マイクロサンプリング普及のためのレギュラトリーサイエンス的解析○高橋 一彰1, 服部 則道2,神野 文宏3, 大塚 博比古4, 中井 惠子1, 宅見 あすか2, 渋井 勇祐2, 田中 庸一5, 齊藤 公亮5, 小林 章男6, 斎藤 嘉朗5Regulatory Scientific Analysis for Increased Use of Microsampling○Kazuaki TAKAHASHI1, Norimichi HATTORI2, Fumihiro JINNO3, Hirohiko OHTSUKA4, Keiko NAKAI1, Asuka TAKUMI2, Yusuke SHIBUI2, Yoichi TANAKA5, Kosuke SAITO5, Akio KOBAYASHI6, Yoshiro SAITO5

1: Mediford Corporation, 2: AJINOMOTO CO., INC., 3: Axcelead Drug Discovery Partners, Inc., 4: SEIKAGAKU CORPORATION, 5: National Institute of Health Sciences, 6: INTERNATIONAL UNIVERSITY OF HEALTH AND WELFARE

This presentation is based on an article by Takahashi et al., published in J Appl Toxicol., 2024. If you have any questions, please contact the following: kazuaki.takahashi@mediford.com

BACKGROUND/PURPOSE
Recently, animal welfare has been attracting worldwide attention, and implementation of 3Rs(replacement, reduction, and refinement) is prioritized in every way possible in drug
development. Microsampling (MS), in which small amounts of blood are collected (typicallyno more than 50 µL per time point), is attracting attention in this context. The toxicokinetic(TK) evaluation of samples collected from the animals in the main study group by MS in thegeneral toxicity study enables the analysis of the relationship between toxicity and drug
exposure levels in the individual values and can contribute to the 3Rs by reducing oreliminating the number of satellite animals and reducing the amount of blood sampled (Fig.).
However, no standard methods have been established for practical use, particularly withregard to the appropriate volume of blood to be collected over a period of time. Therefore,
for increased use of MS in non-clinical studies, providing a guideline for appropriate MS bloodcollection volume is necessary while taking its toxic effect into account. The most importantparameter to consider concerning toxicological effects of MS would be the percentage of thetotal blood collection volume to circulating blood volume in animals, when circulating blood
volume/body weight in rats is estimated to be 64 ml/kg [1].

In previous reports on MS, the cervical and tail veins have been mainly selected as the site ofTK blood collection, and it was considered that the amount of blood to be collected could
vary according to the site of blood collection. Similarly, it was considered possible that theeffects of MS might vary between conditions where animals are under the stress of drug
treatment and conditions without drug treatment.
In this presentation, a regulatory scientific analysis based on a systematic review wasconducted with the aim of obtaining a blood collection volume that would have no effect on
the animals or would be generally acceptable, based on the estimated circulating bloodvolume.
[1] Diehl et al., J Appl Toxicol. 2001

RESULTS

Fig. Advantages of MS

Appropriate volume of blood samples taken from the cervical vein of drug-free animals

BASICSReferenceAnimal

SexDrugYokoyama et al., Rats
Males/ None
2020
Crl:CD(SD) females
Hattori et al.,
2020

Rats
Males/ None
Crl:CD(SD) females

Blood sampling points
Days 1-2: 0.5, 1, 2, 4, 8, and 24 hrs*2Days 27-28: 0 , 0.5, 1, 2, 4, 8, and 24 hrs
Days 1-2: 0.5, 1, 2, 4, 8, and 24 hrs*3Days 27-28: 0 , 0.5, 1, 2, 4, 8, and 24 hrs

Percentage of blood collection volume*1
to circulating blood volume

Blood sampling
volume50 µL/point

Days 1-2: 2.1%-2.6% in males and 2.9%- 3.3% in females

Days 27-28: 1.3%-1.5% in males and 2.2%-2.5% in females
0 µL/point or Days 27-28*4100 µL/point 100 µL/point group: 2.8% in males and 4.5% in females*1: Mean circulating blood volume is regarded as 64 mL/kg for rats (Diehl et al., 2001).

Number ofBloodevaluatedsampling
organizations
site4
Cervical vein
1

Evaluation
pointsToxicological effect

Day 29No or minimal influences on body weight gain,
food consumption, hematological and blood
clinical chemistry parameters, and organ weights.
Decreases in red blood cells, hemoglobin
concentration, and hematocrit in females and anincrease in fibrinogen in males.

Cervical vein

Day 29Red square : There were toxicological effects due to blood collection.

*2: Time 0 was set in the morning.
*3: Time 0 refers to the point before dosing.
*4: The 100 µL/point do not fall into the MS category.

Blue square : Maximum percentage in which there were no toxicological effects

When blood samples are taken from the cervical vein of drug-free animals, it is recommended that 3% of
the circulating blood volume should be the upper limit of the total blood collection within 24 hours, with arecovery period of at least 1 day between the end of the transit blood collection and the evaluation point.

Appropriate volume of blood samples taken from the tail vein of drug-free animals
Reference

AnimalSex

Drug

Blood sampling points

Blood
Percentage of blood collection volume*1
samplingto circulating blood volume
volumeExp 2: 0 µL/point,
Male/None Days 1-2: 0.5, 1, 2, 4, 8, and 24 hrs
Days 29-30
Hattori et al., Rats
*250 µL/point group: 1.5% in males and 2.5% in females
2020
Crl:CD(SD) females
Days 29-30: 0 , 0.5, 1, 2, 4, 8, and 24 hrs 50 µL/point, or*3100 µL/point group: 2.9% in males and 4.7% in females
100 µL/point
Powles-Glover Rats
Males/ None Days 1-2: 0.5, 1, 2, 4, 8, and 24 hrs
Group 1: Sham*3et al., 2014
Crl:WI(HAN) females
Days 14-15: 0.5, 1, 2, 4, 8, and 24 hrs
Group 2: 200 µL/point Group 2 Males: 5.7% on day 1 and 5.5% on day 15
Group 2 Females: 8.5% on day 1 and 8.2% on day 15
Group 3: 32 µL/point
Group 3 Males: 0.9% on day 1 and 0.8% on day 15*4Group 3 Females: 1.4% on day 1 and 1.3% on day 15*1: Mean circulating blood volume is regarded as 64 mL/kg for rats (Diehl et al., 2001).
*2: Time 0 was set in the morning.
*3: The 100 µL/point and 200 µL/point do not fall into the MS category.
*4: The body weight range for day 1 and day 15 was regarded as about 330 g and 340 g for Group 2 males,340 g and 360 g for Group 3 males, and 220 g and 230 g for Group 2 and 3 females, respectively,from the figures and tables in the article.

Number ofevaluatedorganizations
11

Evaluation
Toxicological effect
Bloodpoints
samplingsite
Tail vein
Day 31 No parameters were affected after sampling of up
to 100 µL.
Tail vein

Day 15 Group 2: Decreases in hemoglobin, hematocrit,and red blood cell count and increases inreticulocytes, monocytes, neutrophils, AST, andGLDH
Group 3: A small increase in monocyte and a slightdecrease in hemoglobin concentration.

Red square : There were toxicological effects due to blood collection.
Blue square : Maximum percentage in which there were no toxicological effects

When blood samples are taken from the tail vein of drug-free animals, it is recommended that 5% of thecirculating blood volume should be the upper limit of the total blood collection within 24 hours, with arecovery period of at least 1 day between the end of the transit blood collection and the evaluation point.

Appropriate volume of blood samples taken from the cervical vein of drug-treated animals

APPLICATIONSReference

AnimalSex

Drug

Blood sampling points

Blood sampling
volumePercentage of blood collection volume*1
to circulating blood volume

Number ofevaluatedorganizations

Bloodsamplingsite

Evaluation
pointsToxicological effectHattori et al., Rats
Female Methapyrilene Days 1-2: 0.5, 1, 2, 4, 8, and 24 hrs*22023
Crl:CD(SD)Days 27-28: 0 , 0.5, 1, 2, 4, 8, and 24 hrs

50 µL/point

Days 1-2: 2.8%- 2.9% in females
Days 27-28: 2.1%-2.3% in females

2

Cervical vein

Day 29The changes observed at each of the two facilities
were not reproducible.

Ohtsuka et al., Rats
Female Phenacetin
2022
Crl:CD(SD)Days 1-2: 0.5, 1, 2, 4, 8, and 24 hrs*2Days 27-28: 0 , 0.5, 1, 2, 4, 8, and 24 hrs

50 µL/point

Days 1-2: 2.8% in females
Days 27-28: 2.3%-2.7% in females

2

Cervical vein

Day 29Minimal effects on a few hematologicalparameters.

Tanaka et al., Rats
Female Azathioprine
2023
Crl:CD(SD)Days 1-2: 0.5, 1, 2, 4, 8, and 24 hrs*2Days 27-28: 0 , 0.5, 1, 2, 4, 8, and 24 hrs

50 µL/point

Days 1-2: 2.7%- 2.9% in females
Days 27-28: 2.2%-2.4% in females

3Cervical vein

Day 29Minimal or no effects on almost all parameters
except for leukocytic parameters.

*1: Mean circulating blood volume is regarded as 64 mL/kg for rats and 72 mL/kg for mice(Diehl et al., 2001).
*2: Time 0 refers to the point before dosing.

Blue square : Maximum percentage in which there were no toxicological effects

When blood samples are taken from the cervical vein of drug-treated animals, it is recommended that 3%of the circulating blood volume should be the upper limit of the total blood collection within 24 hours, witha recovery period of at least 1 day between the end of the transit blood collection and the evaluation point.

CONCLUSIONS
1. Appropriate volume of blood samples from the cervical vein or tail vein of drug- 2. Appropriate volume of blood samples taken from the cervical vein of drugfree animals was ≤3% or ≤5%, respectively, of the circulating blood volume
treated animals was ≤3% of the circulating blood volume within 24 hours.
within 24 hours.
Blood sampling from the cervical vein is a common and rapid method in Japan. However, it is Although the effects of MS on the azathioprine induced immunotoxicity on leukocytic
speculated that blood leakage from the sampling site may explain why toxic effects due toparameters could not be completely ruled out, other studies conducted under drug treatment
MS are more frequently observed compared to blood sampling from the tail vein of drug-free conditions have shown that MS has minimal toxicological effects.
animals. Therefore, continuous improvement of blood sampling techniques, especially fromthe cervical vein, is considered crucial.

With the blood sampling volumes described above, the application of MS in non-clinical toxicity studies in rats is deemed feasible.