100IU/mL penicillin and 100 µg/ml
streptomycin. The lymphocytes were stimulated to enter cell division by addition of phytohaemagglutinin (PHA; at 5% v/v) and the cultures were maintained at approximately 37°C for 48 hours with gentle daily mixing where possible.

Prior to treatment, the cultures to be treated for a 20 hour period were centrifuged and the culture medium was replaced with fresh supplemented RPMI-1640 culture medium. The cultures to be treated for a 3 hour period did not receive a medium change. Treatment started approximately 48 hours after culture initiation. Aliquots of the test substance, solvent control or positive controls were administered to duplicate cultures as appropriate to the experiment design. In addition, 200μl of a 1:1 mix of S9 and co-factor solution was added to each culture to be treated in the presence of S9-mix. Cultures from both experiments in the presence of S9-mix and Experiment 1 in the absence of S9-mix were treated for a period of 3 hours at 37°C, after which the culture medium was removed following centrifugation and replaced with fresh supplemented RPMI-1640 culture medium. The cultures were re-incubated at 37°C for the remainder of the 68 hour growth period. Cultures from Experiment 2 in the absence of S9-mix were treated for a period of 20 hours until the end of the 68 hour growth period.

A single sampling time, 20 hours after the start of treatment was used. The sampling time of 20 hours after the start of treatment was absed on a measured mean cell cycle time for cultured human peripheral blood lymphocytesof 13.5 hours in the laboratory.



Evaluation criteria:
For the study to be valid the following criteria must be met:
1.The vehicle control mean should be within, or close to, the historical control range.
2. The positive control group should show an appropriate increase, compared to vehicle control values, in percentage of aberrant cells.
Statistics:
The Fisher Exact Probability Test (one-sided) is used to evaluate statistically the percentage of metaphases showing aberrations (excluding cells with only gap-type aberrations).

Results and discussion

Test results
Species / strain:
lymphocytes: human, primary culture
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
Additional information on results:
No significant reductions in the mean mitotic activity compared to the solvent control values were observed in cultures treated with sodium terephthalate.

Treatment of the culture medium with sodium terephthalate up to 10 mM had no significant effect on pH. Some increases in the osmolality of the culture medium treated with sodium terephthalate were observed in experiment 2. As these values were within the normal range of osmolality values used for the in vitro cytogenetic assay, these were considered acceptable for use. No significant increase in osmolality was observed in Experiment 1.

Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Two independent cytogeneticity experiments were conducted initially using a range of sodium terephthalate concentrations and are detailed below. The highest concentration selected for chromosome aberration analysis was 10mM (2100μg/ml) which is the limit concentration for the assay. No significant reductions in mean mitotic activity, compared to the solvent control values, were observed in cultures treated with sodium terephthalate.

Experiment 1

Experiment 2

Treatment

Mitotic Index %

Mean % Mitotic Index

Treatment

Mitotic Index, %

Mean % Mitotic Index

Solvent Control (10 µl/mL)

4.5

3.7

4.1

Solvent Control (10 µl/mL)

8.8

9.0

8.9

Sodium terephthalate (µg/mL)

 

Sodium terephthalate (µg/mL)

 

2100

3.8

3.9

3.9

2100

10.3

9.1

9.7

1800

4.2

3.7

4.0

1800

9.8

10.1

10.0

1500

4.8

5.2

5.0

1500

9.8

9.9

9.9

1000

4.8

5.5

5.2

1000

9.7

9.6

9.7

 750

a

a

   750

a

a

 
 500

a

a

 

 500

a

 
 250

a

   250

a

a

 
 125

a

a

   125

a

a

 
 63

a

a

   63

a

a

 
 31

a

a

   31

a

a

 

a       Mitotic index not required for selection of concentrations for chromosome aberration analysis

Table 1 Mitosis Indices in the Absence of Metabolic Activation

Experiment 1

Experiment 2

Treatment

Mitotic index,%

Mean mitotic index, %

 Treatment

Mitotic index, % 

Mean mitotic index, % 

Solvent Control (10 µl/mL)

3.7

4.8

4.3

Solvent Control (10 µl/mL)

9.8

8.8

9.3

Sodium terephthalate (µg/mL)

 

Sodium terephthalate (µg/mL)

 

2100

8.5

7.2

7.9

2100

10.3

4.7

7.5

1800

9.2

8.0

8.6

1800

8.8

9.5

9.2

1500

5.1

5.6

5.4

1500

10.4

10.6

10.5

1000

7.4

7.1

7.3

1000

9.1

8.4

8.8

750

a

a

  750 

a

a

 
500

a

a

  500

a

a

 
250

a

a

  250

a

 
125

a

a

  125

a

a

 
63

a

a

  63

a

a

 
31

a

  31

a

a

 

 a       Mitotic index not required for selection of concentrations for chromosome aberration analysis

Mitotic Indices in the Presence of Metabolic Activation (S9-mix)

From the previous results, the following concentrations (1000, 1500 and 2100 µg/mL) were selected for chromosomal aberration analysis. In experiment 1, the cultures were tested with sodium terephthalate in the presence and absence of the S9 mix for 3 hours (table 4) and in experiment 2, the cultures were tested with sodium terephthalate in the presence of S9 mix for 3 hours and in the absence of S9 mix for 20 hours (table 3).

Small but statistically significant increases in the percentage of aberrant cells, compared to the solvent control values, were observed in cultures treated for 3 hours with sodium terephthalate in the presence and absence of S9-mix. No statistically or biologically significant increases in the percentage of aberrant cells were recorded in cultures treated for 20 hours with sodium terephthalate in the absence of S9-mix. The positive control materials, mitomycin C and cyclophosphamide induced statistically and biologically significant increases in the percentage of aberrant cells, compared to the solvent control cultures.

Treatment

Mean % Aberrant Cells excluding Gaps

Mean % Mitotic Index

Experiment 1- 3 hour treatment

 

Solvent Control 10 µL/mL

0.00

4.1

Mitomycin C 0.5 µg/mL

55.00**

1.1 Δ

Sodium terephthalate

2100 µg/mL

1500 µg/mL

1000 µg/mL

 

3.00*

4.00**

2.50*

 

3.9

5.0

5.2

Experiment 2- 20 hour treatment

 

Solvent Control 10 µL/mL

1.00

8.9

Mitomycin C 0.5 µg/mL

28.89**

6.0Δ

Sodium terephthalate

2100 µg/mL

1500 µg/mL

1000 µg/mL

 

1.00

2.00

0.50

 

9.7

9.9

9.7

*Statistically significant increase of aberrant cells at p<0.05 using Fisher’s Exact Test (one-sided)

**Statistically significant increase in the percentage of aberrant cells at p<0.01 using Fisher’s Exact Test (one sided)

Δ Positive control mitotic index and % aberrant cells are determined from a single culture.

Table 3 Mean Chromosomal Aberrations and Mitotic Indices in the Absence of Metabolic Activation (S9 -mix)  

Treatment

Mean % Aberrant Cells excluding Gaps

Mean % Mitotic Index

Experiment 1- 3 hour treatment

 

Solvent Control 10 µL/mL

0.50

4.3

Mitomycin C 0.5 µg/mL

40.00**

3.8 Δ

Sodium terephthalate

2100 µg/mL

1500 µg/mL

1000 µg/mL

 

0.50

4.00*

1.00

 

7.9

5.4

7.3

Experiment 2- 3 hour treatment

 

Solvent Control 10 µL/mL

0.00

9.3

Mitomycin C 0.5 µg/mL

40.00**

4.9Δ

Sodium terephthalate

2100 µg/mL

1500 µg/mL

1000 µg/mL

 

3.00*

3.50**

0.50

 

7.5

10.5

8.8

*Statistically significant increase of aberrant cells at p<0.05 using Fisher’s Exact Test (one-sided)

**Statistically significant increase in the percentage of aberrant cells at p<0.01 using Fisher’s Exact Test (one sided)

Δ Positive control mitotic index and % aberrant cells are determined from a single culture.

Table 4 Mean Chromosomal Aberrations and Mitotic Indices in the Presence of Metabolic Activation (S9-mix)

 

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

It is concluded that, under the conditions of this assay, sodium terephthalate is not clastogenic to cultured human lymphocytes treated in vitro in either the presence or absence of S9-mix.
Executive summary:

The test substance, sodium terephthalate, was assessed in an in vitro cytogenicity assay in cultured human peripheral blood lymphocytes to assess clastogenic activity. Sodium terephthalate was tested over a range of concentrations, both in the presence and absence of S9-mix, in two independent tests. Small but statistically significant increases in the percentage of aberrant cells, compared to the solvent control values, were observed in cultures treated for 3 hours with sodium terephthalate in the presence and absence of S9-mix. These increases were not concentration related, were within the historical control range and were therefore considered to be of no biological significance. In the second experiment, the cultures were also exposed for 20 hour in the absence of S9 -mix. No statistically or biologically significant increases in the percentage of aberrant cells were recorded in cultures treated for 20 hours with sodium terephthalate in the absence of S9-mix. It is concluded that, under the conditions of this assay, sodium terephthalate is not clastogenic to cultured human lymphocytes treated in vitro in either the presence or absence of S9-mix.