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Toxicological information

Genetic toxicity: in vitro

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Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1994-05-23 to 1994-07-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Please refer to Section 13.2 for full read-across justification

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1994
Report Date:
1994

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): Purified Isophthalic Acid, received from Amoco Corporation- Physical state: White Powder- Storage condition of test material: Stored at ambient temperature and protected from lightThe purity was not stated. However, the batch of terephthalic acid used in this study is considered to have been representative of commercial production and to have been at least 99% pure.

Method

Target gene:
Thymidine kinase locus (TK +/-)
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
L5178Y mouse lymphoma cells were obtained from Dr. D Clive, Burroughs Wellcome, Research Triangle Park, North Carolina, USA in 1982. They were then stored in liquid nitrogen (-196°C).
Metabolic activation:
with and without
Metabolic activation system:
Mixed-function oxidase activity (S9 mix)
Test concentrations with justification for top dose:
Toxicity test: The concentrations of P.I.A. Purified Isophthalic Acid tested were as follows: 7.5, 25, 75, 250, 750 and 2500 µg/mlMutation Assays: The concentrations of P.I.A. Purified Isophthalic Acid tested were as follows: Assay 1 (in the absence of S9 mix): 150, 300, 450, 600, 750 and 900 µg/mlAssay 2 (in the presence of S9 mix): 150, 300, 450, 600, 750 and 900 µg/ml Assay 3 (in the absence of S9 mix): 600, 650, 700, 750, 800, 850 and 900 µg/ml Assay 4 (in the presence of S9 mix): 650, 700, 750, 800, 850, 900 and 950 µg/ml
Vehicle / solvent:
Dimethylsulphoxide (DMSO).
Controls
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Remarks:
Two positive controls were used
Positive control substance:
ethylmethanesulphonate
Remarks:
The positive controls were dissolved and diluted in DMSOMigrated to IUCLID6: 3-methylcholanthrene
Details on test system and experimental conditions:
A toxicity test was performed in the absence and presence of S9 mix to establish the range of concentrations of test material for the mutation assays.Mutation Assay: Two independent mutation assays were performed in the absence of S9 mix, and 2 in the presence of S9 mix. Each experiment consisted of the following groups: Vehicle control - 4 cultures, Postive control - 2 cultures, P.I.A Purified Isophthalic Acid - 6 or 7 concentrations, 2 cultures per concentration. The positive control cultures received 250 µg EMS.ml-1 in the absence of S9 mix and 2.5 µg MC.ml-1 in its presence. 3-MC was also tested in the absence of S9 mix, as proof of S9 dependency. Volumes of cell cutlure (5 ml) in F10P, each containing 6 x 10e6 exponentially growing tk+tk- cells, were dispensed to sterile plastic universal bottles. Freshly prepared S9 mix or F0P (1ml) was added, followed by test material or positive control solution ( in 0.1 ml DMSO). Vehicle control cultures received 0.1 ml DMSO. After incubation on a roller-drum for 4 hour at 37°C, the cells were harvested and washed by 2 centrifugations for 5 min at 200 g. The cultures were resuspended in F10P (20ml) to give concentraion of 3 x 10e5 cells.ml-1 and were then returned to the roller drum. pH was measured in one of each pair of P.I.A.-treated cultures (plus one vehicle control culture). Cells were incubated for 2 days after exposure. Cells counts were recorded after one day and cultures were then diluted to 3 x 10e5 cells.ml-1.On day 2, cell desities were determined, then adjusted to 2 x 10e5 cells.ml-1 and samples for the viability assay were removed. Three samples of cell suspension (5ml) were then dispensed into universal bottles for mutant colony selection. The volume in each universal bottle was adjusted to give 25 ml cloning medium supplemented with trifluorothymidine (TFT) (3 µg.ml-1). Each sample was then left dispensed into a petri dish (90 mm) and left to gel at room temperature. The plates were incubated at 37°C in 5 % Co2:95% air v/v, until colonies were fully developed (usually 14 days). The numbers of colonies were then counted using an Artek 880 automated colony counter. Viability Assay: On day 2 , after the cell numbers had been adjusted, a 0.1 ml sample was taken and 9.9 ml F10P was added. Cloning medium (25 ml) was added to each of 3 x 0.1 ml samples of this dilution. The suspensions were poured into 3 x 90 mm Petri dishes and left to gel at room temperature. The plates were then processed as for the mutation assay. Where a positive response was combined with a meaningful increase in absolute mutant numbers, colony size distribution was performed. Cell survival from the day of treatment (Day 0) to the day of expression of genetic damage (Day 2) was assessed by calculating the relative total growth (RTG) of treated groups. For the viability assay, 0.1 ml of 2 x 10e5 cells.ml-1 + 9.9 ml F10P was used. Three 0.1 ml samples of this dilution were plated out, giving 200 cells per dish. To estimate the number of TFT resistant mutants, 3 samples, each of 5 ml of 2 x 10e5 cell.ml-1 were added to cloning medium containing TFT, giving 1 x 10e6 cells per dish. The mean number of mutants per set of 3 dishes is therefore the mean number of mutants per 10e6 cells plated. To adjust for cloning efficiency, each value is multiplied by 200 (200 cells plated per viability plate) and divided by the mean count from the corresponding viability plates. The resultant value is the mutant fraction per 10e6 viable cells.
Evaluation criteria:
i) A negative response was recorded if responses from, the test substance were not higher than those of the vehicle control and the chemical had been tested to pre-set limits that included either a reduction of relative total growth to 20 %, or precipitation of the test compound, or a maximum acceptable dose of 5 mg/ml. ii) The response at a single dose was classified as significant if the cloning efficiency was at least 10 % and the mean mutant fraction was at least 1.7-fold higher than the mean control value.iii) An experiement was positive if the response in at least the highest acceptable dose was significant by the criteria described in ii) and was associated with an increase in mutant numbers or an upward trend in the remaining doses.iv) A compound was positive if 2 positive experiments were recorded within the same activation conditions.
Statistics:
Analyses were not conducted beyond comparison of means.

Results and discussion

Test results
Species / strain:
mouse lymphoma L5178Y cells
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:
Toxicity Test:The toxicity test performed on P.I.A. Purified Isophthalic acid, showed that the test material was toxic to a portion of the cell population at 750 µg.ml-1, in both the absence (RSG = 26%) and presence (RSG = 55%) of S9 mix. Whilst the highest concentration of 2500 µg.ml-1 did not cause complete killing of the populations, there was evidence that the exposed cell cultures were not recovering after treatment. P.I.A. precipitated on dosing at the highest concentrations of 2500 µg.ml-1. It was noted that on adding P.I.A. to the cell cultures, the pH was reduced (observation of phenol red indicator in tissue culture medium.Vehicle Control Groups:The solvent control values were within the normal ranges experienced in the laboratory and reported in the literature. Positive Control Groups:The high mutant fractions obtained with EMS and 3-MC were within the normal ranges for the laboratory.P.I.A. Purified Isophthalic acid (Assays 1 - in the absence of S9 mix and Assays 2 - in the presence of S9 mix).In the absence of S9 mix, no evidence of mutagenic activity was obtained at any tested concentration of P.I.A. Results were not obtained, however from a critically toxic dose level. The mean RTG at 750 µg.ml-1 was 77%. The difference in pH at this concentration was 7.36 in the vehicle control, compared to 6.21 in the P.I.A. -treated culture.In the presence of S9 mix, a small increase in mutant fraction was obtained over the vehicle control values at 750 µg.ml-1. The mean increase of 1.55-fold was not considered significant. Again, results were not obtained from a critically toxic dose level: the mean RTG at 750 µg.ml-1 was 75 %. The difference in pH at this concentration was 7.17 in the vehicle control, compared to 6.22 in the P.I.A.-treated culture.P.I.A. Purified Isophthalic acid (Assays 3 - in the absence of S9 mix and Assays 4 - in the presence of S9 mix).In the absence of S9 mix, the highest concentration of 750 µg.ml-1 gave a mean increase in mutant fraction of 1.85-fold over the vehicle control values. The 2 cultures differed slightly, probably due to differences in the level of toxicity; one culture gave a 1.6 fold increase at 12 % RTG, while the other culture gave a 2.1-fold increase at 8% RTG.The size of the increase at 750 µg.ml-1 was marginal, by the stated criteria. Additionally, it was on the limit with respect to level of toxicity, that mutagenic effetcs obtainable only at < 10% RTG are of questionable biological significance.The pH was significantly reduced at 750 µg.ml-1 (vehicle = 7.21, P.I.A.-treated = 5.98). Although pH-related mutagenic effects are more usually associated with experiments conducted in the presence of S9 mix, Cifone et al (1987) observed a 1.9 fold increase (24% RTG) at a pH of 6.3 in the absence of S9 mix. This latter finding makes it feasible that the effect seen at 750 µg. P.I.A..ml-1 is due to the reduction in pH.In the presenc of S9 mix, the steepness of the toxicity curve again prevented results from being obtained at a criticially toxic dose level. No evidence of mutagenicity was obtained from any of the concentraions assessed. The highest dose level (750 µg.ml-1) resulted in a mean RTG of 54.5 %.Colony Size Distribution with P.I.A. Purified Isophthalic Acid:The following cultures were selected for analysis of colony size distribution:Assay 3 (in the absence of S9 mix) - Second vehicle control culture, First EMS-treated culture, Second 750 µg.P.I.A. Purified Isophthalic Acid.ml-1 -treated culture.The analysis produced good separation between large and small colony populations in the positive control and P.I.A.-treated cultures (less so in the vehicle control culture). The results from vehicle and positive controls were typical of those obtained in the laboratory. EMS induced predominantly large colonie (point mutations or small chromosomal aberrations), giving a ratio of 0.5 small colonies for every large colony. P.I.A. Purified Isophthalic Acid gave a sufficiently high ratio of small to large colonies (2.02 small to every large) to indicate that the induced increase in mutant fraction was entirely the result of clastogenic activity. Similiar results were obtained by Clifone et al (1987), when performing colony sizing analyses on ppH-induced increases in L5178Y cells. Literature exists to substantiate these results, whereby pH-induced positive responses were obtained in the in vitro chromosomal aberrations assay (Morita et al (1992) and others).
Remarks on result:
other: strain/cell type: L5178Y
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Mouse Lymphoma in the absence of S9 Mix - Suspensions Counts (Assay 1)

Chemical

Dose Level

pH after 4 h

*Daily Suspension Count x 105.ml-1         (ml Cells Kept)

Total Suspension Growth

Day 1

Day 2

Dimethylsulphoxide

100 µl added

7.36

8.8

(6.8)

16.4

(1.8)

16.0

10.0

(6.0)

14.8

(2.0)

16.4

9.6

(6.2)

13.2

(2.3)

14.1

10.0

(6.0)

10.4

(2.9)

11.6

Ethyl methanesulphonate

250

-

8.2

(7.3)

12.0

(2.5)

10.9

8.4

(7.1)

12.2

(2.4)

11.4

3-Methylcholanthrene

2.5

-

11.2

(5.4)

10.0

(3.0)

12.4

12.0

(5.0)

11.6

(2.6)

12.9

Purified Isophthalic Acid

150

7.17

8.0

(7.5)

12.8

(2.3)

11.4

7.6

(7.9)

10.8

(2.8)

9.1

300

6.90

8.0

(7.5)

13.0

(2.3)

11.6

7.2

(8.3)

14.4

(2.1)

11.5

400

6.74

10.8

(5.6)

10.8

(2.8)

13.0

7.6

(7.9)

11.4

(2.6)

9.6

600

6.48

9.6

(6.2)

10.0

(3.0)

10.7

7.6

(7.9)

1.0

(2.7)

9.3

750

6.21

7.2

(8.3)

13.8

(2.2)

11.0

10.0

(6.0)

8.4

(3.6)

9.3

900

5.77

0

(-)

-

 

NP

0

(-)

-

 

NP

* = Adjusted to 20 ml of 3 x 105.ml-1 after counting on Day 1

  = Adjusted to 20 ml of 2 x 105.ml-1 after counting on Day 2

NP = Not plated - too toxic

Table 2: Mouse Lymphoma in the presence of S9 Mix (FLI 075) - Suspensions Counts (Assay 2)

Chemical

Dose Level

pH after 4 h

*Daily Suspension Count x 105.ml-1         (ml Cells Kept)

Total Suspension Growth

Day 1

Day 2

Dimethylsulphoxide

100 µl added

7.17

9.0

(6.7)

13.2

(2.3)

13.2

10.0

(6.0)

12.0

(2.5)

13.3

10.6

(5.7)

10.0

(3.0)

11.8

8.8

(6.8)

11.0

(2.7)

10.8

3-Methylcholanthrene

2.5

-

3.5

(17.1)

7.5

(4.0)

2.9

4.4

(13.6)

7.2

(4.2)

3.5

Purified Isophthalic Acid

150

6.91

9.4

(6.4)

13.0

(2.3)

13.6

10.4

(5.8)

12.0

(2.5)

13.9

300

6.74

10.6

(5.7)

12.6

(2.4)

14.8

9.8

(6.1)

11.6

(2.6)

12.6

400

6.59

11.4

(5.3)

11.2

(2.7)

14.2

11.0

(5.4)

12.0

(2.5)

14.7

600

6.39

10.4

(5.8)

12.0

(2.5)

13.9

8.6

(7.0)

12.8

(2.3)

12.2

750

6.22

8.2

(7.3)

11.0

(2.7)

10.0

9.2

(6.5)

11.0

(2.7)

11.2

900

5.90

0

(20)

1.8

 

NP

0

(20)

0.5

 

NP

* = Adjusted to 20 ml of 3 x 105.ml-1 after counting on Day 1

  = Adjusted to 20 ml of 2 x 105.ml-1 after counting on Day 2

NP = Not plated - too toxic

Table 3: Mouse Lymphoma in the absence of S9 Mix - Suspensions Counts (Assay 3)

Chemical

Dose Level

pH after 4 h

*Daily Suspension Count x 105.ml-1         (ml Cells Kept)

Total Suspension Growth

Day 1

Day 2

Dimethylsulphoxide

100 µl added

7.17

10.2

(5.9)

10.0

(3.0)

11.3

10.2

(5.9)

10.8

(2.8)

12.2

10.0

(5.4)

12.0

(2.5)

13.3

11.6

(5.2)

11.0

(2.7)

14.2

Ethyl methanesulphonate

250

-

8.0

(7.5)

11.0

(2.7)

9.8

10.8

(5.6)

9.4

(3.2)

11.3

3-Methylcholanthrene

2.5

-

10.2

(5.9)

16.4

(1.8)

18.6

10.4

(5.8)

13.0

(2.3)

15.0

Purified Isophthalic Acid

600

6.43

9.6

(6.2)

11.2

(2.7)

11.9

9.2

(6.5)

10.0

(3.0)

10.2

650

6.40

8.2

(7.3)

10.8

(2.8)

9.8

10.2

(5.9)

9.4

(3.2)

10.7

700

6.03

4.9

(12.2)

8.8

(3.4)

4.8

6.5

(9.2)

11.0

(2.7)

7.9

750

5.98

1.0

(20)

4.7

(6.4)

1.6

1.0

(20)

3.6

(8.3)

1.2

800

5.82

0.1

(20)

0.0

 

NP

0.1

(20)

0.0

 

NP

850

5.58

0

 

-

 

NP

0

 

-

 

NP

900

4.92

0

 

-

 

NP

0

 

-

 

NP

* = Adjusted to 20 ml of 3 x 105.ml-1 after counting on Day 1

  = Adjusted to 20 ml of 2 x 105.ml-1 after counting on Day 2

NP = Not plated - too toxic

Table 4: Mouse Lymphoma in the presence of S9 Mix (FLI 075) - Suspensions Counts (Assay 4)

Chemical

Dose Level

pH after 4 h

*Daily Suspension Count x 105.ml-1         (ml Cells Kept)

Total Suspension Growth

Day 1

Day 2

Dimethylsulphoxide

100 µl added

7.03

10.2

(5.9)

12.6

(2.4)

14.3

12.0

(5.0)

10.0

(3.0)

13.3

11.4

(5.3)

11.8

(2.5)

14.9

10.0

(6.0)

11.0

(2.7)

12.2

3-Methylcholanthrene

2.5

-

7.0

(8.6)

7.0

(4.3)

5.4

5.8

(10.3)

7.8

(3.8)

5.0

Purified Isophthalic Acid

650

6.30

10.8

(5.6)

9.6

(3.1)

11.5

10.0

(6.0)

10.2

(2.9)

11.3

700

6.15

7.5

(8.0)

10.4

(2.9)

8.7

8.6

(7.0)

9.6

(3.1)

9.2

750

6.09

6.8

(8.8)

10.0

(3.0)

7.6

5.9

(10.2)

9.8

(3.1)

6.4

800

5.90

0.1

(20)

0.2

 

NP

0.1

(20)

1.0

 

NP

850

5.78

0

 

-

 

NP

0

 

-

 

NP

900

5.60

0

 

-

 

NP

0

 

-

 

NP

950 pptn

5.39

0

 

-

 

NP

0

 

-

 

NP

* = Adjusted to 20 ml of 3 x 105.ml-1 after counting on Day 1

  = Adjusted to 20 ml of 2 x 105.ml-1 after counting on Day 2

NP = Not plated - too toxic

pptn = precipitation

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):negativeThe weight of evidence suggests that purified isophthalic acid is not mutagenic in mouse lymphoma L5178Y cells.
Executive summary:

Purified Isophthalic Acid (PIA) was tested for mutagenic activity in the mouse lymphoma L5178Y assay in both the absence and presence of a rat liver preparation and the co-factors required for mixed-function oxidase activity (S9 mix). A preliminary toxicity test showed that the test substance was toxic to a portion of the exposed cell population at 750 µg/mL in the absence and presence of S9 mix. It was noted that on adding PIA to the cell cultures, the pH was reduced (observation of phenol red indicator in tissue culture medium). It is known that pH of treated cultures was monitored throughout the study. In 4 independent mutation assays (2 in the absence and 2 in the presence of S9 mix), results were obtained where the final concentrations of PIA in the treatment medium ranged between 150 -750 µg/ml in the absence and presence of S9 mix. Positive controls demonstrated the sensitivity of the assay and the effectiveness of the S9 mix. In both assays in the presence of S9 mix, and the first assay in the absence of S9 mix, the steepness of the toxicity curve results prevented results from being obtained at critically toxic dose levels. No indication of mutagenic activity was obtained, however in any of these assays. In the second assay in the absence of S9 mix results were obtained in the region of 10% cell survival. These results gave a marginal increase (1.85-fold) in mutant fraction over the vehicle control values. The biological significance of such a weak effect at such high concentrations is doubtful.