Isophthalic acid

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:

1990-08-14 to 1991-03-15

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1991

Report date:

1991

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

: test material purity is not reported

GLP compliance:

yes

Type of assay:

other: in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

Isophthalic acid

EC Number:

204-506-4

EC Name:

Isophthalic acid

Cas Number:

121-91-5

Molecular formula:

C8H6O4

IUPAC Name:

isophthalic acid

Test material form:

other: white solid

Details on test material:

- Name of test material (as cited in study report): Isophthalic acid
- Physical state: White solid
- Lot/batch No.: MIL-62
- Storage condition of test material: Room temperature, protected from light

Specific details on test material used for the study:

Name of test material (as cited in study report): Isophthalic acid
- Physical state: White solid
- Lot/batch No.: MIL-62
- Storage condition of test material: Room temperature, protected from light

Method

Target gene:

The observation of chromosome aberrations in Chinese hamster ovary (CHO) cells is an acceptable clastogenic activity.

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media:
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no, in order to assure the karyotypic stability of the cell line, cells were not used beyond passage 20
- Periodically "cleansed" against high spontaneous background: not stated

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Arocolor-induced S-9

Test concentrations with justification for top dose:

Test concentrations 625, 1,250, 2,500 and 5,000 ug/ml were used.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: No justification was provided.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

activated and non-activated

Positive control substance:

triethylenemelamine

cyclophosphamide

Details on test system and experimental conditions:

METHOD OF APPLICATION: In medium


DURATION
- Preincubation period:16-24 hours
- Exposure duration: 6 hours exposure
- Expression time (cells in growth medium): 24 hours
- Selection time (if incubation with a selection agent):
- Fixation time (start of exposure up to fixation or harvest of cells):12 hours (due to observations of a slight delay in cell cycle kinetics in the absence of S-9 mix and 10 hours in the absence of any observed delay in cell cycle kinetics in the presence of S-9 mix

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 100 per duplicate

DETERMINATION OF CYTOTOXICITY
- Method: The toxic effects of treatment were based upon mitotic inhibition relative to the solvent-treated control and were presented for the toxicity and aberration study.

OTHER: A minimum of 200 metaphase spreads (100 duplicate flask) were examined and scored for chromatid-type and chromosome-type aberrations. chromatid-type aberations inculded chromatid and isochromatid breaks and exchange figures such as dicentrics and rings. Fragments observed with an exchange figure were not scored as an aberaion but instead were considered part of the incomplete exhange. Chromatid and isochromatid gaps were recorded but not included in the anylaisis. Cells were arrested in metaphase by the addition of colcemid two hours prior to harvest.

Evaluation criteria:

The frequency of the cells with structural chromosome aberrations in either the untreated or solvent control must be no greater than 6%. the percentage of cells with chromosome aberations in the positive control must be statistically increased relative to the untreated control.

Statistics:

Statistical analysis of the percent aberrant cells was performed using the Fisher's exact test. The Fisher's exact test was used to compare pairwise the percent aberrant cells of each treatment group with that of the solvent control. In the event of a positive Fisher's test article dose level, the Cochran-Armitage test was used to measure dose-responsiveness.

Results and discussion

Test results

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Reduction in mitotic index in the absence of S-9 mix only.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Test concentrations 150, 500, 1500 and 5000 ug/ml were pH adjusted to approximately pH 7 in treatment medium prior to addition of the treatment flasks in order to maintain the neutrality of the test system.

Any other information on results incl. tables

Cytogenetic analysis of CHO cells with isophthalic acid in the absence of exogenous metabolic activation

Treatment^1	Flask	Mitotic Index^2(%)	Cells Scored	Aberrant Cells^3(%)	Total Number of Structural Aberrations							Average Aberrations Per Cell^3,7
					Chromatid-type^4			Chromosome-type^5			Severely Damaged Cells^6
					Gaps	Breaks	Exch	Breaks	Dic	Ring
Untreated cells	A	4.8	100	2	3	0	0	1	1	0	0	0.020
	B	4.6	100	2	1	1	0	1	0	0	0	0.020
DMSO	A	5.0	100	0	3	0	0	0	0	0	0	0.000
	B	4.8	100	1	1	1	0	0	0	0	0	0.010
Isophthalic acid
625 µg/ml	A	4.0	100	1	1	0	0	1	0	0	0	0.010
	B	3.6	100	2	0	1	0	1	0	0	0	0.020
1250 µg/ml	A	3.0	100	1	1	1	0	0	0	0	0	0.010
	B	3.4	100	1	0	0	0	0	1	0	0	0.010
2500 µg/ml	A	2.4	100	3	1	1	0	2	0	0	0	0.030
	B	3.0	100	3	1	2	0	1	1	0	0	0.040
5000 µg/ml	A	4.4	100	2	1	0	0	1	0	0	1	0.110
	B	4.0	100	1	0	1	0	0	1	0	0	0.020
TEM 0.5 µg/ml	A	2.2	100	18	6	13	3	2	2	0	1	0.300
	B	2.6	100	18	7	13	3	2	0	0	2	0.400

¹ CHO cells treated for 10 hours at 37±1 °C in the absence of an exogenous source of metabolic activation.

² Mitotic index is number mitotic figures x 100/500 cells counted.

³ Excluding cells with only gaps.

⁴ Chromatid breaks include chromatid and isochromatid breaks and fragments; chromatid exchange figures include quadriradials, triradials and complex rearrangements.

⁵ Chromosome breaks include breaks and acentric fragments; dic, dicentric chromosome.

⁶ Severely damaged cells includes cells with one or more pulverized chromosome and cells with 10 or more aberrations.

⁷ Severely damaged cells and pulverizations were counted as 10 aberrations.

 

 

 

Cytogenetic analysis of CHO cells with isophthalic acid in the presence of exogenous metabolic activation

 

Treatment^1	Flask	Mitotic Index^2(%)	Cells Scored	Aberrant Cells^3(%)	Total Number of Structural Aberrations							Average Aberrations Per Cell^3,7
					Chromatid-type^4			Chromosome-type^5			Severely Damaged Cells^6
					Gaps	Breaks	Exch	Breaks	Dic	Ring
Untreated cells	A	8.4	100	2	0	0	0	0	2	0	0	0.020
	B	8.8	100	0	0	0	0	0	0	0	0	0.000
DMSO	A	8.0	100	3	1	0	0	3	0	0	0	0.030
	B	9.8	100	1	0	0	0	1	0	0	0	0.010
Isophthalic acid
625 µg/ml	A	8.2	100	0	0	0	0	0	0	0	0	0.000
	B	9.2	100	2	0	0	1	1	0	0	0	0.020
1250 µg/ml	A	9.6	100	0	0	0	0	0	0	0	0	0.000
	B	9.4	100	0	0	0	0	0	0	0	0	0.000
2500 µg/ml	A	9.2	100	1	0	1	0	0	0	0	0	0.010
	B	8.6	100	0	0	0	0	0	0	0	0	0.000
5000 µg/ml	A	9.6	100	2	1	1	0	0	0	1	0	0.020
	B	9.6	100	2	0	1	0	0	1	0	0	0.020
TEM 0.5 µg/ml	A	2.6	100	17	3	15	3	2	0	0	1	0.300
	B	2.2	100	15	4	11	2	2	1	1	2	0.370

¹ CHO cells treated for 10 hours at 37±1 °C in the presence of an exogenous source of metabolic activation.

² Mitotic index is number mitotic figures x 100/500 cells counted.

³ Excluding cells with only gaps.

⁴ Chromatid breaks include chromatid and isochromatid breaks and fragments; chromatid exchange figures include quadriradials, triradials and complex rearrangements.

⁵ Chromosome breaks include breaks and acentric fragments; dic, dicentric chromosome.

⁶ Severely damaged cells includes cells with one or more pulverized chromosome and cells with 10 or more aberrations.

⁷ Severely damaged cells and pulverizations were counted as 10 aberrations.

 

 

 

 

Summary of results

Treatment	S-9 Activation	Harvest Time	Mitotic Index	Cells Scored	Aberrations Per Cell^1(Mean ± SD)	Cells with Aberrations (%)
Untreated cells	-	12	4.7	200	0.020 ± 0.140	2.0
DMSO	-	12	4.9	200	0.005 ± 0.071	0.5
Isophthalic acid
625 µg/ml	-	12	3.8	200	0.015 ± 0.122	1.5
1250 µg/ml	-	12	3.2	200	0.010 ± 0.100	1.0
2500 µg/ml	-	12	2.7	200	0.035 ± 0.210	3.0
5000 µg/ml	-	12	4.2	200	0.065 ± 0.723	1.5
TEM 0.5 µg/ml	-	12	2.4	200	0.350 ± 1.291	18.0**
Untreated cells	+	10	8.6	200	0.010 ± 0.100	1.0
DMSO	+	10	8.9	200	0.020 ± 0.140	2.0
Isophthalic acid
625 µg/ml	+	10	8.7	200	0.010 ± 0.100	1.0
1250 µg/ml	+	10	9.5	200	0.000 ± 0.000	0.0
2500 µg/ml	+	10	8.9	200	0.005 ± 0.071	0.5
5000 µg/ml	+	10	9.6	200	0.020 ± 0.140	2.0
CP 50 µg/ml	+	10	2.4	200	0.335 ± 1.293	16.0**

1 Severely damaged cells were counted as 10 aberrations

** p≤0.01 Fisher’s exact test

Applicant's summary and conclusion

Conclusions:

Under the conditions of the assay described in this report, isophthalic acid was concluded to be negative under the conditions of this study

Executive summary:

The potential clastogenicity of isophthalic acid was investigated in vitro in CHO cells. Duplicate cultures were exposed to the test material (in DMSO) in the presence and absence of an exogenous metabolic activation system (Aroclor 1254 -induced male Sprague-Dawley rat liver S9 fraction) at concentrations of 625, 1250, 2500 and 5000 µg/ml. Cells were arrested in metaphase by the addition of Colcemid two hours prior to harvest. Cells were exposed for 10 hours (-S9) or 2 hours (+S9) and harvested after 12 hours (-S9) or 10 hours (+S9) and 100 cells/duplicate flask assessed for chromosomal aberrations. Exposure to the test material did not result in any significant increase in the levels of chromosomal aberrations; appropriate responses were seen with the positive control compounds TEM and CPS. No evidence of clastogenicity was seen under the conditions of this study.