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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Remarks:
Type of genotoxicity: DNA damage and/or repair
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Recent experimental result performed to the appropiate guideline and to GLP.
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

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

Materials and methods

Test guideline
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

Test material

Constituent 1
Chemical structure
Reference substance name:
Terephthalic acid
EC Number:
202-830-0
EC Name:
Terephthalic acid
Cas Number:
100-21-0
Molecular formula:
C8H6O4
IUPAC Name:
terephthalic acid

Test animals

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




Administration / exposure

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.
Doses / concentrations
Remarks:
Doses / Concentrations:
2000 mg/kg
Basis:
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); 3 rats per time point
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.

Examinations

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 than
zero and should show less than 15% cells in repair.
c) The positive control animals should give a mean net nuclear grain count of five or
greater.
d) There should be a minimum of 3 valid test substance treated animals per group.

Results and discussion

Test results
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 I

Purified 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 II

No 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.

Any other information on results incl. tables

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

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
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.
Executive summary:

Purified terephthalic acid was tested for the indiction of unscheduled DNA synthesis (UDS) in rat liver in vivo at the limit dose of 2000 mg/kg bw. The data from the study do not indicate any DNA repair induced in the rat liver following treatment with purified terephthalic acid. The sensitivity of the test system was clearly demonstrated by the marked increases in DNA repair, as measured by UDS, induced by the positive control substance N-DMA.