Tris(oxiranylmethyl) benzene-1,2,4-tricarboxylate

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

April 16, 1991 to November 15, 1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study performed according to OECD 483

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1991

Report date:

1991

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 483 (Mammalian Spermatogonial Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

chromosome aberration assay

Test material

Test material information

Constituent 1

Reference substance name:

Tris(oxiranylmethyl) benzene-1,2,4-tricarboxylate

EC Number:

230-638-7

EC Name:

Tris(oxiranylmethyl) benzene-1,2,4-tricarboxylate

Cas Number:

7237-83-4

Molecular formula:

C18H18O9

IUPAC Name:

1,2,4-tris[(oxiran-2-yl)methyl] benzene-1,2,4-tricarboxylate

Constituent 2

Reference substance name:

tris(oxiranylmethyl)benzene-1,2,3-tricarboxyl

IUPAC Name:

tris(oxiranylmethyl)benzene-1,2,3-tricarboxyl

Details on test material:

- Name of test material (as cited in study report): Trimelitic acid-triglycidylester (TKK 30009)
- Physical state: Viscous yellow liquid
- Analytical purity: no data
- Lot/batch No.: Co 69/23
- Expiration date of the lot/batch: no data
- Storage condition of test material: at room temperature in the dark
- Other: Delivery on 8 April 1991

Test animals

Species:

mouse

Strain:

other: B6D2F1 (hybrid of C57B16 x DBA/2 origin)

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK Ltd, Margate, UK
- Age at study initiation: 56 to 63 days for the range-finder and 57 to 61 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 5 days for the main study


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 24°C
- Humidity (%): 30 to 56%
- Air changes: at least 20 fresh air changes per hour
- Photoperiod: 12-hour dark/light cycle.


IN-LIFE DATES: From: 22 April 1991 To: 3 July 1991

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

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 TKK 30009 in 1% (w/v) methyl cellulose to give the highest concentrations (15.08 mg/mL for the range-finder study and 3.6 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. 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

Remarks:

Doses / Concentrations:
0 (vehicle control), 90, 180, 360 and 720 mg/kg
Basis:
nominal conc.

No. of animals per sex per dose:

5 males

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).

Examinations

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 cool 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 (720 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.

Statistics:

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 in category 2 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

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

based on the pre-test

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RANGE FINDER STUDY

Dose (mg/kg) x 5 Number of treated animals Observed death Cytotoxic ratio (Mean)
Vehicle 3 0 2.8
377.1 3 0 3.1
580.1 3 0 2.9
892.5 3 0 6.6
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. 1107 mg/kg
720 mg/kg (approx 65% of the LD50) was selected for the upper dose level in the chromosome study. 360, 180 and 90 mg/kg were also selected.

There were no statistically significant differences between the negative control groups and groups receiving the substance TKK 30009 and frequencies of aberrant cells fell within the normal range of animals.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
It is concluded that trimelitic acid-triglycidylester (TKK 30009) was unable to induce structural chromosome aberrations in the spermatogonia of
treated mice following oral administration.

Executive summary:

Trimelitic acid-triglycidylester (TKK 30009) 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 TKK 30009, 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

1107 mg/kg. For the cytogenetics assay TKK 30009 was made up as described and administered as 5 daily doses of 90, 180, 360 and 720 mg/kg (equivalent to approximately 65% of the calculated LD50) to groups of 5 animals killed 6 hours after the final treatment.

The vehicle (negative) control was 1% (w/v) methyl cellulose, also administered orally. Groups 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 TKK 30009 had frequencies of aberrant cell which were similar to those observed in concurrent vehicle controls. There were no statistically significant differences between the negative control groups and groups receiving TKK 30009 and frequencies of aberrant cells fell within the normal

range in all animals.

It is concluded that Trimelitic acid-triglycidylester (TKK 30009) was unable to induce structural chromosomal aberrations in the spermatogonia of treated mice following oral administration.