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17

533

FIGURE LEGENDS

534

Fig. 1 Experimental schedule

535

536

CLO was orally administered to ICR mice on postpartum day 10 or 11 (n=13). Breast milk and blood

samples were collected at 1, 3, 6, 9, 12, and 24 h after the administration.

537

538

Fig. 2 Milking device

539

(A) The milking device was a one-handed milking device for mice and rats (KN-591; Natsume

540

Seisakusho, Tokyo), which consists of a pressure adjustment pipe, sampling tube, exhaust pipe, and

541

milking pipe. The milking pipe was replaced with the straight part of a 10 µL tip, a pipette connecting

542

part of a 10 µL tip (110-207C; WATSON Co., Tokyo), and a silicon tube (10 mm long, 4 mm in outer

543

diameter, 2 mm in inner diameter). The exhaust pipe was connected to an aspirator and milk was

544

collected from a 10 µL tip in contact with the teat into a sampling tube. The milk collection method was

545

based on that in a previous study (Kawakami et al., 2015).

546

(B) The 10 µL tip was approximately 20 mm in length at the pipette-connecting part and at the straight

547

part. The colors of the parts in (A) correspond to those in (B).

548

549

Fig. 3 CLO and dm-CLO concentrations in breast milk and blood

550

(A) The CLO concentration in milk was highest at 1 h after the administration and was gradually

551

decreased at 3, 6, and 9 h. The blood CLO concentration was highest at 1 h after the administration and

552

was gradually decreased at 3, 6, and 9 h. The CLO concentration in the milk and blood was below the

553

detection limit at 12 and 24 h. CLO concentrations in the milk were significantly higher than those in

554

the blood at 1 and 3 h after the administration. The values are expressed as mean ± SE. The numbers of

555

samples were as follows: 1 h (milk, blood = 9), 3 h (milk, blood = 9), 6 h (milk = 4, blood = 6), 9 h (milk

556

= 7, blood = 9), 12 h (milk = 5, blood = 6), and 24 h (milk = 3, blood = 12). **P < 0.01.

557

(B) The milk dm-CLO concentrations were highest at 3 h after the administration and were gradually

558

decreased at 6, 9, and 12 h. Dm-CLO concentrations in the blood were highest at 1 h or 3 h after the

559

administration and were gradually decreased at 6 and 9 h after the administration. The milk dm-CLO

560

concentrations were below the detection limit at 24 h and the blood dm-CLO concentrations were below

561

the detection limit at 12 and 24 h after the administration. The concentrations of dm-CLO in the milk

562

were significantly higher than those of dm-CLO in the blood at 1, 3, 6, and 9 h after the administration.

563

The values are expressed as mean ± SE. The numbers of samples were as follows: 1 h (milk, blood = 9),

564

3 h (milk, blood = 9), 6 h (milk = 5, blood = 6), 9 h (milk = 7, blood = 9), 12 h (milk = 5, blood = 6),

565

and 24 h (milk = 5, blood = 12); *P < 0.05, **P < 0.01.

566

18

567

Fig. 4 Ratio of CLO and dm-CLO concentrations in the breast milk to those in the blood

568

(A) The distribution of CLO concentrations in the milk relative to CLO concentrations in the blood

569

showed that CLO concentrations in the milk were higher. The numbers of samples were as follows: 1 h

570

= 9, 3 h = 9, 6 h = 4, and 9 h =4.

571

(B) The distribution of dm-CLO concentrations in the milk relative to dm-CLO concentrations in the

572

blood showed that dm-CLO concentrations in the milk were higher. The numbers of samples were as

573

follows: 1 h = 9, 3 h = 9, 6 h = 5, and 9 h = 7.

574

575

Fig. S1 Changes in CLO and dm-CLO concentrations in the breast milk and blood

576

In the milk, the highest CLO concentration (2,663 ± 191 ng/mL) was observed at 1 h after the

577

administration, and the highest dm-CLO concentration (1,084 ± 126 ng/mL) was observed at 3 h after

578

the administration. In the blood, the CLO concentration was highest at 1 h after the administration (1,656

579

± 225 ng/mL), and the dm-CLO concentration was highest at 1 h after the administration (397 ± 73

580

581

ng/mL). The values are expressed as mean ± SE.

19

Fig. 1

1h 3h

6h

9h

: Administration

: Milk and blood collection

12 h

24 h

Fig. 2

A)

pressure

adjustment pipe

teats

10 µL tip

(connecting part)

silicon tube

B)

10 µL tip

aspirator

exhaust

pipe

connecting part

sampling tube

10 µL tip

(straight part)

straight part

Fig. 3

A)

B)

＊＊

3,000

1,400

2,500

1,200

dm-CLO (ng/mL)

CLO (ng/mL)

＊＊

2,000

1,500

1,000

500

1h

3h

6h

9h

12h

12

24h

24

blood

(h)

＊＊

＊＊

1h

3h

6h

9h

1,000

800

600

400

200

milk

12h

12

24h

24

(h)

Fig. 4

A)

B)

CLO

4,000

dm-CLO

4,000

3,500

3,500

3,000

3,000

2,500

2,500

milk (ng/mL)

milk (ng/mL)

y=x

2,000

1,500

2,000

1,500

1,000

1,000

500

500

500

1,000

1,500

2,000

2,500

3,000

3,500

y=x

4,000

500

3h

1,500

2,000

2,500

3,000

blood (ng/mL)

blood (ng/mL)

1h

1,000

6h

9h

12 h

24 h

3,500

4,000

Fig. S1

concentration (ng/mL)

3,000

milk: CLO

2,500

milk: dm-CLO

2,000

blood: CLO

1,500

blood: dm-CLO

1,000

500

1h

3h

6h

9h

12h

12

24h

24

(h)

Table 1 CLO concentration in the maternal milk and blood

dam

1h

3h

6h

9h

12 h

24 h

3428.8

1678.8

1519.6

492.7

74.5

< LOD

2299.7

1318.6

2546.1

1095.8

23.2

< LOD

3249.2

1471.2

532.9

2924.7

861.2

192.7

1474.5

1322.7

977

668.9

1032.4

1431.3

326.3

< LOD

2974.6

1336

1769.8

676.4

166.2

19.2

< LOD

2170.8

1195.6

1014

222.5

< LOD

< LOD

2409.9

1344.9

82.5

103.9

< LOD

1640.5

89.6

9.9

< LOD

< LOD

< LOD

3302.3

518

44.9

25.5

1722.2

360.5

9.3

< LOD

< LOD

< LOD

2659.3

3022.2

736.6

545.3

< LOD

10

28.9

< LOD

< LOD

38.4

< LOD

< LOD

11

10.3

< LOD

27.2

< LOD

< LOD

12

18

< LOD

< LOD

32.3

< LOD

< LOD

13

7.6

< LOD

< LOD

26.8

< LOD

< LOD

－ : Not examined or samples not taken;

LOD: Limit of detection;

Upper cell: Breast milk; Lower cell: Maternal blood (ng/mL).

Table 2 dm-CLO concentration in the maternal milk and blood

dam

1h

3h

6h

9h

12 h

24 h

935.2

975.6

243.1

282.7

253.3

< LOD

546.3

1144.5

638.6

678.3

55.7

< LOD

790.9

1133.5

590.6

768.6

646.2

388.1

143.7

473.6

568.5

158.5

520.6

216.4

< LOD

744.5

925.7

602.5

62.1

368.6

376.7

179.4

54

< LOD

580

1370.7

230.1

219.8

29.3

< LOD

940.2

1860.9

334

155.9

< LOD

< LOD

521.3

122

101.8

47.6

< LOD

< LOD

1089.8

672.2

227.2

148.4

535.7

324

80.5

44.2

< LOD

< LOD

443

1196.6

< LOD

104.6

217.2

< LOD

10

187.8

49.7

< LOD

117.6

< LOD

< LOD

11

173

48

74.8

< LOD

< LOD

12

218.7

40.3

< LOD

81.5

< LOD

< LOD

13

96.6

22.2

< LOD

62.6

< LOD

< LOD

－ : Not examined or samples not taken;

LOD: Limit of detection;

Upper cell: Breast milk; Lower cell: Maternal blood (ng/mL).

Table S1 CLO ratio of the maternal milk vs. blood

dam

1h

3h

6h

9h

12 h

24 h

2.3

3.4

0.9

1.2

1.1

1.7

2.8

1.4

0.9

3.0

1.7

2.0

2.1

5.4

1.5

15.0

8.3

1.9

1.4

4.8

3.6

5.5

10

0.8

11

0.4

12

0.6

13

0.3

－ : Not examined or samples not taken

Table S2. dm-CLO ratio of the maternal milk vs. blood

dam

1h

3h

6h

9h

12 h

24 h

3.8

3.5

0.9

1.7

1.0

1.8

1.5

0.9

0.9

2.6

2.0

2.5

3.4

1.2

2.5

6.2

1.8

15.3

3.3

3.3

2.0

2.1

2.8

3.4

4.2

5.5

10

1.6

11

2.3

12

2.7

13

1.5

－ : Not examined or samples not taken

Table S3 CLO concentration in the maternal milk and blood after administration of NOAEL doses of CLO

dam

CLO-1

CLO-2

CLO-3

CLO-4

CLO-5

Ctrl-1

Ctrl-2

Ctrl-3

Ctrl-4

Ctrl-5

1h

6h

31919.6

5517.0

151.6

2069.7

10922.3

< LOD

< LOD

35045.3

1247.3

< LOD

< LOD

9353.6

507.3

< LOD

< LOD

44963.2

2644.0

< LOD

< LOD

13900.1

700.5

< LOD

< LOD

147255.7

26980.2

2969.8

< LOD

9054.8

7439.3

753.2

< LOD

49791.8

4493.3

59.7

< LOD

14700.3

1562.3

31.5

< LOD

< LOD

< LOD

< LOD

< LOD

< LOD

< LOD

< LOD

< LOD

< LOD

< LOD

< LOD

< LOD

< LOD

< LOD

< LOD

< LOD

< LOD

< LOD

< LOD

< LOD

< LOD

< LOD

< LOD

< LOD

< LOD

< LOD

< LOD

< LOD

< LOD

< LOD

－ : Not examined or samples not taken;

12 h

24 h

< LOD

< LOD

LOD: Limit of detection;

Upper cell: Breast milk; Lower cell: Maternal blood (ng/mL).

Table S4 dm-CLO concentration in the maternal milk and blood after administration of NOAEL doses of CLO

dam

CLO-1

CLO-2

CLO-3

CLO-4

CLO-5

Ctrl-1

Ctrl-2

Ctrl-3

Ctrl-4

Ctrl-5

1h

6h

12 h

24 h

20273.8

13545.0

4267.6

1487.7

5956.0

855.7

< LOD

35611.8

3857.2

605.1

9736.3

1989.7

< LOD

< LOD

30322.4

16262.8

1608.0

< LOD

10954.7

5136.3

761.0

< LOD

157056.6

37224.2

10363.5

186.8

7731.8

10767.5

2869.4

< LOD

44303.9

15088.1

3007.8

< LOD

13115.4

< LOD

934.2

< LOD

< LOD

< LOD

< LOD

< LOD

< LOD

< LOD

< LOD

< LOD

< LOD

< LOD

< LOD

< LOD

< LOD

< LOD

< LOD

< LOD

< LOD

< LOD

< LOD

< LOD

< LOD

< LOD

< LOD

< LOD

< LOD

< LOD

< LOD

< LOD

< LOD

< LOD

－ : Not examined or samples not taken;

< LOD

< LOD

< LOD

LOD: Limit of detection;

Upper cell: Breast milk; Lower cell: Maternal blood (ng/mL).

...