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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:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to O.E.C.D. Testing Guideline No. 486 with GLP compliance.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1994
Report Date:
1994

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)
GLP compliance:
yes
Type of assay:
unscheduled DNA synthesis

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
As per IUCLID5 Sections 1.1. - 1.4.

Test animals

Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals and environmental conditions:
The animals were acquired from Charles River Wiga GmbH, Sulzfeld and were 6 - 10 weeks old. Mean aniaml body weight was 203 grams at study initiation. Healthy animals underwent quarantine in the animal house of CCR for at least five days after their arrival. During this period the animals did not show signs of illness or altered behavior. The animals were distributed into the test groups at random and identified by cage number. Animals were kept at 21 +/- 3 degrees Celcius, relative humidity 30-76 %, and artificial light from 6:00 a. - 6:00 pm. The animals were housed singly in Makrolon Type 1 cages and ofered pelletd standard feed and tap water ad libitum.

Administration / exposure

Route of administration:
oral: unspecified
Vehicle:
Arachis oil, 10 ml/kg
Details on exposure:
The animals were fasted overnight for approximately 6 hr. Body weights were taken before dosing and the dose volume calculated for oral exposure.
Duration of treatment / exposure:
Two or 16 hr to detect short and long-patch DNA repair.
Frequency of treatment:
Once
Post exposure period:
None.
No. of animals per sex per dose:
5 male
Control animals:
yes
Positive control(s):
100 mg/kg 2-acetylaminofluorene

Examinations

Tissues and cell types examined:
Liver hepatocytes
Details of tissue and slide preparation:
The liver was perfused in vivo via the vena portae with Hanks' Balanced Salt Solution (HBSS) supplemented with collagenase (0.0% wt/v Boehringer Mannheim) adjusted to pH 7.4 at approximately 37 degrees C. The hepatocytes were brushed free and washed twice with HBSS by centrifugation and resuspension. The cell suspension was filtered through steel mess to yield a single suspension. Viability was determined by trypan dye exclusion. The hepatocye suspension was again centrifuged and resuspended in complete Williams medium E with 10% v/v fetal calf serum.

2.5 mL of hepatocyte suspension in complete Williams medium E at 100,000 viable cells/mL were seedd onto 25 mm gelatinized Thermanox round plastic coverslips in 35 mm six-well culture dishes. After attachment for approximately 1.5 hr at 37 C the culture medium was removed by aspiration. The attached cells were washed once with phosphate buffered saline. DNA radiolabeling was initiated by adding 2.0 mL of WME with 1% v/v FCS and 3H-thymidine (5 uCi/mL, specific activity 20 Ci/mmol.) The cultures were incubated for 4 hr with 3H-thymidine and then washed twice with WME supplemented with 1% FCS and 0.25 mM unlabeled thymidine. Culture were then incubated overnight with the same medium. After overnight incubation the medium was removed by aspiration and the coverslips treated with a hypotonic solution of 1% sodium citriate for 10 minutes to swell the hepatopcyte nuclei. The cells were then fixed to the coverslips by three changes of methanol:acetic acid 3:1 for 20 minutes each and then rinsed with with 95% ethanol before air drying.

For autoradiography the coverslips were fixed to microscope slids and treated with molten KOAK NTB2 photographic emulsion in the dark. After drying the coated slides were stored in the dark for 12 - 14 days at 4 degrees C. The photographic emulsion was developed with KODAK D-19 developer at room temperature and fixed in TETENAL. The slides were stained with hematoxylin/eosin.
Evaluation criteria:
The test substance is considered to be positive in the study if a mean net nuclear grain count of 5 or greater is observed at one treatment point. A dose-related increase in mean net nuclear grain count provides further evidence of a positve genotoxic resoponse.
Statistics:
Non-parametric Mann-Whitney test as needed.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Treatment of rats with the positive control 2-acetylaminofluorene for approximately 16 hr at 100 mg/kg induced a mean net nuclear grain count of 44.5. The vehicle control mean net nuclear grain count was -5.34. Treatment with 1,6-Hexanediol Diglycidylether (HDDGE) at the high dose of 2000 mg/kg for 2 or 16 hr produced mean net nuclear grain counts of -4.99 and -5.20 respectively.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
1,6-Hexanediol Diglycidylether (HDDGE) did not induce evidence of repairable DNA damage in hepatocytes following oral treatment with upto 2000 mg/kg of body weight. Therefore, HDDGE is not genotoxic under the conditions of the study.
Executive summary:

1,6-Hexanediol Diglycidylether (HDDGE) was accessed for the potential to induce repairable DNA damage in an in vivo/in vitro rat hepatocyte O.E.C.D. 486 UDS Testing Guideline study with GLP compliance. HDDGE was tested upto a high oral dose of 2000 mg/kg of body weight. 1,6-Hexanediol Diglycidylether (HDDGE) did not induce evidence of repairable DNA damage in hepatocytes following oral treatment with upto 2000 mg/kg of body weight. Therefore, HDDGE is not genotoxic under the conditions of the study.