5-amino-2,4,6-triiodo-1,3-benzenedicarbonyldichloride

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: OECD (1986) N° 482 genetic toxicology: DNA damage and repair/unscheduled DNA synthesis in mammalian cells in vitro

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: BUTTERWORTH, B.E. et a1 ASTM task group (1987) A protocol and guide for the in vivo rat hepatocyte DNA-repair assay. Mutation Research, 189, 123-133.

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: KENNELLY, J.C. et a1 (1993) In vivo rat liver UDS assay. In: KIRKLAND, D.J. and FOX, M. (Eds) Supplementary Mutagenicity Tests: UKEMS recommended procedures Part I1 revised, p. 52. Cambridge University Press, England.

Principles of method if other than guideline:

OECD (May 1994) revised draft proposal for a new guideline, DNA damage and repair/unscheduled DNA synthesis in mammalian cells in vivo

GLP compliance:

yes

Type of assay:

other: in vivo mammalian bone marrow cytogenetics test

Test material

Test material information

Constituent 1

Reference substance name:

5-amino-2,4,6-triiodo-1,3-benzenedicarbonyldichloride

EC Number:

417-220-1

EC Name:

5-amino-2,4,6-triiodo-1,3-benzenedicarbonyldichloride

Cas Number:

37441-29-5

Molecular formula:

C8H2Cl2I3NO2

IUPAC Name:

5-amino-2,4,6-triiodobenzene-1,3-dicarbonyl dichloride

Test material form:

solid: particulate/powder

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Olac UK Ltd, Bicester, Oxon, England
- Age at study initiation: five weeks old
- Weight at study initiation: 140 - 149 grams
- Assigned to test groups randomly: yes
- Housing: Each group of rats was kept in a plastic disposable cage with a stainless steel grid top
- Diet: free access to pelleted SDS LAD 1 rodent diet
- Water: free access to tap water
- Acclimation period: four to five days prior to dosing

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C
- Humidity (%): 55%
- Air changes (per hr): 20
- Photoperiod (hrs dark / hrs light): artificial light for 12 hours per day

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Duration of treatment / exposure:

2 hours and 14 hours

Frequency of treatment:

once orally

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

200 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day

Doses / concentrations 4

Dose / conc.:

2 000 mg/kg bw/day

No. of animals per sex per dose:

male: 0 mg/kg, 5 animals, sacrifice time: 14h
male: 200 mg/kg, 5 animals, sacrifice time: 14h
male: 1000 mg/kg, 5 animals, sacrifice time: 14h
male: 2000 mg/kg, 5 animals, sacrifice time: 14h

Positive control(s):

Dimethylnitrosamine (DMN) > 99% pure was used as the positive control compound for the 2 hour expression. It was prepared as a solution in distilled water just prior to use at a concentration of 0.4 mg/ml.
2-Acetylaminotluorene (2AAF) 95 - 97% pure, was used as the positive control compound for the 14 hour expression. It was prepared as a suspension in aqueous 1% methylcellulose at a concentration of 5 mg/ml.

Examinations

Tissues and cell types examined:

The slides were randomised, encoded and grain count analysis performed using a Zeiss Photomicroscope II connected to an AMS 40-10 image analyser via a high resolution camera fitted with a 1" chalnicon tube. A Tulip AT compact 3 microcomputer (IBM compatible personal
computer) linked to the image analyser was used to provide a direct data capture system to record grain counts. Three slides per animal were examined using high-magnification, oil-immersion optics; the remaining autoradiographs prepared from each animal were held as reserves in case of any technical problems with the three slides initially examined.

The image analyser was used in the area count mode and the count obtained was automatically converted to an equivalent grain count using a constant conversion factor of 0.15 grains per pixel.

This method is believed to give the most accurate assessment of labelling levels because actual grain counting methods do not take into account variation in grain size or overlapping of grains at the high density seen in the hepatocyte UDS system (Kennelly et a1 1993).

Usually, fifty hepatocytes over several widely-separated, randomly chosen fields of view, from each of three cultures per animal were analysed. Only results from hepatocytes not in S-phase with a normal morphology (ie not pyknotic or lysed) without staining artifacts or debris were recorded. For each cell the number of silver grains overlying the nucleus was estimated using the image analysis system, then the number of silver grains in an equivalent-sized and most heavily-grained, adjacent area of cytoplasm was estimated. The cytoplasmic grain count was subtracted from the gross nuclear grain count to give the net nuclear grain count. Mean grain counts were calculated for each slide examined. For slides showing a strong response, ie where the mean net grain count was in excess of 10, only 25 cells were examined.

Details of tissue and slide preparation:

HEPATOCYTE ISOLATION AND CULTURE
Hepatocytes were isolated from each rat by enzymatic dissociation of the liver using the perfusion procedure. The isolated cells were suspended in Williams' medium E supplemented with 10% foetal calf serum (WEC) at a density of approximately 0.2 x 10 6 cells per ml. This cell suspension was dispensed in 2 ml aliquots into the 35 mm diameter wells of multi-well tissue culture plates, each well containing a sterile 22 mm diameter No. 1 1/2 glass coverslip. Twelve replicate cultures were initiated per animal. The cultures were incubated at 37°C in a humid atmosphere containing 5% carbon dioxide for 90 minutes to allow hepatocytes to attach to the coverslips. After this attachment period the supernatant medium was removed and the cells were gently rinsed with one wash of Williams' medium E without serum (WEI).
The medium was then replaced with WE1 containing high specific activity (methyl-3H)thymidine (Amersham International, batch numbers 153 and 154; specific activities 94 and 87 Cilmmol respectively) at a final activity of 10 µCi/ml. The cultures were incubated in this medium for a period of 4 hours. After this labelling period, the supernatant medium was removed and replaced by WEI containing 250 µM cold (unlabelled) thymidine. The cultures were then incubated for a 'chase'  period of 24 hours. This additional culture period helps to wash out excess radiolabel and improves cell morphology thus facilitating subsequent grain count analysis of autoradiographs.

CELL HARVEST
After the 24 hour cold chase with thymidine, coverslips with attached cells were removed from the culture medium, given three 5 minute washes in Hanks' balanced salts solution then fixed in 2.5% v/v acetic acid in ethanol and allowed to dry: They were mounted on glass microscope slides, with the cell layer uppermost, using DPX mountant. The mountant was allowed to harden at approximately 37°C.

Statistics:

A positive response is normally indicated by a substantial dose-associated statistically significant increase in the net nuclear grain count which is accompanied by a substantial increase in the gross nuclear grain count over concurrent control values.

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

doses producing toxicity: not found up to 2000 mg/kg bw.

Vehicle controls validity:

not specified

Negative controls validity:

not specified

Positive controls validity:

not specified

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative
The test substance did not cause unscheduled DNA synthesis in the rat liver in this in vivo test system.

Executive summary:

The test substance was assessed for induction of DNA repair in hepatocytes following acute oral administration to Specific Pathogen Free outbred albino Hsd/Ola Sprague-Dawley male rats at dosages of 200, 1000 and 2000 mg/kg bodyweight. The high dose of 2000 mg/kg chosen for the DNA repair test is the maximum level recommended by the UKEMS and by draft OECD guidelines for this test and is also the OECD and EEC limit dose for acute oral toxicity testing; it is therefore considered to be an appropriate limit for use in this system.

A negative control group was treated with the vehicle (corn oil) and a positive control group was treated with dimethylnitrosamine at 4 mg/kg (for the 2 hour expression) or 2-acetylaminofluorene at 50 mg/kg (for the 14 hour expression). Hepatocytes were isolated by enzymatic dissociation at 2 or 14 hours after exposure of the animals to the test substance. Four animals were assessed at each experimental point with the exception that only two animals from the positive control group were assessed at each expression time.

The isolated hepatocytes were allowed to attach to glass coverslips and were cultured in vitro with (methyl-3H)thymidine at 10 µCi/ml for four hours to 'radiolabel' replicating DNA. The hepatocytes were 'chased' for 24 hours with unlabelled thymidine then they were fixed and processed for autoradiography .

DNA repair was assessed by comparing the labelling levels of hepatocyte nuclei from treated animals with control values and with the accompanying cytoplasmic labelling levels (usually a total of 150 cells per animal were examined).

The test substance did not cause any substantial increases in either the gross nuclear grain count or the net nuclear grain count (ie the gross nuclear grain count minus the cytoplasmic grain count) at any dose level at either sampling time.

Positive control group animals showed a large and highly significant increase (P < 0.001) in the net nuclear grain count which was accompanied by a large increase in the gross nuclear grain count.

It is concluded that the test substance did not cause unscheduled DNA synthesis in the rat liver in this in vivo test system.