thResponse to the Issues Raised at the 7 IWGT Meeting:

P-55

Effect of Aging on the Repeated Dose Liver Micronucleus Assay○ Miyuki Shigano1, Kiyoko Nakadate1, Hironao Takasawa1, Shuichi Hamada1
1LSI Medience Corporation, Kamisu, Ibaraki, JapanAt the 6th IWGTin Iguazu 2013

Abstract
The liver micronucleus (MN) assay is an effective and important in vivo test for
detecting genotoxic compounds, particularly for those that require metabolic
activation to show genotoxicity. We have developed a repeated dose liver MN(RDLMN) assay which greatly facilitates incorporation of a liver MN assay into ageneral toxicity study. Usefulness of the RDLMN assay was appraised highly inthe 7th International Workshops on Genotoxicity Testing (7th IWGT, 2017 inTokyo). The working group members agreed that the RDLMN assay issufficiently validated for an OECD guideline in terms of numbers and types ofchemicals studied, and the easy integration into general toxicity studies ispreferred from the 3R’s point of view; however, it is necessary to evaluate theimpact of dosing animals of different ages (6- versus 8-weeks old). This isbecause the age of animals at the start of administration in a 4-week repeateddose general toxicity study often differs between Japan (6 weeks) and Westerncountries (8 weeks). Our data were for animals at 6 weeks of age at the start ofadministration only, but not for animals at 8 weeks of age. In this study, we usedclofibrate, a weak inducer of the liver MN, to compare the age difference
between 6 and 8 weeks at the start of administration to examine effects of aging
on the results of RDLMN assays, and the results are to be reported at theACEM/JEMS meeting.
.

Background 1【 Effects of repeated dosing on micronucleus assay using hepatocytes 】
2,4-DAT is a hepatocarcinogen but difficult to detect by a bone marrow
micronucleus assay. Even in juvenile rats which have high mitotic activity,
MNHEP induction did not increase more than 2-fold that of the negative
controls in a double treatment. On the other hand, a 28-day repeated treatment
showed remarkable acceleration in micronucleus induction by theaccumulation of MNHEP even in mature rats which have low mitotic activity.
1.8
v ehicle
2,4-DAT (25m g/kg)
2,4-DAT (50m g/kg)
2,4-DAT (100m g/kg)
2,4-DAT (200m g/kg)

1.6
1.4

2,4-Diaminotoluene(2,4-DAT)

2.5

2

MNHEP (%)

MNHEP (%)

1.2

3

10.8

Diethyl nitrosamine(DEN)v ehicle
DEN (6.25m g/kg)
DEN (12.5m g/kg)
DEN (25m g/kg)
DEN (50m g/kg)

1.5

10.6
0.4

0.5

0.2

Juvenile rats

0double dose

Mature rats

Juvenile rats

0

Mature rats

double dose

28-da y r epea t ed dose

28-da y r epea t ed dose

Fig. 11 Double
Double treatment
treatment vs.
vs. Repeated
Repeated treatment
treatment
Fig.

2.5

5-day repeated dosing
14-day repeated dosing
28-day repeated dosing

MNHEP (%)

21.5

10.5

Mature rats

Mature rats
2,4-DAT(50mg/kg/day)Time course of BM micronucleus induction

180 to 200 days
life span of hepatocytes

Single highdose treatment
to juvenile rats

DEN(12.5mg/kg/day)Single highdose treatment
to mature rats

Repeated low dose treatment
to mature rats

Difference of sensitivity
between single treatment
Difference of sensitivity and 28-day repeatedbetween mature rats and treatment
juvenile rats

Mature rats

0ControlTime course of liver micronucleus induction

MN induction in BM cells

3MN induction in hepatocytes【 Accumulation and time course of liver micronucleus induction 】
In both the 2,4-DAT and DEN administration, MNHEP(%) was the highest in the28-day repeated dose, followed by the 14-day and 5-day repeated dose in thissequence, which suggesting accumulation of the MNed hepatocytes. Theseresults suggest that MNHEP is not selectively eliminated, but accumulated after
repeated dose for a general cellular life span, about 200 days. The liver MN
assay is extremely effective when integrated into repeated dose general toxicity
studies, taking advantages of hepatocytes long-term viability (180 to 200 days)
that brings about accumulation of MNed hepatocytes and good compatibility
with repeated treatment even in a low top dose (not depend on Cmax).

At the 7th IWGT
in Tokyo 2017

Fig. 3 The performance of the repeated-dose liver MN assay【The agreements and the issues pointed out at the 7th IWGT meeting】
1. The working group members agreed that the liver MN assay is sufficiently
validated in terms of numbers and types of chemicals studied to develop thistest into an OECD Test Guideline.
2. From a 3R’s point of view, combination with other genotoxicity assays and/or
integration into general toxicity studies would be preferred.
3. Regarding integration potential, it should be noted that the OECD 407
Guidance (rat one month study) indicates that dosing should begin as soon
as feasible after weaning, in any event before 9 weeks of age.
4. This recommendation therefore supports the use of 6-week old rats, the agethat was used to develop the majority of one month repeated dose liver MN
assays have been accomplished to date. However, given the tendency ofindustry to conduct their general toxicology studies with rats older than 6weeks at start of treatment, the impact of age at the time of dosing warrants
further study.

Effects of aging【 Animals 】
We used male Crl:CD(SD) rats that were purchased from Charles River
Laboratories Japan, Inc. The animals were 6 weeks old and 8 weeks old at thebeginning of dosing.
【 Test substance 】
We used clofibrate (CAS No.: 637-07-0) that act through nuclear receptor.
【 Dose levels and treatment 】
Clofibrate was administered orally to the rats once a day for 28 consecutive
days. For control, the vehicle was administered in the same way as clofibrate.
Clofibrate was administered to the animals at 125 and 500 mg/kg/day.
【 Results and discussions 】
In animals aged 6 weeks at the start of dosing, a significant increase in liver
micronucleus induction was observed in the 125 and 500 mg/kg/day groups. Inanimals aged 8 weeks at the start of dosing, although a significant increase inliver micronucleus induction was observed in the 500 mg/kg/day group, nosignificant increase was observed in the 125 mg/kg/day group. As for clofibrateused in this study, it was possible to detect liver micronucleus induction evenin animals aged 8 weeks at the start of dosing. However, liver micronucleus
induction was lower in 8-week-old animals at the start of dosing compared to 6week-old animals at the start of dosing receiving the same dose of clofibrate.
We believe that further examination is necessary to identify usefulness of 8week-old animals at the start of dosing in detecting hepatocarcinogens in a 28day repeated liver micronucleus assay and the difference in detection capability
compared to 6-week-old animals at the start of dosing.

Single high dose treatment

28-day repeated low dose treatment(Single low dose treatment)

Difference of sensitivity
between single dosetreatment and repeateddose treatment････････････････

Sensitivity of single treatment to juvenile rats
28 days

Duration

1 dayDuration

Fig. 2 Accumulation of the micronucleated hepatocytes by repeated dosing

Background 2【The performance of liver MN assay 】
Follow-up studies to the test performance have been performed after the 6thIWGT, and the results were reported at the 7th IWGT. Four aneugens(carbendazim, colchicine, vinblastine and docetaxel), 2 genotoxic noncarcinogens (amaranth and 2,6-diaminotoluene), and 3 non-genotoxic noncarcinogens (alpha-naphthyl isothiocyanate (as a hepatotoxicant), sodium
chloride and sucrose) were newly examined with the repeated dose method ofliver MN assay (Fig. 3). Carbendazim, vinblastine, and docetaxel were foundpositive, and colchicine was equivocal in the 4 days treatment but negative inthe 28 days treatment. It was considered that colchicine failed to increase liver
MN because it showed strong mitotic inhibition under the test conditions (i.e.,dosing period and dose levels) of the 28 days treatment. Overall, these results
suggest that aneugenic chemicals can be detected by the liver MN assay withappropriate dosing period and dose levels. The two genotoxic non-carcinogens,and the 3 non-genotoxic non-carcinogens, including a hapatotoxicant, werefound negative in the MN assays, indicating that the liver MN assay would havehigh specificity for carcinogenicity.

Fig. 4 Effects of aging of the repeated dose liver MN assay

Summary
1. The working group members of 7th IWGT agreed that the liver MN assay issufficiently validated in terms of numbers and types of chemicals studied todevelop this test into an OECD Test Guideline.
2. This recommendation therefore supports the use of 6-week old rats, the agethat was used to develop the majority of one month repeated dose liver MNassays have been accomplished to date. However, given the tendency ofindustry to conduct their general toxicology studies with rats older than 6weeks at start of treatment, the impact of age at the time of dosing warrants
further study.
3. In this study, we used clofibrate, a weak inducer of the liver MN, to compare
the age difference between 6 and 8 weeks at the start of administration toexamine effects of aging on the results of liver MN assays.
4. As for clofibrate used in this study, it was possible to detect livermicronucleus induction even in animals aged 8 weeks at the start of dosing.
5. We believe that further examination is necessary to identify usefulness of 8week-old animals at the start of dosing in detecting hepatocarcinogens in a
28-day repeated liver micronucleus assay.