Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test: Negative (DuPont; 1979)


Clastogenicity: Positive (Fox; 2006a)

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
Please refer to Section 13.2 for full read-across justification
Reason / purpose for cross-reference:
read-across source
Key result
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
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.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
No study dates available
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline- and GLP-compliant study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
Not applicable
Species / strain / cell type:
lymphocytes: primary culture (human)
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Two independent cytogenetic experiments using a range of concentrations were conducted initially to determine the limit concentration. The three concentrations tested in the chromosomal aberration analysis were 500, 250 and 50 µg/mL.
Vehicle / solvent:
The test substance was diluted in dried dimethylsulphoxide. Both positive control substances were prepared as solutions in sterile double deionised water.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Cyclophosphamide, Mitomycin C
Details on test system and experimental conditions:
Duplicate human peripheral blood cultures were exposed to the solvent, test substance or positive control substance at appropiate concentrations in two experiments;1. A cytogenetic test was conducted using a sample of pooled blood. Cells were exposed to the test substance and control substances for a period of 3 hours both in the presence and absence of S9 mix. Solvent, untreated and positive control cultures were included.2. In a second, independent cytotoxic test, 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.Treatment of the cultures started approximately 48 hours after culture initiation. A single sampling time, 20 hours after the start of treatment (68 hours after culture initiation), was used.Cultures were established from blood samples from healthy volunteers. At 0 hours, cultures (10ml) were established by the addition of 0.5ml of whole blood to RPMI-1640 (Dutch modification) tissue culture medium supplemented with approximately 10% foetal bovine serum (FBS), 1.0IU/ml heparin, L-glutamine (2mM), 100IU/ml penicillin and 100μg/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 which were used in the 20 hour study in experiment 2 were centrifuged and the culture medium changed with fresh supplemented RPMI-1640 culture medium. After approximately 48 hours from culture establishment, aliquots of the test substance, solvent control or positive control were administered to duplicate cultures as appropiate. 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. In experiment, all cultures were kept at 37 degC for 3 hours after which the culture medium as replaced with fresh RPMI01640 culture medium and then the cells were reincubated at 37 degC for the remainder of the 68 hour 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.At 68 hours, the cells were harvested which involved centrifugation, removal of the supernatent and suspension in 0.075M KCl. The cells were fixed using a methanol/glacial acetic acid fixative (3:1 v/v). After at least two subsequent changes of fixative the cell suspension was mounted onto a microscope slide. The slides were examined and the mitotic index determined by examining 1000 lymphocytes per culture and calculating the percentage of cells in metaphase. For each experiment, both in the presence and absence of S9-mix, duplicate cultures treated with terephthalic acid at three concentrations were selected for chromosomal aberration analysis along with the appropriate solvent and positive control cultures.
Evaluation criteria:
The percentages of aberrant metaphases and the number of aberrations per cell were calculated for each treatment scored, both including and excluding cells with only gap-type aberrations.The Fisher Exact Probability Test (one-sided) was used to evaluate statistically the percentage of metaphases showing aberrations (excluding cells with only gap-type aberrations). Data from each treatment group, in the presence and absence of S9-mix, was compared with the respective solvent control group value. The data have been interpreted as follows:a) No statistically significant increase in the percentage of aberrant cells (at any concentration) above concurrent solvent control values indicates the study to be negative (non-clastogenic).b) A statistically significant increase in the percentage of aberrant cells above concurrent solvent control values, which falls within the laboratory solvent control range indicates the study to be negative (non-clastogenic).c) An increase in the percentage of aberrant cells, at least at one concentration, which is substantially greater than the laboratory historical solvent control values indicates the study to be positive (clastogenic).d) A statistically significant increase in the percentage of aberrant cells which is above concurrent solvent values and which is above the historical solvent control range upper value but below that described in (c) may require further evaluation.
Statistics:
Results were analysed using Fisher's exact test
Species / strain:
lymphocytes:
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
DETERMINATION OF MITOTIC INDICES AND SELECTION OF CONCENTRATIONS (see tables 1 and 2 below)It was discovered that the pH limited the concentration at which the chromosomal aberration could be performed. The concentration of test substance above 500 µg/mL decreased the pH of the culture medium by 1.1 pH units. No significant effect on osmolality was observed.A reduction in the mean mitotic activity, compared to the control, was observed in cultures from both experiment 1 (34% +S9-mix; 26% -S9 mix) and Experiment 2 (35% +S9-mix, 59% -S9 mix) treated with the highest concentrations of terephthalic acid selected for chromosomal aberration analysis. CHROMOSOMAL ABERRATION ANALYSESStatistically and biologically significant increases in the percentage of aberrant cells were observed in Experiment 2 following 20 hour treated in the absence of S9-mix. Small increases in the percentage of aberrant cells were observed following 3 hour treatment in Experiment 1 in the presence and absence of S9-mix and in Experiment 2 in the presence 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.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Mitotic Indices in the Absence of Metabolic Activation

Experiment 1

Experiment 2

Treatment

Mitotic Index %

Mean % Mitotic Index

Treatment

Mitotic Index, %

Mean % Mitotic Index

Solvent (10 µl/mL)

11.9

9.4

10.7

Solvent (10 µl/mL)

7.0

6.5

6.8

TPA (µg/mL)

 

TPA (µg/mL)

 

1660

b

b

1660

 b

b

1000

b

b

1300

b

b

500

7.6

8.2

7.9

1000

b

b

250

9.7

8.4

9.1

500

3.0

2.5

2.8

 175

a

a

  250

5.5

4.4

 5.0
100

a

a

 

175

a

a

 
50

8.1

10.2

 9.2  100

a

a

 
 10

a

a

  50

7.8

5.0

 6.4

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

b       Large reduction in pH of culture medium

Mitotic Indices in the Presence of Metabolic Activation

Experiment 1

Experiment 2

Treatment

Mitotic Index %

Mean % Mitotic Index

Treatment

Mitotic Index, %

Mean % Mitotic Index

Solvent Control (10 µl/mL)

12.6

15.2

13.9

Solvent Control (10 µl/mL)

8.0

7.5

7.8

Terephthalic acid (µg/mL)

 

Terephthalic acid

(µg/mL)

 

1660

b

b

1660

 b

b

1000

b

b

1300

b

b

500

8.6

9.8

9.2

1000

b

b

250

9.2

9.0

9.1

500

3.0

2.5

5.1

 175

a

a

  250

5.5

4.4

5.1
100

a

a

 

175

a

a

 
50

8.8

12.4

 10.6  100

a

a

 
 10

a

a

  50

7.8

5.0

7.4

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

b       Large reduction in pH of culture medium

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

3.50

10.7

Mitomycin C 0.5 µg/mL

36.00**

10.4 Δ

Sodium terephthalate

2100 µg/mL

1500 µg/mL

1000 µg/mL

 

7.00

4.00

3.00

 

7.9

9.1

9.2

Experiment 2- 20 hour treatment

 

Solvent Control 10 µL/mL

4.0

6.8

Mitomycin C 0.5 µg/mL

20.00**

2.5Δ

Sodium terephthalate

2100 µg/mL

1500 µg/mL

1000 µg/mL

 

23.43**

12.00**

4.00

 

2.8

5.0

6.4

**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.

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

 

Treatment

Mean % Aberrant Cells excluding Gaps

Mean % Mitotic Index

Experiment 1- 3 hour treatment

 

Solvent Control 10 µL/mL

1.00

13.9

Mitomycin C 0.5 µg/mL

44.00**

8.6 Δ

Sodium terephthalate

2100 µg/mL

1500 µg/mL

1000 µg/mL

 

4.50*

2.00

2.50

 

9.2

9.1

10.6

Experiment 2- 20 hour treatment

 

Solvent Control 10 µL/mL

4.50

7.8

Mitomycin C 0.5 µg/mL

38.00**

2.9Δ

Sodium terephthalate

2100 µg/mL

1500 µg/mL

1000 µg/mL

 

7.00**

2.00**

3.50

 

5.1

5.1

7.4

**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.

 

Conclusions:
Interpretation of results (migrated information):positive without metabolic activationUnder the conditions of the assay, terephthalic acid is clastogenic to cultured human lymphocytes treated in vitro in the absence of S9-mix.
Executive summary:

The clastogenicity of terephthalic aicd was investigated in primary cultures of human peripheral blood lymphocytes. The data obtained in this study shows that the test sample of terephthalic acid induced chromosomal damage in human peripheral blood lymphocytes following in vitro treatment in the absence of S9 mix. This observation however, is different to that of a separate study of the same design on sodium terephthalate which was concluded to be non-clastogenic when tested at concentrations up to 2100 μg/ml (10 mM). This would suggest that the clastogenicity observed with terephthalic acid in this study is not associated with the terephthalate anion itself, but may be secondary to pH effects in the culture medium.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
No information: study reported in 1979
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Older study, guideline compliant but not conducted under GLP
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
: E. coli strain or S. typhimurium TA102 not used, as specified in the current guideline
GLP compliance:
no
Remarks:
: older study
Type of assay:
bacterial reverse mutation assay
Target gene:
Reversion to histidine independence
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat-liver homogenate
Test concentrations with justification for top dose:
The test concentration used were in the range 500-10000 µg/plate.
Vehicle / solvent:
DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2AA, MNNG, 9-AA, 2-NF
Details on test system and experimental conditions:
The assay was performed in the presence and absence of a rat liver homogenate activation system (S9 fraction). In the absence of an activation system, 0.1 mL of a solution of the test sample and the bacteria added to 2 mL of top agar (0.6% agar, 0.6% NaCl, 0.05 mM L-histidine and 0.05 M biotin). These components were mixed and poured onto a plate containing 20 mL of Davis minimal agar. In the presence of an activation system, 0/5 Ml of S9 mix was also added to the plate. The revertant colonies are counted after the plates are incubated at 37 degC for 48 hours.The cytotoxicity of the test sample in the presence and absence of an activation system was measured in strain TA 1535 and was the basis for selecting concentrations to be used in the mutagenesis test.
Evaluation criteria:
A chemical is classified as non mutagenic if the reversion frequency is less than two times the spontaneous frequency and if less than 0.02 revertants/nmole are observed.
Statistics:
Not stated in report; not required for this study type
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
10,000 ug/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
An initial cytotoxicity experiment with strain TA 1535 was performed which found that terephthalic acid was not toxic at the concentrations tested (10,000 µg/plate). Results from the mutagenesis experiment showed that the chemical is not mutagenic for these strains of bacteria with and without an activation system.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Mutagenic Activity in Salmonella Typhimurium Strains TA 1535, TA 1537, TA 98 and TA 100 with Metabolic Activation (Trial 1) 

Exposure

µg/plate

Histidine Revertants per plate (average of 2 plates)

TA1535

TA1537

TA98

TA100

DMSO

 

21

9

38

176

TPA

500

19

11

37

170

TPA

1000

18

5

33

161

TPA

2500

15

6

30

178

TPA

5000

19

16

39

156

TPA

10000

19

7

34

131

 

 

 

 

 

 

2AA

5

 -

-

 -

2236

 

10

887

620

2124

 -

DMSO = Dimethylsulfoxide (solvent control)

TPA = terephthalic acid

2AA = 2-Aminoanthracene (positive control)

 

Mutagenic Activity in Salmonella Typhimurium Strains TA 1535, TA 1537, TA 98 and TA 100 without Metabolic

Exposure

µg/plate

Histidine Revertants per plate (average of 2 plates)

TA1535

TA1537

TA98

TA100

DMSO

 

22

8

23

115

TA

500

17

6

15

119

TA

1000

18

6

27

123

TA

2500

22

8

24

137

TA

5000

19

6

16

116

TA

10000

21

6

22

123

 

 

 

 

 

 

MNNG

2

2250

 

 

1782

9AAc

50

 

1120

 

 

2NF

25

 

 

1851

 

DMSO = Dimethylsulfoxide (solvent control)

TA = terephthalic acid

MNNG = N-methyl-N’-nitro-N-nitrosoguanidine (positive control)

9AAc = 9-Aminoacridine (positive control)

2NF = 2-Nitrofluorene

Mutagenic Activity in Salmonella Typhimurium Strains TA 1535, TA 1537, TA 98 and TA 100 with Metabolic Activation (Trial 2) 

Compound added

µg/plate

Histidine Revertants per plate (average of 2 plates)

TA1535

TA1537

TA98

TA100

DMSO

 

21

5

32

168

TPA

500

22

7

38

 

TPA

1000

25

7

36

192

TPA

2500

25

9

38

188

TPA

5000

34

10

32

185

TPA

10000

18

10

31

175

 

 

 

 

 

 

2AA

5

 -

 -

 -

2210

2AA

10

772

576

2492

 -

DMSO = Dimethylsulfoxide (solvent control)

TPA = terephthalic acid

2AA = 2-Aminoanthracene (positive control)

 

Mutagenic Activity in Salmonella Typhimurium Strains TA 1535, TA 1537, TA 98 and TA 100 without Metabolic Activation (Trial 2) 

Exposure

µg/plate

Histidine Revertants per plate (average of 2 plates)

TA1535

TA1537

TA98

TA100

DMSO

 

19

9

21

129

TPA

500

27

8

18

135

TPA

1000

18

7

20

130

TPA

2500

33

9

26

127

TPA

5000

32

14

24

145

TPA

10000

27

6

20

142

 

 

 

 

 

 

MNNG

2

728

 -

 -

1312

9AA

50

 -

1334

 -

 -

2NF

25

 -

 -

1981

 -

DMSO = Dimethylsulfoxide (solvent control)

TPA = terephthalic acid

MNNG = N-methyl-N’-nitro-N-nitrosoguanidine (positive control)

9AA = 9-Aminoacridine (positive control)

2NF = 2-Nitrofluorene

Mutagenic Activity in Salmonella Typhimurium Strain TA 1537 with Metabolic Activation (Trial 3)

Exposure

µg/plate

Histidine Revertants Per Plate (average of 2 plates)

TA1537

DMSO

 

14

TPA

500

10

TPA

1000

12

TPA

2500

13

TPA

5000

15

TPA

10000

17

2AA

10

506

DMSO = Dimethylsulfoxide (solvent control)

TPA = terephthalic acid

2AA = 2-Aminoanthracene (positive control)

 

Conclusions:
Interpretation of results (migrated information):negativeTerephthalic acid was tested in Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100. This chemical is not mutagenic for these strains of bacteria in the presence or absence of an activation system.
Executive summary:

The mutagenicity of terephthalic acid was investigated in an Ames testusing Salmonella typhimurium strains TA98, TA100, TA1535 and TA 1537. Duplicate cultures of the bacterial strains were exposed to concentrations of terephthalic acid (dissolved in DMSO) of up to 10,000 µg/plate in the absence and presence of a metabolic activation system (Aroclor 1254-induced rat-liver homogenate).  Results were confirmed in an independently repeated assay. Exposure to terephthalic acid did not result in any increase in the mutation frequency, with the exception of small increases in strain TA1537 at the intermediate concentration of 5000 µg/plate in the absence of metabolic activation. This finding was not confirmed in an additional assay.  Response to appropriate positive control compounds confirmed the sensitivity of the assay. The results of this study indicate that terephthalic acid is not mutagenic.

Endpoint conclusion
Endpoint conclusion:
no study available (further information necessary)

Genetic toxicity in vivo

Description of key information

Rat liver UDS: Negative  (Fox; 2006c)


Mouse micronucleus: Negative (Gudi & Krsmanovic; 2001)

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 February to 27 March 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Proprietary GLP and guideline-compliant study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes (incl. QA statement)
Type of assay:
micronucleus assay
Species:
mouse
Strain:
ICR
Sex:
male/female
Details on test animals or test system and environmental conditions:
Male and female ICR mice were obtained from Harlan Sprague Dawley, Inc. in two batches. At study initiation mice were 6-8 weeks old. Pilot study mouse weights were: males 29.5-34.1 g; females 25.2-27.4 g. Micronucleus assay mouse weights were: males 25.6-30.4 g; females 23.9-28.1 g.Mice were quarantined for 5 days. The animal room was maintained at a temperature of 72±3°F, 50±20% relative humidity and a 12 hour light/dark cycle. Mice were housed in same sex groups of 5 in polycarbonate cages on racks. Hardwood chips were used as bedding. Food (Harlan TEKLAD certified Rodent 7012C) and tap water (Washington Suburban Sanitary Commission) were provided ad libitum.Mice were numbered and identified by ear tag.
Route of administration:
intraperitoneal
Vehicle:
Corn oil. The vehicle was chosen as it permitted preparation of the highest soluble or workable stock solution, up to 100 mg/ml (compared to 0.5% CMC in water).
Details on exposure:
Mice were given a single intraperitoneal injection of the test substance, or vehicle alone. All mice were weighed immediately prior to dose administration and the dose volume based on individual body weights. Injections were kept to a constant volume of 20 ml/kg body weight.
Duration of treatment / exposure:
Pilot study & Toxicity study: mice were observed for signs of toxicity for 3 days after adminstration.Micronucleus study: single injection, mice were sacrificed 24 hours later. Additional mice from the vehicle control and high dose groups were sacrificed 48 hours after dose administration.
Frequency of treatment:
Single injection.
Post exposure period:
Pilot study & Toxicity study: 3 days.Micronucleus study: 24 or 48 hours.
Remarks:
Doses / Concentrations:1, 10, 100, 1000 and 2000 mg/kgBasis:nominal conc.Pilot study
Remarks:
Doses / Concentrations:1200, 1400, 1600 and 1800 mg/kgBasis:nominal conc.Toxicity study
Remarks:
Doses / Concentrations:0, 200, 400 and 800 mg/kgBasis:nominal conc.Main micronucleus study
No. of animals per sex per dose:
Pilot study: 5 mice/sex received 2000 mg/kg, and 2 males/dose received either 1, 10, 100 or 1000 mg/kg.Toxicity study: 5 mice/sex/doseMicronucleus study: controls - 10 mice/sex; 200 and 400 mg/kg - 5 mice/sex/dose, 800 mg/kg - 15 mice/sex/per dose, positive controls - 5 mice/sex/dose.
Control animals:
yes, concurrent vehicle
Positive control(s):
In the micronucleus study, 5 males and 5 females were injected with cyclophosphamide dissolved in distilled water at a dose of 50 mg/kg.
Tissues and cell types examined:
Bone marrow cells obtained from the femurs.
Details of tissue and slide preparation:
Immediately following sacrifice, the femurs were exposed, cut just above the knee, and the bone marrow was aspirated into a syringe containing foetal bovine serum. The cells were transferred to a capped centrifuge tube containing approximately 1 ml foetal bovine serum. The cells were pelleted by centrifugation at 100 x g for 5 minutes and the supernatant drawn off. The cells were resuspended and a small drop of bone marrow suspension was spread onto a clean glass slide. Two slides were prepared from each mouse. The slides were fixed in methanol and stained wiith May-Gruenwald-Giemsa and permanently mounted. Slides were coded prior to analysis.
Evaluation criteria:
2000 polychromatic erythrocytes were scored per slide for the presence of micronuclei. The number of micronucleated normochromatic erythrocytes per 2000 polychromatic erythrocytes was recorded, and the proportion of polychromatic erythrocytes to total erythrocytes oer 1000 was recorded.The test article was considered to induce a positive response if a dose responsive increase in micronucleated polychromatic erythrocytes was observed, and one or more doses doses were statistically elevated relative to the vehicle control.
Statistics:
Kastenbaum-Bowman tables were used to determine statistical significance at the 5% level.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
1000 mg/kg and above
Vehicle controls validity:
not examined
Negative controls validity:
not examined
Positive controls validity:
not examined
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
lethargy and piloerection at all doses
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Pilot study: 3/5 males and 5/5 females died within 2 days administration of 2000 mg/kg. Lethargy and piloerection were noted in males at 1000 mg/kg and males and females at 2000 mg/kg. Tremors were observed in the males at 2000 mg/kg, and convulsions, prostation and crusty eyes were observed in females at this dose. All other animals appeared normal.Toxicity study: mortality occurred within 3 days of dosing as follows: 2/5 males and 1/5 females at 1200 mg/kg; 4/5 males and 3/5 males at 1400 mg/kg; 2/5 males and 4/5 females at 1600 mg/kg; and 4/5 males and 2/5 females at 1800 mg/kg. Lethargy, piloerection and crusty eyes were seen in all mice at all doses. Tremors were seen in females at 1200 mg/kg, and in males and females at 1400 and 1800 mg/kg. Convulsions were seen in males and females at 1600 and 1800 mg/kg, and prostration in males at 1200 and 1400 mg/kg and males and females at 1600 and 1800 mg/kg. The maximum tolerated dose chosen for the micronucleus assay was 800 mg/kg.Micronucleus study: 1 male mouse in the 800 mg/kg group was found dead the day after administration, but was replaced. Lethargy and piloerection were seen at all 3 doses in both sexes. Mice treated with vehicle alone and the positive control substance appeared normal throughout the study. Reductions of 2% to 9% in the ratio of polychromatic erythrocytes to total erythrocytes were observed in some of the treated groups compared to controls suggesting that erythropoiesis was not inhibited. There were no significant increases in the number of micronucleated polychromatic erythrocytes per 2000 polychromatic erythrocytes in treated groups compared to controls, irrespective of sex and time of bone marrow collection. The positive control substance induced a significant increase.

Administration of terephthalic acid did not induce any increase in the incidence of micronucleated PCEs in any of the treated groups.

Conclusions:
Interpretation of results (migrated information): Negative: Under the conditions of this study, terephthalic acid did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in bone marrow and was concluded to be negative in the mouse micronucleus test.
Executive summary:

Terephthalic acid was tested in the mouse micronucleus assay. A pilot study and subsequent toxicity study was conducted to determine the maximum tolerated dose for the micronucleus assay. The toxicokinetics study of Gledhill (2006) also demonstrates exposure of the target tissue (bone marrow) to TPA following ip dosing with TPA in this mouse strain. Terephthalic acid was administered in corn oil by a single intraperitoneal injection, at a constant injection volume of 20 ml/kg. Based on mortality and clinical signs observed during the pilot and toxicity studies, the maximum tolerated dose set for the micronucleus study was 800 mg/kg bw. Clinical signs included lethargy, piloerection, tremors and crusty eyes. In the micronucleus assay, terephthalic acid was administered i.p. at doses of 0, 200, 400 and 800 mg/kg. Bone marrow cells were harvested 24 or 48 hours later. The positive control was cyclophosphamide. No significant increase in micronucleated polychromatic erythrocytes in test article treated groups relative to vehicle controls was observed.

Under the conditions of this study, was concluded to be negative in the mouse micronucleus test.

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Remarks:
Type of genotoxicity: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
key study
Study period:
No information
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Recent experimental result performed to the appropiate guideline and to GLP.
Qualifier:
according to guideline
Guideline:
OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)
GLP compliance:
yes (incl. QA statement)
Type of assay:
unscheduled DNA synthesis
Species:
rat
Strain:
other: Alpk:APfSD
Sex:
male
Details on test animals or test system and environmental conditions:
The test animals were male and female Alpk:APfSD rats in the age range of 6-7 were used for Phase I and male rats in the age range 5-7 weeks were used for Phase II of the study. The animals were supplied by the Rodent Breeding Unit (RBU), AstraZeneca, Alderley Park, Macclesfield, Cheshire and acclimatised for at least 5 days on arrival.On arrival the rats were housed on mobile rat cage racks and given food, Rat and Mouse No.1 Maintenance Diet (Special Diets Services, Stepfield, Witham, Essex, UK) and water ad libitum.The rooms used for phase I and II were maintained at the following conditions;- Temperature 19-25 °C - Humidity 30-70% - 12 hours of light, 12 hours of darkness.- 15 air changes per hour
Route of administration:
oral: gavage
Vehicle:
An individual stock suspension of the test substance was prepared in corn oil. The control substance was also a sample of corn oil.
Details on exposure:
In phase I a test was conducted to confirm the test substance was not toxic to the rats and there was no difference in toxicity between the sexes. In phase II, a single oral dose was given to a group of male rats at a dose level of 2000 mg/kg. This dose level is the limit dose level of the assay. Two sampling times, 2 hours and 16 hours post-dose were used.
Duration of treatment / exposure:
A single oral dose was administered was given to a group of male rats and samples were collected at 2 and 16 hours post-dose.
Frequency of treatment:
Only one single oral treatment.
Post exposure period:
The rats were monitored for 16 hours. At 2 and 16 hours a sample was collected.
Remarks:
Doses / Concentrations:2000 mg/kgBasis:nominal conc.
No. of animals per sex per dose:
In the preliminary study (phase I) 3 rats per sex per dose were used.In the main study (phase II);For the positive and vehicle control, 2 rats were used for each control. For the treated group, 6 rats were used.
Control animals:
yes, concurrent vehicle
Positive control(s):
The positive control, N-DMA was supplied by Sigma Chemical Company Ltd, UK. The positive control substance was prepared as a solution in sterilised double deionised water.
Tissues and cell types examined:
Rat liver hepatocytes
Details of tissue and slide preparation:
An initial experiment (phase I) was performed in order to determine the maximum dose level which would not produce a toxic effect.In phase II, the rats received a single dose of terephthalic acid. Samples were taken at 2 and 16 hours. Sampling involved taking the animal and examination of the dead animal for signs of colouration or abnormalities to organ/tissues. The hepatocytes were isolated and suspensed in Williams' E complete medium to a dilution of 1.5 x 10(+5) cells/mL. The solution was transferred onto coverslips placed etched side up in six well plates. The cultures were placed in a humidified 37 degC incubator with a 95% air: 5% CO2 (v/v) atmosphere for at least 90 minutes to enable cell attachment. The culture medium was aspirated using aseptic technique and the hepatocytes washed with Williams’ E incomplete medium. Williams’ E incomplete medium containing 3H-thymidine was added to each well and the dishes were incubated for approximately 4 hours in a 37°C incubator (humidified, 37°C, 95% air: 5% CO2 v/v atmosphere). Cultures were then washed three times with Williams’ E incomplete medium plus thymidine. This ‘cold chase’ procedure removed excess radiolabel from the cultures. The cultures were then incubated overnight (at least 12 hours) with the same culture medium.The cultures were then washed once with Williams’ E incomplete medium or cold chase medium prior to being fixed at least three times with freshly prepared 1:3 glacial acetic acid:absolute alcohol (v/v) followed by four washes with double deionised water. The coverslips were mounted, cell side up, on labelled microscope slides.After the exposure period, slides were developed using Kodak D19 developer and Ilford Hypam fixer. The cells were stained using Meyers Haemalum and eosin.Slides from one vehicle control and one positive control animal were selected for UDS analysis from each experiment along with the slides from the animals treated with the test substance. For each cell, the number of silver grains over the nucleus [N] was determined by autoradioagraphy. Then an equivalent area of cytoplasm tangential to the nucleus and with the highest apparent number of silver grains was analysed [C]. The difference between these two values [N-C] was the net nuclear grain count. Sixty cells were scored from each animal (Kennelly et al, 1995).
Evaluation criteria:
The validity of the study was assessed according to the following criteria:a) The cells isolated from the vehicle control animals should show a viability of greater than 50%.b) The vehicle control animals should give a mean net nuclear grain count of less thanzero and should show less than 15% cells in repair.c) The positive control animals should give a mean net nuclear grain count of five orgreater.d) There should be a minimum of 3 valid test substance treated animals per group.
Statistics:
no data
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF PHASE IPurified terephthalic acid was administered as a single oral dose to groups of male and female rats at a dose level of 2000 mg/kg. No clinical signs or mortalities were observed in either male or females, therefore the limit dose of 2000 mg/kg was administered to males only in phase II.RESULTS OF PHASE IINo clinical signs were observed for rats dosed with purified terephthalic acid. Examination of the internal organs/tissues showed no abnormalities. Hepatocytes prepared from all animals were examined microscopically. No apparent signs of excessive cytotoxicity were observed on slides from animals dosed with purified terephthalic acid.The test substance did not cause any significant increases in mean net nuclear grain count or percentage of cells in repair compared to the control. Hepatocytes from all purified terephthalic acid treated animals had mean net nuclear grain values of less than zero. This data therefore suggests that there is no evidence for induction of UDS by purified terephthalic acid. The positive control substance , N-DMA, induced marked increases in the mean net nuclear grain counts and percentage of cells in repair.

Phase I-Determination of the maximum dose

Group

Test Substance

Sex

Dose (mg/kg)

Animal identification

No. of deaths/no. dosed

1

Purified Terephthalic Acid

Male

Female

2000

2000

101-103

104-106

0/3

0/3

Summary Data - Phase II

Treatment

Dose

No. of Animals

Mean

N±SD

Mean

C±SD

Mean (N-C) ±SD

Mean % Cells in Repair

2 Hours

Corn Oil (Dried)

10 mL/kg

1

2.2

4.3

-2.1

0.0

Purified Terephthalic Acid

2000 mg/kg

3

2.1 ±0.3

3.6 ±0.5

-1.5 ±0.3

0.0

N-DMA

10 mg/kg

1

10.2

3.0

7.2

66.7

16 Hours

Corn oil (Dried)

10 mL/kg

1

2.2

4.0

-1.8

0.0

Purified Terephthalic Acid

2000 mg/kg

3

2.6 ±0.3

4.0 ±0.6

-1.4 ±0.3

0.0

N-DMA

10 mg/kg

1

9.1

3.1

6.0

66.7

Conclusions:
Interpretation of results (migrated information): negativeUnder the conditions of test, purified terephthalic acid did not induce DNA repair, as measured by unscheduled DNA synthesis, in the rat liver in vivo.
Executive summary:

Purified terephthalic acid was evaluated, using an autoradiographic technique, for its ability to induce unscheduled DNA synthesis (UDS) in the liver of Alpk:APfSD rats. A single oral dose was given to a group of male rats at a dose level of 2000 mg/kg bw; this dose level is the limit dose for the assay. Two sampling times, 2 hours and 16 hours post-dose were used. The values recorded for the mean net nuclear grain counts and the percentage of cells in repair clearly show that purified terephthalic acid did not induce DNA repair, as measured by UDS, at either dose level or time point investigated. The test system positive control, N-nitrosodimethylamine (N-DMA), induced marked increases in UDS, compared to the vehicle control values, thus demonstrating the sensitivity of the test system to a known genotoxin. It is concluded that, under the conditions of test, purified terephthalic acid did not induce DNA repair as measured by unscheduled DNA synthesis in the rat liver in vivo.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

Not applicable as no effect observed.

Additional information

Studies in vitro

No evidence of mutagenicity was seen in a guideline-comparable Ames test (DuPont, 1979) performed with terephthalic acid. The clastogenicity of terephthalic acid was investigated in a study using cultured peripheral human lymphocytes (Fox, 2006a). This study reports a positive result in the absence of metabolic activation; however negative results are reported in studies using sodium terephthalate (Fox, 2006b; 2007a,b), indicating that the terephthalate anion itself is not clastogenic. Findings in the study with terephthalic acid are therefore likely to be a consequence of the acidity of the test substance. The results of a mouse lymphoma assay (Riach & Willington, 1994) with the read-across substance (structural isomer) isoterephthalic acid report a weak positive response; however this was non-reproducible and was associated with marked cytotoxicity and pH changes in the culture medium. The biological significance of this effect is therefore considered to be doubtful.

Studies in vivo

No evidence of unscheduled DNA synthesis (UDS) was seen in a study in the rat liver at the limit dose of 2000 mg/kg bw (Fox, 2006c). No evidence of clastogenicity was seen in a mouse bone marrow micronucleus assay (intraperitoneal dosing) at dose levels sufficient to cause toxicity (Gudi & Krsmanovic, 2001).

Although the results of the studies in mammalian cells in vitro are not universally negative, the positive or equivocal findings seen in some assays are considered likely to be due to pH changes caused by the acidic nature of the test substance (terephthalic acid or isophthalic acid) are associated with marked toxicity. The clear negative results in the two higher tier assays in vivo demonstrate that the substance is not genotoxic.

COM opinion

The available data on the genetic toxicity of terephthalic acid has also been reviewed by the independent UK Government Committee on Mutagenicity (COM). The COM concluded, on the basis of the data available, that the two in vivo studies of genotoxicity performed with terephthalic acid were adequate and negative, and indicated that terephthalic acid is not an in vivo mutagen. The COM further concluded that the available evidence supported their previous conclusion of a non-genotoxic mechanism for the bladder tumours seen in the rat carcinogenicity study.


Justification for selection of genetic toxicity endpoint
A weight of evidence approach is taken to this endpoint; there is no single key study.

Short description of key information:
An Ames test is available for terephthalic acid. Studies of clastogenicity in mammalian cells (cultured human lymphocytes) in vitro are available for terephthalic acid and its sodium salt. A study of mammalian cell mutagenicity (mouse lymphoma assay) is available for the structural isomer isophthalic acid.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Terepthalic acid is not classified for genotoxicity under CLP, based on the negative results in studies in vivo.