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Toxicological information

Genetic toxicity: in vivo

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Administrative data

in vivo mammalian germ cell study: cytogenicity / chromosome aberration
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
September 09, 1991 to December 05, 1991
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study performed according to OECD 483

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 483 (Mammalian Spermatogonial Chromosome Aberration Test)
GLP compliance:
yes (incl. QA statement)
Type of assay:
chromosome aberration assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Bis(2,3-epoxypropyl) terephthalate
EC Number:
EC Name:
Bis(2,3-epoxypropyl) terephthalate
Cas Number:
Molecular formula:
1,4-bis[(oxiran-2-yl)methyl] benzene-1,4-dicarboxylate
Details on test material:
- Name of test material (as cited in study report): TK 12103
- Physical state: White powder
- Analytical purity: 89%
- Lot/batch No.: Co 188
- Stored at room temperature in the dark
- Test Item received on 09 August 1991

Test animals

other: B6D2F1 (hybrid of C57B16 x DBA/2 origin)
Details on test animals or test system and environmental conditions:
- Source: Charles River UK Ltd, Margate, UK
- Age at study initiation: 62 to 73 days for the range-finder and 63 to 77 days for the main study
- Weight at study initiation: 24 to 30g on day 1 of treatment
- Assigned to test groups randomly: yes, using a system of random numbers
- Fasting period before study: 3 hours prior to dosing
- Housing: in groups of no more than 3 in polypropylene cages with wire mesh lids and solid floors containing wood shavings (washed in detergent, rinsed and dried before use).
- Diet: Special Diets Services Ltd, RM1.(E). SQC. diet ad libitum
- Water: Bottled mains tapwater ad libitum
- Acclimation period: 6 days for the range-finder study and main study

- Temperature (°C): 20 to 22°C
- Humidity (%): 36 to 58%
- Air changes: at least 20 fresh air changes per hour
- Photoperiod: 12-hour dark/light cycle.

Administration / exposure

Route of administration:
oral: gavage
1% (w/v) methyl cellulose as aqueous solution (1% MC)
Details on exposure:
Animals were weighed before dosing and the volume of vehicle, test chemical preparation or positive control solution to be administered was
calculated based on a dose volume of 25 ml/kg.

Dosing preparations were made by freshly suspending TK 12103 in 1% (w/v) methyl cellulose to give the highest concentrations (15.08 mg/mL for the range-finder study and 2.35 mg/mL for the main study). Dilutions were then made using 1% methyl cellulose to reach the lower concentrations and the test chemical preparations used within 2 hours.
For the positive control Mitomycin C was freshly dissolved in saline at 0.012 mg/mL and administered intraperitoneally at 0.3 mg/kg. The negative control (1% Methyl cellulose) was administered orally.
Duration of treatment / exposure:
5 consecutive days for the test groups and vehicle control group
1 single application for the positive control group
Frequency of treatment:
One daily application for the test groups and vehicle control group
One single application for the positive control group
Post exposure period:
6 hours after the last dose for the test groups and vehicle control group.
24 hours after treatment for the positive control group.
Doses / concentrations
Doses / Concentrations:
0 (vehicle), 45, 90, 180 and 360 mg/kg
nominal conc.
No. of animals per sex per dose:
5 male animals
Control animals:
yes, concurrent vehicle
Positive control(s):
Mitomycin C (0.3 mg/kg bw in saline, injected once intraperitoneally on day 4 of main experiment).


Tissues and cell types examined:
Testes and seminiferous tubules
Details of tissue and slide preparation:
Selection of doses for main study
Slides from surviving animals treated in the range-finder were analysed for cytotoxic ratio or the ratio between mitotic and primary and secondary
meiotic metaphases. Fifty cells were analysed per animal and the group mean ratios calculated. These data and the pattern of mortality were used
to determine the maximum dose for the main study.

Four hours prior to killing, animals were injected with colchicine (2 mg/kg) to arrest dividing cells in mitosis. Test article and vehicle treated mice were killed in groups of 5, 6 hours after the last dose; MMC-treated animals were killed after 24 hours. Animals were killed by asphyxiation with carbon dioxide and cervical dislocation following the same sequence used for dosing.
In the main study both testes were dissected from each animal and placed in petri dishes in a small volume of 1% (w/v) trisodium citrate.
Seminiferous tubules were teased out using dissecting needles and the preparation transferred to a plastic centrifuge tube labelled with the ear tag number and A or B (for right and left testis although the designation of right and left was arbitrary) . The tubes were left for approximately 45 minutes and the supernatant discarded.
The cells were fixed by addition of 4 ml of fresh, ice-cold methanol/glacial acetic acid (3.1, v/v) for 5 minutes. The fixative was changed by aspiration and resuspension, and the tubes of fixed cells placed in a refrigerator.
After storing overnight in a refrigerator the fixative was removed and the cells resuspended in 3 ml 45% (v/v) acetic acid in water. The contents
were allowed to settle for approximately 5 minutes and centrifuged at 200 x 'g' for 5 minutes. This procedure was repeated twice, in each case
the cells were resuspended in a further 4 ml fresh fixative. The tubes were stored in the refrigerator for a further 2 days then centrifuged (800 x 'g', 2-3 minutes). The cells were then resuspended in a minimal amount of fresh fixative to give a milky suspension. Several drops of 45% (v/v) acetic
acid in water were added to each suspension to enhance spreading, and 5 or 6 drops of this suspension dropped onto clean, wet microscope slides. Two slides (1 in range-finder) were made from each testis and the slides dried. The slides were labelled with the study number, sampling time and the individual tag number plus A or B (right or left testis). The tag number served as the code so analysis could be carried out "blind".
When the dried slides were dry the cells were stained for 5 minutes in freshly prepared, filtered 5% (v/v) Giemsa stain in pH 6.8 buffer. The
slides were then rinsed, dried and mounted with coverslips.

Scoring of aberrations
Slides from the positive control mice treated with MMC were checked first to ensure the system was operating satisfactorily. The slides from all
dose groups were arranged according to sequential tag number and taken for microscope analysis. Five animals from groups 5 and 6 (360 mg/kg) were chosen for analysis on the basis of slide quality.
Where possible, 50 metaphases from each testis were analysed for chromosome aberrations. Cells with 38 (ie 2n-2) or more chromosomes were considered acceptable for scoring. Aberrations were classified according to the scheme described by Scott et al (5) which is detailed in
Appendix 1 . Cytotoxicity was assessed by determining the cytotoxic ratio on samples of 50 cells per animal.
Evaluation criteria:
3 categories of results were developed: cells with structural aberrations with gaps, cells with structural aberrations without gaps, and
endoreduplicated and hyperdiploid cells.
The acceptance criteria defind by the laboratory were fulfilled for this assay.

A test chemical was to be considered as clearly positive in this assay if:
1) a statistically significant increase in the frequency of cells with structural aberrations occurred for at least one dose
2) the incidence of cells with structural aberrations at such a point exceeded accepted vehicle control ranges established from published literature.
The proportion of cells with structural aberration excluding gaps for each treatment group was compared with the proportions in negative controls by using a 2 x 2 chi-square test. Probability values of p ≤O.05 were to be accepted as significant. The proportions of cells with structural aberration excluding gaps were examined in relation to published control ranges. A further statistical analysis (linear trend test) was used to evaluate possible dose-response relationships.
Lovell D P, Anderson D, Albanese R, Amphlett G E, Clare G, Ferguson R, Richold M, Papworth D G and Savage J R K (1989)
Statistical analysis of in vivo cytogenetic assays. In "Statistical Evaluation of Mutagenicity Test Data", (UKEMS Guidelines Subcommittee Report, Part III), Ed. D J Kirkland, Cambridge University Press, pp 184-232.

Results and discussion

Test results
not specified
Vehicle controls validity:
Negative controls validity:
not applicable
Positive controls validity:
Additional information on results:

Dose (mg/kg) x 5 Number of treated animals Observed death Cytotoxic ratio (Mean)
Vehicle 3 0 1.9
377.1 3 0 2.3
580.1 3 1 3.1
892.5 3 2 1.9
1373 3 3 -
2113 3 3 -
3250 3 3 -
5000 3 3 -

The cytotoxic ratio measures the rate of cell proliferation in the testis and is the ratio between mitotic and primary and secondary meiotic
metaphases. No decrease in ratio was observed in any surviving groups of animals. (An increase in ratio was, in fact, apparent at the highest dose
level scored). This, however, may be of no biological significance insofar as there was no evidence of an increase in cytotoxic ratio at any
dose level in the main study.
The LD50 calculated was found to be approx. 720 mg/kg
360 mg/kg (approx 50% of the LD50) was selected for the upper dose level in the chromosome study (trial 2). 180, 90 and 45 mg/kg were also selected. In a first trial 470, 235, 117.5 and 58.75 mg/kg were selected but due to unexpected high level of mortality among the animals receiving 470 mg/kg trial 2 was performed.

Treatment with TK 12103 at all dose levels resulted in mean frequencies of aberrant cells which were similar to those in concurrent negative controls. Group mean frequencies of cells with aberrations feil within the normal historical control ranges.
These data appeared to be indicative of a clearly negative result.
Closer examination, however, revealed that 2 chromatid exchanges had been recorded on slides from 1 mouse receiving 360 mg/kg/day. These aberrations are very rarely seen in preparations from negative control mice and their appearance was considered to be of some significance. The observation of an exchange in cells from one of the negative control animals in this assay was, however, also surprising. This was the only exchange to have been observed in this laboratory in a total of 50 vehicle control animals.
In order to clarify these data and determine whether the two animals could be considered as an "outlier", that is, unrepresentative of the expected response, additional metaphase analysis was performed as follows:
1) as many cells as possible were scored from slides from the two animals.
2) a further 100 cells, where possible, were analysed from mice which had been treated with 360 mg/kg/day but not previously analysed (reserve animals).
The additional analysis indicated that animal of the test group could well be an outlier. One further exchange was found and a large number of gaps.
In contrast, low numbers of aberrant cells comparable with and not significantly different from, those in vehicle controls were recorded in the preparations from the additional TK 12103 treated animals. No further exchanges were seen in these mice. It was concluded that the chromatid exchanges seen in cells from the test animal were not related to treatment with TK 12103.

Applicant's summary and conclusion

Interpretation of results (migrated information): negative
It is concluded that Terephthalic acid diglycidyl ester (TK 12103) was unable to induce structural chromosome aberrations in the spermatogonia of treated mice following oral administration.
Executive summary:

Terephthalic acid diglycidyl ester (TK 12103) was tested in an in vivo cytogenetics assay in the spermatogonial cells of B6D2F1 mice at 4 dose levels. These were chosen on the basis of data from an initial toxicity range-finder study where TK 12103, made up in 1% (w/v) methyl cellulose, was administered to mice orally. Three male mice each received the test article on 5 consecutive days at doses covering the range 377.1 to 5000 mg/kg. The LD50 calculated from the pattern of mortality was found to be approximately 720 mg/kg. For the cytogenetics assay, TK 12103 was made up as described and administered as 5 daily doses of 45, 90, 180 and 360 mg/kg (equivalent to approximately 50% of the LD50) to groups of 5 animals killed 6 hours af ter the final treatment.

The vehicle (negative) control was 1% (w/v) methyl cellulose, also administered orally. A group of 5 mice treated with this were killed and

sampled 6 hours after the final dose. These animals had low incidences of aberrant cells, with group means within the accepted historical vehicle control range. Mitomycin C (MMC), the positive control, was dissolved in saline and administered intraperitoneally at 0.3 mg/kg to one group of 5 mice killed 24 hours later. All MMC-treated animals showed clear increases in chromosomal aberrations such that the frequency of aberrant cells in the positive control group was significantly greater than that observed in concurrent controls.

Slides from all dose groups were analysed for chromosome aberrations. Animals treated with TK 12103 had frequencies of aberrant cells (excluding gaps) which were similar to those observed in concurrent vehicle controls. There were no statistically significant differences between negative control groups and groups receiving TK 12103 and frequencies of aberrant cells feil within normal ranges in all animals. The presence of 2 chromatid exchanges in preparations from one animal receiving the highest dose level was shown, by additional

microscope slide analysis, to have not been due to TK 12103 treatment.

It is concluded that Terephthalic acid diglycidyl ester (TK 12103) was unable to induce structural chromosome aberrations in the spermatogonia of treated mice following oral administration.