# ELECTRONIC SUPPLEMENTARY MATERIAL

to

Endocrine effects of hexabromocyclododecane (HBCD) in a one

- generation reproduction study in Wistar rats

Leo TM van der Ven, Ton van de Kuil, Wout Slob, Pim EG Leonards, Hellmuth Lilienthal, Theo J Visser, Rocío F Can tón, Mar t in van den Berg,

Sabina Litens, Helen Håkansson, Henk van Loveren, Josephus G Vos, Aldert H Piersma

Table 1

– P food intake

................................ ................................ ................................ ................................ ................................ ................................ .. 2

Table 1

– P food intake (continued)

................................ ................................ ................................ ................................ ................................ ............... 3

Table 2

– P body weights

................................ ................................ ................................ ................................ ................................ ............................... 4

Table 2

– P body weights (continued)

................................ ................................ ................................ ................................ ................................ ............ 5

Table 3

– Reproduction

................................ ................................ ................................ ................................ ................................ ................................ .. 6

Table 4

– Sexual development, F1 animals

................................ ................................ ................................ ................................ ................................ .... 7

Tabl e 5

– Body weights during juvenile growth (g), F1 animals

................................ ................................ ................................ ................................ ... 8

Table 6

– F1 Food intake during juvenile growth

................................ ................................ ................................ ................................ .......................... 9

Table 7

– Ca uda epididymis sperm characteristics at necropsy of F1 males

................................ ................................ ................................ ............... 10

Table 8

– Body and organ weights at necropsy (grams) , F1 animals

................................ ................................ ................................ ........................... 11

Table 9

– Clinical chemistry of blood plasma after HBCD in F1 animals

................................ ................................ ................................ ................... 12

Table 10

– Endocrine parameters after HBCD (P, F1)

................................ ................................ ................................ ................................ .................. 13

Table 11

– Apolar Liver Retinoids

................................ ................................ ................................ ................................ ................................ ................. 14

Table 12

– Bone parameters (tibia)

................................ ................................ ................................ ................................ ................................ ................ 15

Table 13

– Antibody titers after immunization against sheep red blood cells, male F1 animals

................................ ................................ ................... 16

Table 14

– Spleen immune cells

- FACS analysis of subpopulations and NK activity in male F1rats after HBCD

................................ ..................... 17

Table 15

– White blood cells in blood and bone marrow of F1 male rats after HBCD

................................ ................................ ................................ . 18

Table 16

– Red blood cell parameters in blood of F1 male rats after HBCD

................................ ................................ ................................ ................. 19

# Endocrine effects of hexabromocyclododecane (HBCD) in a one

- generation reproduction study in Wistar rats

Leo TM van der Ven, Ton va n de Kuil, Wout Slob, Pim EG Leonards, Hellmuth Lilienthal, Theo J Visser, Rocío F Cantón, Martin van den Berg, Sabina Litens , Helen Håkansson, Henk van Loveren, Josephus G Vos, Aldert H Piersma



## Supplementary Tables



# Table 1

– P food intake

female rats

premating continued on next page

HBCD dose

mg/kg bw w1 w2 w3 w4 w5 w6 w7 w8 HBCD

dose

mg/kg bw w9 w 10

standard na na na na na na na 127.2 ± 16.3 standard 126.6 ± 11.4 108. 7 ± 7.7

0 114.1 ± 5.6 119.7 ± 11.3 123.1 ± 14.2 110.5 ± 16.6 99.3 ± 14.2 108.9 ± 9.7 120.8 ± 46.1 106.7 ± 10.4 0 127.0 ± 12.2 95.0 ± 8.7

0 124.8 ± 11.6 128.0 ± 9.2 136.9 ± 15.4 123.6 ± 23.5 110.7 ± 11.8 116.2 ± 11.8 118.0 ± 8.1 120.4 ± 9.4 0.1 142. 8 ± 13.7 104.9 ± 7.3

0 127.2 ± 14.7 133.3 ± 11.8 140.9 ± 12.7 131.3 ± 29.7 113.1 ± 10.8 129.8 ± 12.8 121.8 ± 16.0 121.2 ± 9.9 0.3 121.2 ± 9.9 114.0 ± 21.5

0 115.7 ± 15.2 119.9 ± 9.0 119.5 ± 34.4 117.7 ± 18.9 104.9 ± 9.6 113.3 ± 6.1 115.7 ± 15.2 110 .5 ± 7.0 1 126.4 ± 8.5 95.9 ± 10.8

0 118.2 ± 7.3 129.8 ± 42.6 128.6 ± 10.8 116.8 ± 22.2 102.4 ± 9.3 113.7 ± 7.2 111.1 ± 6.1 108.7 ± 7.0 3 121.9 ± 11.0 99.6 ± 16.4

0 118.0 ± 9.6 120.4 ± 10.9 129.7 ± 15.8 117.2 ± 16.2 107.0 ± 12.5 117.0 ± 9.2 115.5 ± 7.7 112.8 ± 8.8 10 138.1 ± 17.0 105.7 ± 10.7

0 123.4 ± 14.4 128.1 ± 12.9 140.0 ± 13.7 129.3 ± 33.5 108.7 ± 9.0 118.1 ± 9.0 116.6 ± 9.6 116.3 ± 9.4 30 136.7 ± 22.8 108.0 ± 10.0

0 116.7 ± 6.5 118.8 ± 7.7 126.6 ± 14.2 128.1 ± 38.7 104.6 ± 12.4 113.6 ± 10.6 114.1 ± 5.7 107.4 ± 8.1 100 137.6 ± 13.2 107.6 ± 9.2



### dose response

- - - - a

- a a

- -



## male rats

standard na na na na na na na 149.6 ± 16.7 158.3 ± 11.0 137.0 ± 14.1

0 167.1 ± 26.0 179.0 ± 16.3 186.3 ± 16.0 172.9 ± 38.5 153.9 ± 15.0 1 59.8 ± 13.8 166.9 ± 33.6 159.0 ± 14.1 163.2 ± 17.6 137.3 ± 15.1

0.1 162.9 ± 25.3 171.6 ± 13.2 181.8 ± 17.3 172.4 ± 26.6 154.8 ± 20.4 164.6 ± 6.2 156.5 ± 5.1 158.3 ± 9.0 165.6 ± 14.2 133.4 ± 9.2

0.3 164.9 ± 30.9 178.3 ± 13.3 186.9 ± 16.5 176.5 ± 3 3.2 163.5 ± 17.8 162.1 ± 10.5 149.1 ± 28.6 160.6 ± 12.4 166.5 ± 9.8 138.9 ± 5.9

1 155.4 ± 23.7 173.3 ± 14.5 178.0 ± 19.8 163.1 ± 28.1 144.0 ± 19.5 154.3 ± 14.3 149.4 ± 16.3 154.4 ± 13.4 156.0 ± 13.6 132.2 ± 13.3

3 167.7 ± 29.9 175.8 ± 15.5 184.6 ± 28.7 175.4 ± 26.4 146.3 ± 10.6 157.2 ± 21.1 154.4 ± 12.4 154.7 ± 11.2 154.4 ± 13.6 135.4 ± 13.4

10 170.3 ± 23.1 177.2 ± 13.3 186.1 ± 19.0 188.2 ± 35.2 154.7 ± 19.8 165.5 ± 14.9 155.3 ± 14.8 169.6 ± 9.0 172.8 ± 11.3 141.0 ± 15.9

30 175.7 ± 24.6 187.8 ± 18.2 191.4 ± 21.0 191.8 ± 45.3 165.2 ± 18.9 172.2 ± 24.5 170.8 ± 30.6 163.7 ± 20.2 172.3 ± 23.7 140.8 ± 22.9

100 159.8 ± 25.6 170.5 ± 11.6 175.4 ± 18.8 167.7 ± 32.0 146.2 ± 18.6 154.6 ± 7.3 155.6 ± 14.7 155.3 ± 18.4 163.0 ± 13.1 133.9 ± 11.3



### dose response

- - - a a a

- a a a

Figures are average ± standard deviation in grams of at 10 animals per dose group in the premating period, and 8

- 10 for females during gestation)

, and represent absolute weekly intake in grams

. Standard, standard fat diet, included as a reference for the effect of high oil in the exposure diets. +, Significant dose response (for results see Table 1 in the text );

- , no significant dose response; a

, absence of effect concluded from average values, dose

- response analy sis not performed. * P<0.0001 for difference between control high oil and control standard fat diet.

# Endocrine effects of hexabromocyclododecane (HBCD) in a one

- generation reproduction study in Wistar rats

Leo TM van der Ven, Ton va n de Kuil, Wout Slob, Pim EG Leonards, Hellmuth Lilienthal, Theo J Visser, Rocío F Cantón, Martin van den Berg, Sabina Litens , Helen Håkansson, Henk van Loveren, Josephus G Vos, Aldert H Piersma



## Supplementary Tables



# Table 1

– P food intake (continued)

female rats

gestation lactation

HBCD dose

mg/kg bw w1 w2 w 3 w1 w2 w 3

standard 142.7 ± 15.3* 158.5 ± 17.4* 16 8.3 ± 19.1* 520.7 ± 48.4 550.1 ± 69.5 *

0 109.0 ± 16.2* 128.4 ± 13.4* 138.6 ± 12.9* 474.8 ± 62.9 448.2 ± 40.1 *

0.1 127.4 ± 12.7 135.1 ± 9.7 144.8 ± 22.7 513.8 ± 53.2 507.8 ± 82.1

0.3 133.7 ± 21.9 145.0 ± 19.2 147.8 ± 29.7 507 .0 ± 78.3 469.1 ± 7.1

1 112.3 ± 9.3 128.0 ± 13.1 141.7 ± 20.5 447.5 ± 119.1 367.8 ± 114.2

3 119.1 ± 12.8 131.8 ± 14.4 142.2 ± 28.0 479.1 ± 57.6 463.7 ± 46.1

10 129.0 ± 12.5 140.1 ± 12.8 144.4 ± 26.9 438.9 ± 84.4 463.4 ± 77.8

30 129.8 ± 14.3 133.3 ± 17.1 148.1 ± 30.0 517 .0 ± 94.8 469.9 ± 11.5

100 124.0 ± 24.6 137.1 ± 48.4 120.6 ± 36.6 429 .0 ± 77.9 508.5 ± 22.7



### dose

response - a + a a

Figures are average ± standard deviation in grams of at 10 animals per dose group in the

premating period, 8

- 10 for females during gestati on , and 4

- 8 during lactation )

, and represent

absolute weekly intake in grams

. Data for the first week of lactation are not available. Standard,

standard fat diet, included as a reference for the effect of high oil in the exposure diets. +,

Significant dos e response (for results see Table 1 in the text );

- , no significant dose response; a

, absence of effect concluded from average values, dose

- response analysis not performed.

* P<0. 01 for difference between control high oil and control standard fat diet.

# Endocrine effects of hexabromocyclododecane (HBCD) in a one

- generation reproduction study in Wistar rats

Leo TM van der Ven, Ton va n de Kuil, Wout Slob, Pim EG Leonards, Hellmuth Lilienthal, Theo J Visser, Rocío F Cantón, Martin van den Berg, Sabina Litens , Helen Håkansson, Henk van Loveren, Josephus G Vos, Aldert H Piersma



## Supplementary Tables



# Ta ble 2

– P body weights

f emale rats

premating continued on next page

HBCD dose

mg/kg bw Wk0 w1 w2 w3 w4 w5 w6 w7 w8 HBCD dose

mg/kg bw w9 w10

standard na na na na na na 266

. 0 ± 15

. 5 266

. 5 ± 17

. 9 274

. 3 ± 22

. 1 standard 280

. 4 ± 18

. 8 287

. 0 ± 21

. 4

0 218.4 ± 22.4 230.2 ± 19.5 241.3 ± 23.0 248.7 ± 22.4 253.5 ± 21.3 258.1 ± 24.2 260.9 ± 24.6 262.9 ± 24.4 267.4 ± 25.1 0 271.4 ± 24.2 274.0 ± 26.2

0 223.9 ± 15.6 237.6 ± 15.5 250.5 ± 19.1 258.9 ± 21.2 263.9 ± 20.7 269.3 ± 23.3 271.8 ± 23.0 274.2 ± 20 .8 278.7 ± 22.0 0.1 285.5 ± 23.5 286.9 ± 26.9

0 225.8 ± 24.2 240.8 ± 27.6 251.8 ± 29.3 262.8 ± 31.4 271.4 ± 33.9 277.8 ± 31.6 282.7 ± 35.1 284.5 ± 37.6 287.8 ± 37.7 0.3 294.0 ± 37.3 298.6 ± 38.4

0 215.0 ± 26.7 227.1 ± 23.7 239.1 ± 26.6 247.4 ± 26.8 254.9 ± 23.6 259.8 ± 24.5 264.1 ± 23.6 266.8 ± 23.3 270.0 ± 22.7 1 274.7 ± 22.6 277.5 ± 24.5

0 214.4 ± 12.8 225.3 ± 13.5 238.0 ± 10.6 242.8 ± 19.0 246.9 ± 17.8 253.3 ± 17.2 258.5 ± 17.7 259.1 ± 14.0 262.7 ± 17.7 3 269.8 ± 17.8 269.7 ± 19.0

0 214.6 ± 29.3 224.6 ± 26.5 237.4 ± 29.6 247.1 ± 28.6 255.0 ± 26.3 259.9 ± 28.9 263.5 ± 27.6 268.0 ± 27.2 270.6 ± 27.8 10 278.4 ± 27.5 284.0 ± 29.0

0 231.1 ± 19.0 243.3 ± 21.7 254.8 ± 23.4 265.5 ± 23.7 272.2 ± 25.3 276.3 ± 26.3 281.0 ± 26.7 282.9 ± 27.2 287.1 ± 27.6 30 296.9 ± 31.1 299.7 ± 33.2

0 219.2 ± 11.2 228.1 ± 12.0 239.3 ± 11.4 246.5 ± 12.9 252.9 ± 10.2 259.1 ± 11.5 259.7 ± 11.8 266.0 ± 11.1 265.6 ± 12.3 100 274.7 ± 13.0 280.0 ± 13.5

dose response 

### a a a a a a a a a a a



## male rats

standard n a na na na na na 467.6 ± 21.1 467.2 ± 27.9 478.3 ± 28.9 standard 483.3 ± 25.7 489.5 ± 28.5

0 344.1 ± 45.2 377.4 ± 36.5 395.6 ± 37.4 417.0 ± 42.9 430.7 ± 45.4 442.2 ± 44.5 451.0 ± 47.5 461.3 ± 49.1 473.6 ± 49.9 0 481.8 ± 52.8 489.0 ± 51.5

0.1 343 .9 ± 40.4 376.1 ± 27.8 393.6 ± 26.9 413.3 ± 26.9 428.6 ± 27.0 438.7 ± 25.3 447.2 ± 24.1 455.9 ± 24.1 468.2 ± 25.2 0.1 475.0 ± 26.2 481.3 ± 25.5

0.3 321.5 ± 17.5 344.5 ± 21.3 365.6 ± 24.2 387.8 ± 28.9 404.3 ± 33.2 416.8 ± 32.3 427.3 ± 34.1 438.1 ± 37.3 445.2 ± 39.0 0.3 456.3 ± 38.9 467.7 ± 44.2

1 333.8 ± 25.2 353.5 ± 24.3 372.5 ± 25.1 389.7 ± 23.9 401.3 ± 21.9 410.0 ± 24.8 418.9 ± 23.7 427.7 ± 25.0 440.3 ± 25.0 1 447.7 ± 24.1 452.6 ± 25.0

3 344.3 ± 41.0 371.8 ± 43.7 390.6 ± 46.1 408.1 ± 49.9 421. 0 ± 51.2 430.8 ± 53.7 433.9 ± 52.3 444.0 ± 57.8 454.4 ± 57.0 3 461.7 ± 59.5 470.7 ± 62.2

10 343.3 ± 32.8 375.1 ± 26.5 393.3 ± 25.7 412.4 ± 26.7 426.6 ± 25.4 439.5 ± 23.8 447.4 ± 24.2 457.2 ± 24.3 468.7 ± 22.7 10 476.8 ± 22.6 484.7 ± 23.2

30 353.7 ± 42.7 376.8 ± 43.3 396.7 ± 43.5 419.0 ± 45.1 438.4 ± 55.5 452.0 ± 56.0 462.7 ± 62.5 473.4 ± 64.8 486.4 ± 69.8 30 497.0 ± 70.9 503.8 ± 76.7

100 318.2 ± 26.0 340.4 ± 25.9 359.1 ± 27.0 376.3 ± 27.9 390.8 ± 27.3 401.4 ± 28.0 409.0 ± 29.2 417.4 ± 28.9 428.7 ± 28.1 100 435.9 ± 27.8 441.9 ± 28.7

dose response 

### a + + +

- - - - - - -

Figures are average ± standard deviation in grams of at 10 animals per dose group in the premating period, and 8

- 10 for females during gestation). Standard, standard fat die t, included as a reference for the effect

of high oil in the exposure diets. +, Significant dose response (for results see Table 1 in the text

- , no significant dose response; a, absence of effect concluded from average values, dose

- response analysis not

p erformed. * P<0.05 for difference between control high oil and control standard fat diet.

# Endocrine effects of hexabromocyclododecane (HBCD) in a one

- generation reproduction study in Wistar rats

Leo TM van der Ven, Ton va n de Kuil, Wout Slob, Pim EG Leonards, Hellmuth Lilienthal, Theo J Visser, Rocío F Cantón, Martin van den Berg, Sabina Litens , Helen Håkansson, Henk van Loveren, Josephus G Vos, Aldert H Piersma



## Supplementary Tables



# Table 2

– P body weights (continued)

female rats

gestation lactation

HBCD dose

mg/kg bw w1 w2 w3 w1 w2 w 3

standard 311

. 2 ± 20

. 6 * 335

. 3 ± 23

. 7 * 402

. 2 ± 4 2

. 5 * 336.4 ± 28.1* 340.0 ± 18.0* 331.4 ± 22.0

0 286.7 ± 28.1 * 306.0 ± 32.4 * 357.0 ± 46.1 * 301.8 ± 27.9* 307.6 ± 37.2* 306.6 ± 28.8

0.1 309.9 ± 21.3 333.7 ± 20.3 394.0 ± 33.3 303.1 ± 11.7 313.3 ± 16.5 309.7 ± 20.0

0.3 319.6 ± 43.8 335.8 ± 42.0 369.6 ± 71.0 314.8 ± 28.6 327.5 ± 29.9 312.3 ± 19.5

1 294.6 ± 24.6 314.3 ± 26.3 359.6 ± 50.0 304.7 ± 29.9 316.2 ± 31.7 311.2 ± 31.6

3 288.3 ± 19.9 310.3 ± 23.6 359.6 ± 50.0 298.9 ± 26.2 296.8 ± 26.7 298.0 ± 17.5

10 309.6 ± 26.2 329.6 ± 35.2 379.0 ± 54.2 313.5 ± 38.3 313.6 ± 39.9 310.4 ± 38.0

30 320.5 ± 29.8 329.6 ± 35.2 379.4 ± 65.1 340.0 ± 8.2 317.8 ± 12.5 321.8 ± 16.1

100 300.9 ± 7.5 316.9 ± 2.6 370.3 ± 32.6 297.7 ± 11.9 297.3 ± 11.3 292.2 ± 11.3



### dose

response a a a a a a

Figures are average ± standard d eviation in grams of at 10 animals per dose group in the premating

period, 8

- 10 for females during gestation , and 4

- 8 during lactation )

, and represent absolute weekly

intake in grams . Standard, standard fat diet, included as a reference for the effect of h igh oil in the

exposure diets. a

, A bsence of effect concluded from average values, dose

- response analysis not

performed. * P<0.0 5 for difference between control high oil and control standard fat diet.

# Endocrine effects of hexabromocyclododecane (HBCD) in a one

- generation reproduction study in Wistar rats

Leo TM van der Ven, Ton va n de Kuil, Wout Slob, Pim EG Leonards, Hellmuth Lilienthal, Theo J Visser, Rocío F Cantón, Martin van den Berg, Sabina Litens , Helen Håkansson, Henk van Loveren, Josephus G Vos, Aldert H Piersma



## Supplementary Tables



# Table 3

– R eproduction

HBCD dose

mg/kg bw n s uccessful

matings

% time to

gestation

days gestation

days n implantations

count n litter size

count sex ratio at

pnd4

f/m sex ratio at

pnd21

f/m rate of litters with

mortality

% n mortality rate during lactation

%

m f m+f

standard 10 90 2.2 ± 0.9 22.2 ± 0.4 9 14.0 ± 2.2 9 12.4 ± 2.4 0.8 ± 0.3 0.8 ± 0.2 11 11 2 1.6 2.1 2.0 *

sum HBCD groups 19.3 14.9 17.1

0 10 80 3.9 ± 1.0 22.0 ± 0.0 1 16.0 8 11.9 ± 2.5 1.0 ± 0.7 1.1 ± 0.6 75 95 28.8 14.0 20 .3 *

0.1 10 80 3.0 ± 2.0 21.9 ± 0.4 8 14.8 ± 1.8 8 12.8 ± 1.6 1.0 ± 0.4 1.0 ± 0.6 50 102 15.1 16.3 16.6

0.3 10 40 2.8 ± 1.6 22.0 ± 0.0 4 17.0 ± 4.2 4 13.3 ± 2.6 0.8 ± 0.7 1.0 ± 0.6 75 53 21.9 0.0 11.8

1 10 70 2.1 ± 1.5 22.7 ± 0.5 6 13.0 ± 3.3 6 8.2 ± 3.4 1.8 ± 1.3 1.5 ± 1.4 50 47 35.0 37.0 35.8

3 10 80 2.5 ± 1.6 21.9 ± 0.4 8 13.3 ± 1.8 8 12.1 ± 2.6 0.8 ± 0.4 1.1 ± 0.8 75 96 24.5 18.6 26.0

10 8 75 5.0 ± 2.1 22.5 ± 0.8 8 11.5 ± 1.6 8 9.9 ± 3.4 1.7 ± 1.4 1.9 ± 1.4 38 77 14.7 7.0 12.8

30 10 60 2.4 ± 1.7 22.5 ± 0.8 6 11.8 ± 2.8 5 9.4 ± 5 .7 1.1 ± 0.6 1.5 ± 1.1 50 46 4.2 13.6 10.9

100 10 60 3.9 ± 2.4 22.3 ± 0.8 1 14.0 6 13.3 ± 0.5 1.6 ± 1.0 1.0 ± 0.4 67 80 6.1 12.8 9.7

dose response a a a a a

- a

- a a

-

Successful matings is the rate of matings that resulted in offspring. The range of sex ratios (f/m) at pnd4 was 0.4

- 2.5 in control litters and 1.0

- 3.7 in top dose litters

. * P<0.05 for difference between high

oil control diet and standard fat diet. There seems to be a difference also in the rate of litters with mortality, but this w as not tested because these figures represent single observations. Standard,

standard fat diet, included as a reference for the effect of high oil in the exposure diets.

- , N o significant dose response; a , absence of effect concluded from average values, dos e

- response analysis not

performed.

# Endocrine effects of hexabromocyclododecane (HBCD) in a one

- generation reproduction study in Wistar rats

Leo TM van der Ven, Ton va n de Kuil, Wout Slob, Pim EG Leonards, Hellmuth Lilienthal, Theo J Visser, Rocío F Cantón, Martin van den Berg, Sabina Litens , Helen Håkansson, Henk van Loveren, Josephus G Vos, Aldert H Piersma



## Supplementary Tables



# Table 4

– S exual development, F1 animals

female rats

anogenital distance (mm) day vagina open weight of

reproductive organs c 

### ( mg)

HBCD dose

mg/kg bw d4 d7 d21 d21

standard 3.7 ± 0.8 5.4 ± 1.2 12

. 5 ± 4

. 7 35.3 ± 2.0 46

. 2 ± 17 .4

0 3

. 5 ± 0

. 6 4

. 7 ± 1

. 0 13

. 4 ± 6

. 7 35

. 4 ± 2

. 3 55

. 4 ± 19

. 5

0.1 3

. 7 ± 0

. 8 5

. 2 ± 1

. 1 12

. 7 ± 3

. 3 35

. 3 ± 2

. 2 45

. 2 ± 8

. 1

0.3 3

. 4 ± 0

. 7 4

. 5 ± 1

. 0 9

. 1 ± 2

. 2 36

. 2 ± 2

. 4 46

. 3 ± 8

. 7

1 3

. 5 ± 0

. 5 5

. 1 ± 0

. 9 13

. 4 ± 0

. 8 36

. 8 ± 4

. 1 47

. 5 ± 0

. 7

3 3

. 5 ± 0

. 6 4

. 5 ± 1

. 0 10

. 8 ± 1

. 8 36

. 8 ± 3

. 3 40

. 7 ± 19

. 4

10 3

. 7 ± 0

. 6 4

. 8 ± 0

. 8 14

. 0 ± 4

. 0 35

. 4 ± 2

. 7 46

. 7 ± 8

. 0

30 3

. 5 ± 0

. 6 4

. 9 ± 0

. 8 12

. 9 ± 5

. 9 34

. 8 ± 1

. 6 43.8 ± 6.7

100 3

. 6 ± 0

. 9 4

. 8 ± 1

. 0 10

. 8 ± 2

. 0 39

. 9 ± 2

. 6 43.8 ± 13.4



### dose response a a a +

-



## male rats

day preputial separation

stage I stage II d21

standard 5.2 ± 1.2 6.9 ± 1.4 19

. 5 ± 4

. 8 39.5 ± 2.0 42.3 ± 2.5 323.5 ± 102.4

0 4

. 6 ± 0

. 8 6

. 2 ± 1

. 2 19

. 0 ± 6

. 0 39

. 3 ± 0

. 5 43

. 6 ± 1

. 2 284

. 3 ± 42

. 0

0.1 5

. 1 ± 1

. 1 6

. 7 ± 1

. 2 19

. 1 ± 4

. 1 39

. 0 ± 0

. 6 42

. 6 ± 1

. 5 298

. 0 ± 51

. 0

0.3 4

. 7 ± 0

. 8 5

. 5 ± 1

. 1 14

. 8 ± 2

. 6 40

. 3 ± 0

. 6 45

. 7 ± 1

. 5 271

. 1 ± 60

. 2

1 4

. 8 ± 1

. 0 6

. 4 ± 1

. 4 na 39

. 0 b 42

. 0 b na

3 5

. 0 ± 0

. 8 6

. 1 ± 1

. 3 18

. 7 ± 2

. 9 39

. 9 ± 0

. 9 43

. 9 ± 2

. 0 318

. 9 ± 128

. 2

10 5

. 0 ± 0

. 9 6

. 0 ± 1

. 3 18

. 3 ± 5

. 5 38

. 4 ± 0

. 8 44

. 0 ± 1

. 1 295

. 8 ± 62

. 9

30 4

. 5 ± 0

. 8 6

. 6 ± 1

. 0 18

. 9 ± 6

. 1 40

. 0 ± 0

. 0 45

. 0 ± 0

. 0 275

. 9 ± 43

. 2

100 5

. 4 ± 1

. 0 6

. 3 ± 1

. 2 16

. 0 ± 2

. 2 40

. 4 ± 1

. 1 45

. 1 ± 1

. 6 254

. 3 ± 51

. 4



### dose response + a a a a a

Figures are average ± st andard deviation of animals per dose group for anogenital distance, for day vagina open / preputial separation, and

animals per dose group for weight of reproductive organs. Standard, standard fat diet, included as a reference for the effect of high oil in

the exposure diets. +, Significant dose response (for results see Table 1 in the text );

- , no significant dose response; a , absence of effect

concluded from average values, dose

- response analysis not performed

. b 

### n =1 c 

### Reproductive organs includes testes and epididymis

combined in male pups, and uterus and ovaries combined in female pups.

# Endocrine effects of hexabromocyclododecane (HBCD) in a one

- generation reproduction study in Wistar rats

Leo TM van der Ven, Ton va n de Kuil, Wout Slob, Pim EG Leonards, Hellmuth Lilienthal, Theo J Visser, Rocío F Cantón, Martin van den Berg, Sabina Litens , Helen Håkansson, Henk van Loveren, Josephus G Vos, Aldert H Piersma



## Supplementary Tables



# Table 5

– B ody weights during juvenile growth (g) , F1 animals

female rats

during lactation after weaning

HBCD dose

mg/kg bw d4 d7 d14 d21 growth

d4

- 21 w4 w5 w6 w7 w8 w9 w10 w11 growth

w4

- 11

standard 11.8 ± 1.5** 16.3 ± 2.4** 28.3 ± 5.2** 46.9 ± 7.8** 35.2 ± 6.4 114.4 ± 12.0 139.4 ± 10.5 165.2 ± 10.4 184.4 ± 8.7 202.6 ± 6.8 212.4 ± 7.0 223.8 ± 7.9 228.5 ± 9.3 113.3 ± 11.4

0 9.5 ± 1.5** 1 2.9 ± 2.6** 23.6 ± 5.3** 40.3 ± 8.6** 30.6 ± 7.6 98.2 ± 21.8 125.0 ± 24.8 150.6 ± 26.7 170.0 ± 26.1 190.8 ± 28.1 205.4 ± 25.8 214.2 ± 29.0 222.4 ± 28.2 124.2 ± 20.5

0.1 9.7 ± 0.8 12.8 ± 1.4 23.1 ± 2.7 40.1 ± 5.9 30.3 ± 5.7 110.2 ± 18.0 136.6 ± 16.0 156.2 ± 18.9 177.4 ± 15.6 193.8 ± 16.8 205.6 ± 15.5 215.6 ± 12.5 221.4 ± 16.1 111.2 ± 7.7

0.3 9.4 ± 1.1 12.4 ± 2.1 21.0 ± 3.8 34.1 ± 5.4 24.7 ± 4.5 96.8 ± 18.8 124.8 ± 20.7 146.6 ± 22.1 164.8 ± 21.1 183.2 ± 25.1 196.4 ± 25.0 206.8 ± 28.4 211.4 ± 28.0 114.6 ± 19 .3

1 10.6 ± 2.7 14.2 ± 5.1 31.1 ± 7.9 50.4 ± 11.9 38.8 ± 10.2 130.4 ± 24.2 159.2 ± 19.9 180.6 ± 21.4 199.0 ± 21.4 213.6 ± 24.6 220.8 ± 17.3 236.2 ± 20.4 241.2 ± 20.7 110.8 ± 18.9

3 9.4 ± 1.5 12.5 ± 2.7 22.4 ± 6.0 37.0 ± 10.3 27.5 ± 9.5 94.2 ± 24.2 121.6 ± 20.9 141.6 ± 22.9 164.0 ± 24.1 179.2 ± 24.9 191.8 ± 26.1 201.4 ± 26.8 208.0 ± 27.6 113.8 ± 16 .0

10 10.8 ± 1.1 14.4 ± 2.2 24.7 ± 5.8 40.0 ± 9.5 29.3 ± 8.8 105.2 ± 23.2 133.2 ± 16.3 152.4 ± 14.9 174.2 ± 13.9 190.6 ± 8.8 207.2 ± 13.2 218.2 ± 10.9 224.0 ± 1 0.7 118.8 ± 21 .0

30 10.7 ± 1.2 14.1 ± 1.7 22.5 ± 4.4 37.5 ± 5.9 26.7 ± 5 .0 107.0 ± 24.2 135.6 ± 21.3 157.0 ± 25.1 178.8 ± 25.6 195.8 ± 29.2 207.0 ± 35.0 224.8 ± 30.5 228.2 ± 29.1 124.3 ± 13.2

100 8.9 ± 0.9 11.9 ± 1.3 20.0 ± 2.9 32.3 ± 3.9 23.2 ± 3.9 81.0 ± 19.1 107.0 ± 19.9 131.2 ± 17.9 151.2 ± 12.1 166.6 ± 10.9 179.4 ± 11.1 191.2 ± 10.7 192.2 ± 12.5 111.2 ± 14.8



### dose response + + + +

- + + + + + + + +

-

BMDL 92.2 90.1 48.9 42.2 25.9 19.6 38.5 45.2 50.9 50.1 55.2 57.9



## male rats

standard 12.0 ± 1.4** 16.5 ± 2.2 29.1 ± 4.8** 47.1 ± 8.4** 36.7 ± 6.3 137.4 ± 30.5 188.4 ± 34.1 216.6 ± 64.4 275.0 ± 30.8 307.6 ± 32.8 330.8 ± 31.9 351.6 ± 30.3 368.4 ± 34.9 231 ± 30.1

0 10.0 ± 1.3** 13.4 ± 2.2 22.3 ± 6.4** 39.3 ± 7.5** 29 .0 ± 6.6 112.8 ± 32.2 158.2 ± 37.5 20 1.8 ± 41.0 239.8 ± 38.5 271.2 ± 40.1 294.8 ± 40.0 315.6 ± 41.7 329.0 ± 41.2 216.2 ± 22.5

0.1 10.2 ± 0.7 13.6 ± 1.6 24.2 ± 5.0 41.8 ± 8.9 31.5 ± 8.6 106.4 ± 19.0 151.6 ± 22.5 198.4 ± 24.7 238.2 ± 21.7 272.2 ± 19.0 298.0 ± 15.3 321.6 ± 18.4 332.8 ± 20.1 226 .4 ± 23.3

0.3 9.8 ± 1.2 12.7 ± 2.0 22.0 ± 4.0 35.1 ± 5.2 25.2 ± 4.6 110.2 ± 25.3 158.8 ± 28.2 203.8 ± 26.5 244.2 ± 20.5 278.2 ± 12.1 305.8 ± 13.1 326.8 ± 10.5 339.6 ± 12.1 229.4 ± 22 .0

1 10.8 ± 1.9 14.7 ± 4.1 33.3 ± 8.6 55.7 ± 14.4 44.4 ± 13.2 160.2 ± 35 .2 213.2 ± 36.3 262.2 ± 35.8 302.8 ± 33.1 334.0 ± 33.1 358.4 ± 36.8 377.0 ± 35.7 390.0 ± 38.5 229.8 ± 17.1

3 10.2 ± 1.7 13.1 ± 3.0 24.1 ± 7.7 39.1 ± 12.0 28.7 ± 10.9 108.4 ± 25.6 150.8 ± 28.3 193.8 ± 31.1 231.6 ± 31.3 264.4 ± 34.8 286.8 ± 38.2 307.4 ± 41. 8 323.2 ± 45.0 214.8 ± 36.4

10 10.8 ± 1.4 13.9 ± 2.7 24.6 ± 6.5 39.5 ± 10.0 28.6 ± 9.1 110.0 ± 26.1 156.8 ± 32.0 202.4 ± 34.3 243.6 ± 26.8 274.8 ± 25.8 301.4 ± 26.3 320.8 ± 24.1 334.6 ± 26.0 224.6 ± 12.8

30 11.0 ± 1.3 14.6 ± 1.7 22.5 ± 3.2 35.6 ± 6.2 24. 6 ± 5.2 126.4 ± 27.1 175.0 ± 34.5 226.0 ± 40.3 269.0 ± 41.0 304.4 ± 43.6 333.8 ± 48.8 354.2 ± 50.8 380.3 ± 49.3 248.3 ± 42.3

100 9.5 ± 0.9 12.6 ± 1.0 20.5 ± 2.2 32.2 ± 3.0 22.6 ± 2.7 74.2 ± 11.4 111.6 ± 11.8 142.2 ± 22.5 188.8 ± 15.0 224.4 ± 10.9 252.6 ± 11.0 274.2 ± 10.5 290.6 ± 11.5 216.4 ± 9.5



### dose response +

- + +

- + + + + + + + +

-

BMDL 96.8 50.1 11.6 30.0 30.0 30.0 44.3 51.1 56.4 59.6 58.6



### Figures are average ± standard deviation based on all available pups during lactation ( at least 14 animal s per sex per dose group )

, and 5 (occasionally 4) animals per sex per dose group during w4

- 1 1

(animals assigned to final necropsy)

. Standard, standard fat diet, included as a reference for the effect of high oil in the exposure diets. +, Significant dose r esponse (for results see Table 1 in the text );

- , no significant dose response. ** P<0.01 for difference between carrier (oil) control and standard diet (T

- test).

# Endocrine effects of hexabromocyclododecane (HBCD) in a one

- generation reproduction study in Wistar rats

Leo TM van der Ven, Ton va n de Kuil, Wout Slob, Pim EG Leonards, Hellmuth Lilienthal, Theo J Visser, Rocío F Cantón, Martin van den Berg, Sabina Litens , Helen Håkansson, Henk van Loveren, Josephus G Vos, Aldert H Piersma



## Supplementary Tables



# Table 6

– F1 Food intake during juvenile growth

female rats

weeks of age

HBCD dose

mg/kg bw 5 6 7 8 9 10

standard 108.8 ± 11.3 118.8 ± 12.7 125.2 ± 9.8 111.0 ± 26.5 122.8 ± 6.3 122.6 ± 8.1

0 127.2 ± 35.5 105.2 ± 29.2 148.2 ± 23.4 135.0 ± 15.5 131.4 ± 10.9 123.6 ± 8.8

0.1 119.6 ± 21.6 122.8 ± 47.2 133.4 ± 26.2 128.4 ± 24.5 136.4 ± 33. 4 121.8 ± 20.0

0.3 121.0 ± 20.4 131.2 ± 31.5 134.2 ± 23.2 129.6 ± 19.0 135.6 ± 11.9 121.4 ± 17.1

1 117.2 ± 10.1 113.4 ± 12.4 130.6 ± 12.8 119.8 ± 20.9 126.8 ± 13.8 122.4 ± 18.1

3 96.2 ± 10.1 96.6 ± 9.9 107.2 ± 12.0 105.6 ± 7.8 108.4 ± 8.6 113.4 ± 20.4

10 109.8 ± 21.7 102.8 ± 10.4 118.4 ± 18.8 124.6 ± 23.5 126.2 ± 24.5 122.2 ± 25.2

30 128.2 ± 30.9 123.2 ± 43.0 137.4 ± 36.6 138.2 ± 47.9 131.8 ± 26.8 133.8 ± 37.5

100 97.2 ± 3.4 93.2 ± 9.4 110.2 ± 10.2 120.4 ± 43.9 104.0 ± 8.1 104.6 ± 11.8

dose response



### - a

- - +

-



## male rats

standard 149.4 ± 17.2 156.8 ± 8.6 172.8 ± 9.0 164.2 ± 8.3 166.4 ± 12.9 160.0 ± 6.3

0 132.0 ± 21.4 139.8 ± 16.0 163.0 ± 21.8 158.2 ± 14.6 155.2 ± 18.1 150.6 ± 16.1

0.1 137.8 ± 13.0 139.0 ± 14.3 173.2 ± 21.6 160.2 ± 8.7 162.4 ± 13.6 145.8 ± 5.1

0.3 161.4 ± 7.5 174.8 ± 10.4 176.0 ± 9.3 180.4 ± 14.8 179.4 ± 13.6 169.8 ± 18.6

1 163.4 ± 7.7 162.2 ± 19.6 190.4 ± 12.7 167.4 ± 8.3 177.0 ± 8.7 166.2 ± 12.2

3 117.8 ± 23.0 126.8 ± 13.3 154.8 ± 24.3 149.8 ± 17.7 146.4 ± 15.5 145.2 ± 15.7

10 133.2 ± 13.1 145.6 ± 15.8 169.4 ± 19.9 164.2 ± 13.6 155.8 ± 9.3 148.2 ± 9.9

30 135.8 ± 49.6 161.2 ± 36.7 181.6 ± 27.6 173.0 ± 28.1 173.4 ± 19.8 156.4 ± 21.0

100 111.2 ± 12.5 122.0 ± 12.9 150.2 ± 11.1 158.4 ± 7.7 160.6 ± 8.0 141.4 ± 10.5

dose response 

### + +

- - a a

Figures are average ± standard deviation of 5 animals per dose group, and represent weekly intake in

grams. Standard, standard fat diet, included as a reference for the effect of high oil in the exposure diets. +,

Significant dose response for results see Table 1 in the text ;

- , no significant dose response; a , absence of

effect concluded from average values, dose

- response analysis not performed

.

# Endocrine effects of hexabromocyclododecane (HBCD) in a one

- generation reproduction study in Wistar rats

Leo TM van der Ven, Ton va n de Kuil, Wout Slob, Pim EG Leonards, Hellmuth Lilienthal, Theo J Visser, Rocío F Cantón, Martin van den Berg, Sabina Litens , Helen Håkansson, Henk van Loveren, Josephus G Vos, Aldert H Piersma



## Supplementary Tables



# Table 7

– Cauda epididymis sperm characteristics at

ne cropsy of F1 males

HBCD dose

mg/kg bw n sperm counts coiled tail broken tail separate heads head defects

% rel. to control % of total

standard 4 93.2 ± 4.8 0.0 ± 0.0 0.9 ± 0.8 3.6 ± 3.4 2.3 ± 1.6

0 5 92.0 ± 3.7 0.2 ± 0.4 1.6 ± 1.2 4.2 ± 1.7 2.0 ± 1. 4

0.1 5 94.0 ± 4.4 0.2 ± 0.4 1.1 ± 1.6 3.8 ± 2.9 0.9 ± 0.7

0.3 5 88.4 ± 9.2 0.2 ± 0.4 2.0 ± 2.6 7.5 ± 8.1 0.7 ± 0.8

1 5 94.4 ± 2.6 0.4 ± 0.5 1.7 ± 2.0 2.2 ± 1.9 1.3 ± 2.1

3 4 93.1 ± 3.7 0.5 ± 0.5 1.3 ± 2.1 4.4 ± 1.9 0.7 ± 0.9

10 4 88.2 ± 5.4 0.0 ± 0.0 4.7 ± 3.3 4.1 ± 2.1 3.0 ± 2.5

30 5 92.7 ± 2.7 0.0 ± 0.0 1.8 ± 1.8 5.0 ± 1.8 0.5 ± 0.8

100 5 96.5 ± 1.6 0.2 ± 0.4 1.0 ± 1.0 0.8 ± 0.8 0.6 ± 0.9



### dose response

- - - +

-

Figures are average ± standard deviation of n replicates per dose group. Standard, standard fat diet,

included as a reference for the effect of high oil in the exposure diets. +, Significant dose response for

results see Table 1 in the text ;

- no significant dose response.

# Endocrine effects of hexabromocyclododecane (HBCD) in a one

- generation reproduction study in Wistar rats

Leo TM van der Ven, Ton va n de Kuil, Wout Slob, Pim EG Leonards, Hellmuth Lilienthal, Theo J Visser, Rocío F Cantón, Martin van den Berg, Sabina Litens , Helen Håkansson, Henk van Loveren, Josephus G Vos, Aldert H Piersma



## Supplementary Tables



# Table 8

– Body and organ weights at necropsy (grams) , F1 animals

female rats

HBCD dose

mg/kg bw body

weight pituitary thyroid adrenal L+R ovary L+R uterus spleen thymus popl ln L+R heart liver kidney

L+R brain

standard 229 ± 9 0.011 ± 0.002 0.022 ± 0.003 0.065 ± 0.005* 0.135 ± 0.039 0.62 ± 0.17 0.43 ± 0. 07 0.41 ± 0.05 0.011 ± 0.005 0.83 ± 0.09 8.7 ± 1.2 1.57 ± 0.16 1.79 ± 0.04

0 222 ± 28 0.013 ± 0.003 0.024 ± 0.005 0.053 ± 0.007* 0.103 ± 0.014 0.53 ± 0.11 0.40 ± 0.04 0.49 ± 0.07 0.008 ± 0.002 0.83 ± 0.09 7.7 ± 0.9 1.58 ± 0.17 1.76 ± 0.14

0.1 221 ± 16 0.012 ± 0.002 0.021 ± 0.003 0.054 ± 0.007 0.125 ± 0.023 0.60 ± 0.20 0.39 ± 0.04 0.41 ± 0.05 0.009 ± 0.002 0.83 ± 0.03 7.9 ± 0.8 1.58 ± 0.14 1.71 ± 0.09

0.3 211 ± 28 0.012 ± 0.003 0.019 ± 0.004 0.058 ± 0.008 0.114 ± 0.021 0.50 ± 0.11 0.37 ± 0.06 0.40 ± 0.04 0.009 ± 0.002 0.78 ± 0.11 7.8 ± 1.4 1.46 ± 0.22 1.71 ± 0.09

1 241 ± 21 0.012 ± 0.001 0.020 ± 0.005 0.057 ± 0.006 0.112 ± 0.003 0.75 ± 0.38 0.56 ± 0.37 0.42 ± 0.05 0.008 ± 0.002 0.89 ± 0.09 8.3 ± 0.5 1.64 ± 0.09 1.77 ± 0.08

3 208 ± 28 0.011 ± 0.00 2 0.022 ± 0.004 0.057 ± 0.008 0.128 ± 0.016 0.71 ± 0.39 0.56 ± 0.42 0.48 ± 0.10 0.008 ± 0.002 0.76 ± 0.08 7.7 ± 0.8 1.41 ± 0.19 1.62 ± 0.23

10 224 ± 11 0.012 ± 0.001 0.020 ± 0.004 0.058 ± 0.003 0.111 ± 0.016 0.94 ± 0.28 0.38 ± 0.05 0.45 ± 0.06 0.008 ± 0.002 0.79 ± 0.06 8.3 ± 0.5 1.52 ± 0.06 1.80 ± 0.06

30 228 ± 29 0.014 ± 0.003 0.019 ± 0.006 0.055 ± 0.004 0.121 ± 0.023 0.48 ± 0.10 0.40 ± 0.04 0.44 ± 0.11 0.009 ± 0.001 0.83 ± 0.10 9.0 ± 1.1 1.59 ± 0.21 1.76 ± 0.08

100 192 ± 13 0.011 ± 0.001 0.022 ± 0.003 0.059 ± 0.008 0.110 ± 0.019 0.49 ± 0.22 0.39 ± 0.07 0.37 ± 0.07 0.007 ± 0.002 0.75 ± 0.06 8.4 ± 0.6 1.36 ± 0.08 1.66 ± 0.07

dose response 

### + a

- a a a a +

- - +

-



## male rats

HBCD dose

mg/kg bw body

weight pituitary thyroid adrenal L+R testis L +R epididymis

L+R sem. ves. prostate spleen thymus popl ln heart liver kidney

L+R brain

standard 368 ± 35 0.012 ± 0.002 0.028 ± 0.007 0.054 ± 0.002* 2.98 ± 0.32 0.78 ± 0.26 1.16 ± 0.38 0.64 ± 0.12 0.52 ± 0.04 0.43 ± 0.04** 0.012 ± 0.003 1.14 ± 0.09 14.2 ± 0.9* 2.72 ± 0.27 1.98 ± 0.08

0 329 ± 41 0.011 ± 0.002 0.026 ± 0.003 0.047 ± 0.006* 3.01 ± 0.17 0.95 ± 0.13 1.00 ± 0.40 0.66 ± 0.18 0.49 ± 0.12 0.62 ± 0.10** 0.009 ± 0.002 1.07 ± 0.13 11.9 ± 1.5* 2.47 ± 0.25 1.84 ± 0.12

0.1 333 ± 20 0.013 ± 0.005 0.024 ± 0.003 0.047 ± 0.004 2.91 ± 0.08 0.88 ± 0.13 1.07 ± 0.22 0.73 ± 0.21 0.53 ± 0.07 0.54 ± 0.12 0.010 ± 0.003 1.08 ± 0.11 12.3± 0.4 2.35 ± 0.26 1.87 ± 0.07

0.3 340 ± 12 0.013 ± 0.003 0.030 ± 0.005 0.051 ± 0.005 3.07 ± 0.42 0.95 ± 0.12 1.32 ± 0.23 0.57 ± 0.15 0.49 ± 0.03 0.53 ± 0.12 0.009 ± 0.004 1.08 ± 0.07 12.7 ± 0.8 2.41 ± 0.16 1.94 ± 0.06

1 390 ± 39 0.011 ± 0.002 0.026 ± 0.003 0.052 ± 0.005 3.18 ± 0.20 1.00 ± 0.06 1.14 ± 0.29 0.73 ± 0.21 0.58 ± 0.07 0.56 ± 0.13 0.015 ± 0.011 1.28 ± 0.09 14.4 ± 2.0 2.75 ± 0.16 1.98 ± 0.07

3 323 ± 45 0.011 ± 0.002 0.026 ± 0.003 0.045 ± 0.008 2.88 ± 0.28 0.90 ± 0.09 1.21 ± 0.09 0.57 ± 0.12 0.49 ± 0.05 0.50 ± 0.09 0.009 ± 0.003 1.07 ± 0.11 12.2 ± 1.7 2.33 ± 0.32 1.91 ± 0.07

10 335 ± 26 0.010 ± 0.002 0.025 ± 0.005 0.047 ± 0.0 04 2.82 ± 0.07 0.85 ± 0.13 1.07 ± 0.29 0.58 ± 0.07 0.50 ± 0.07 0.55 ± 0.08 0.008 ± 0.001 1.13 ± 0.09 12.1 ± 0.8 2.35 ± 0.18 1.88 ± 0.05

30 380 ± 49 0.010 ± 0.001 0.025 ± 0.005 0.050 ± 0.003 2.97 ± 0.25 0.98 ± 0.14 1.21 ± 0.25 0.67 ± 0.09 0.58 ± 0.09 0.48 ± 0.14 0.010 ± 0.005 1.22 ± 0.15 14.0 ± 2.8 2.58 ± 0.43 1.92 ± 0.06

100 291 ± 12 0.010 ± 0.001 0.026 ± 0.001 0.041 ± 0.002 2.60 ± 0.06 0.82 ± 0.06 1.09 ± 0.27 0.42 ± 0.13 0.48 ± 0.06 0.45 ± 0.06 0.021 ± 0.016 0.97 ± 0.03 12.0 ± 0.5 2.11 ± 0.09 1.78 ± 0.06

dose response 

### + a a + +

- - + a + + + a + +

Seminal vesicle (sem ves) includes coagulation gland; popl ln: popliteal lymph node. Figures are average ± standard deviation of 5 replicates per dose group, occasionally 4. Standard, standard fat diet, includ ed

as a reference for the effect of high oil in the exposure diets. +, Significant dose response (for results see Table 1 in the text );

- , no significant dose response; a , absence of effect concluded from average values,

dose

- response analysis not performe d. *,** P<0.05, 0.01 between carrier (oil) control and standard diet (T

- test).

# Endocrine effects of hexabromocyclododecane (HBCD) in a one

- generation reproduction study in Wistar rats

Leo TM van der Ven, Ton va n de Kuil, Wout Slob, Pim EG Leonards, Hellmuth Lilienthal, Theo J Visser, Rocío F Cantón, Martin van den Berg, Sabina Litens , Helen Håkansson, Henk van Loveren, Josephus G Vos, Aldert H Piersma



## Supplementary Tables



# Table 9

– Clinical chemistry of blood plasma after HBCD in F1 animals

female rats

HBCD dose

mg/kg bw 

## n ALP

U/L ALAT

U/L CHOL

mmol/L CREA

μmol/L GLU

mmol/L UREA

mmol/L to tal protein

g/L albumin g/L

standard 5 2.32 ± 0.95 30.5 ± 5.6 2.15 ± 0.19** 24.3 ± 2.9 10.30 ± 1.48 6.81 ± 0.36 60.4 ± 1.9 41.4 ± 2.1

0 5 3.78 ± 1.97 34.7 ± 3.3 1.70 ± 0.14** 25.6 ± 2.3 9.36 ± 0.99 6.78 ± 0.48 58.2 ± 2.0 41.5 ± 1.5

0.1 5 2.70 ± 2.37 37. 5 ± 6.5 1.89 ± 0.08 25.1 ± 4.0 9.22 ± 2.17 6.62 ± 0.85 62.9 ± 5.2 45.9 ± 4.2

0.3 5 3.82 ± 3.23 39.7 ± 12.6 1.84 ± 0.11 30.3 ± 10.8 9.48 ± 2.29 7.84 ± 1.25 60.2 ± 0.9 42.6 ± 1.1

1 5 2.64 ± 0.95 37.3 ± 4.8 1.89 ± 0.16 24.5 ± 3.0 8.17 ± 1.03 6.39 ± 0.64 62. 2 ± 1.6 43.8 ± 1.0

3 5 1.14 ± 0.53 33.5 ± 6.2 1.84 ± 0.21 25.2 ± 1.8 8.47 ± 2.10 6.74 ± 0.92 59.5 ± 1.5 42.0 ± 1.3

10 5 3.82 ± 1.64 30.7 ± 6.2 1.87 ± 0.09 23.8 ± 1.3 9.14 ± 1.48 6.50 ± 0.64 61.6 ± 2.3 43.3 ± 1.8

30 5 2.66 ± 1.55 33.9 ± 10.4 1.91 ± 0.10 24.5 ± 3.5 10.73 ± 0.60 7.05 ± 0.63 63.7 ± 1.9 44.3 ± 1.4

100 5 1.28 ± 0.59 34.0 ± 4.6 1.80 ± 0.13 23.7 ± 3.7 7.50 ± 1.44 6.95 ± 1.93 62.6 ± 2.4 42.6 ± 1.4



### dose response + a

- a

- a a a



# male rats

HBCD dose

mg/kg bw 

## n ALP

U/L ALAT

U/L CHOL

mmol/L CREA

μmol/L GLU

mmol/L UREA

mmol/L total protein

g/L albumin g/L

standard 5 4.00 ± 0.77 41.8 ± 4.1 1.76 ± 0.21 23.5 ± 2.1 10.42 ± 1.72 7.74 ± 0.13 61.5 ± 0.8 39.2 ± 0.6

0 5 3.22 ± 2.24 37.3 ± 1.8 1.77 ± 0.14 21.4 ± 1.7 9.20 ± 1.39 6.79 ± 0.71 59.2 ± 1.5 39.3 ± 0.5

0.1 5 4.40 ± 2.31 33.6 ± 4.7 1.88 ± 0.19 23.2 ± 2.4 11.10 ± 0.70 6.94 ± 0.71 59.3 ± 2.5 39.5 ± 1.5

0.3 4 3.28 ± 1.76 43.6 ± 7.8 1.92 ± 0.20 26.1 ± 3.0 9.88 ± 1.03 8.27 ± 0.84 59.7 ± 1.0 39.3 ± 1.1

1 4 4.80 ± 2.79 43.1 ± 4.2 1.71 ± 0.20 22.6 ± 5.2 10.37 ± 1.13 7.05 ± 1.00 60.5 ± 1.8 39.4 ± 0.6

3 5 3.38 ± 1.90 43.3 ± 4.4 1.96 ± 0.12 23.6 ± 0.8 8.02 ± 2.54 7.55 ± 0.84 61.9 ± 3.4 40.5 ± 2.5

10 5 3.20 ± 0.85 40.3 ± 6.8 1.78 ± 0.13 23.9 ± 4.0 8.96 ± 1.22 7.12 ± 0.42 59.2 ± 1.7 39.3 ± 0.8

30 4 4.60 ± 2.43 38.2 ± 4.7 1.66 ± 0.12 21.7 ± 1.6 12.64 ± 2.28 6.80 ± 0.64 58.1 ± 2.8 38.4 ± 1.3

100 5 3.76 ± 1.90 37.2 ± 2.6 1.71 ± 0.17 23.4 ± 2.2 9.37 ± 1.96 6.97 ± 0.24 58.8 ± 2.1 38.9 ± 1.5



### dose response a

- a a

- a a a

Figures are average ± standard deviatio n of n replicates per dose group

. ALP, alkaline phosphatase; ALAT, alanine amino transferase; CHOL, cholesterol; CREA,

creatinin; GLU, glucose. ALP values are low compared to standard references, because of erroneous use of EDTA as anticoagulan t, which app eared to inhibit the

measurements. Standard, standard fat diet, included as a reference for the effect of high oil in the exposure diets. +, Signi ficant dose response (for results see

Table 1 in the text );

- , no significant dose response; a , absence of eff ect concluded from average values, dose

- response analysis not performed. ** P<0.01 for

difference between carrier (oil) control and standard feed control.

# Endocrine effects of hexabromocyclododecane (HBCD) in a one

- generation reproduction study in Wistar rats

Leo TM van der Ven, Ton va n de Kuil, Wout Slob, Pim EG Leonards, Hellmuth Lilienthal, Theo J Visser, Rocío F Cantón, Martin van den Berg, Sabina Litens , Helen Håkansson, Henk van Loveren, Josephus G Vos, Aldert H Piersma



## Supplementary Tables



# Table 10

– Endocrine parameters after HBCD (P, F1)

females males

HB CD dose

mg/kg bw n TT4

nmol/L TT3

nmol/L n CYP17

pmol/h/mg protein n CYP19

pmol/h/mg protein n TT4

nmol/L TT3

nmol/L n CYP17

pmol/h/mg protein

P animals 0 5 44

. 4 ± 9

. 3 0

. 8 ± 0

. 2 5 62.0 ± 4

. 7 0

. 9 ± 0

. 1

100 5

(4) 38.0 ± 17

. 6

( 31

. 3 ± 10.4) b 0

. 9 ± 0

. 3 5

(4) 54

. 2 ± 13

. 8

(48.5 ± 6.2) b 0

. 8 ± 0

. 1



### F1 animals standard 5 36.8 ± 9.9 1.0 ± 0.1 5 76.4 ± 17.3 4 10.7 ± 5.1 5 36.8 ± 9.9 1.0 ± 0.1 5 73.3 ± 57.6

0 5 50.6 ± 16.6 1.1 ± 0.3 5 162.5 ± 151.1 4 4.6 ± 4.0 5 44.8 ± 4.55 0.9 ± 0.1 5 131.5 ± 71.2

0.1 5 37.8 ± 13.4 1.2 ± 0.2 5 56.5 ± 30.7 4 5.7 ± 4.9 5 48.6 ± 7.6 1.2 ± 0.2 5 45.6 ± 28.5

0.3 5 38.8 ± 8.2 1.1 ± 0.2 5 60.6 ± 53.2 5 2.9 ± 2.6 4 46.3 ± 8.2 1.0 ± 0.1 5 53.4 ± 54.3

1 5 49.6 ± 11.1 1.1 ± 0.1 5 125.4 ± 70.2 4 4.2 ± 3.5 4 47.2 ± 3.4 1.0 ± 0.1 5 28.0 ± 15.0

3 3 44.8 ± 13.5 1.2 ± 0.2 4 114.0 ± 94.9 5 6.8 ± 6.8 5 42.6 ± 6.6 1.0 ± 0.1 5 145.4 ± 127.2

10 5 59.7 ± 4.9 1.4 ± 0.1 5 53.4 ± 42.0 4 12.6 ± 9.2 3 45.0 ± 4.3 0.9 ± 0.1 5 60.3 ± 57.1

30 5 41.4 ± 12.1 1.0 ± 0. 1 5 164.4 ± 118.5 4 11.7 ± 14.7 4 46.6 ± 5.1 0.9 ± 0.1 5 83.0 ± 59.0

100 5 47.0 ± 10.8 1.0 ± 0.1 4 110.2 ± 57.8 5 3.8 ± 1.3 5 47.6 ± 12.4 1.0 ± 0.1 5 38.8 ± 30.8

dose response



### - a a

- a a a

Figures are average ± standard deviation of n animals pe r dose group

. b 

### The relatively high SD in this group is produ ced by a single outlier; figures in brackets are values without that

outlier. Standard, standard fat diet, included as a reference for the effect of high oil in the exposure diets

. - , N o signifi cant dose response; a , absence of effect concluded from average

values, dose

- response analysis not performed.

# Endocrine effects of hexabromocyclododecane (HBCD) in a one

- generation reproduction study in Wistar rats

Leo TM van der Ven, Ton va n de Kuil, Wout Slob, Pim EG Leonards, Hellmuth Lilienthal, Theo J Visser, Rocío F Cantón, Martin van den Berg, Sabina Litens , Helen Håkansson, Henk van Loveren, Josephus G Vos, Aldert H Piersma



## Supplementary Tables



# Table 11

– Apolar Liver Retinoids

female rats

HBCD dose

mg/kg bw n sum apolar retinoids 1



### mg/g liver sum apolar retinoids 1



### mg

standard 5 0.7 3 ± 0.13 6.3 ± 1.0

0 5 0.83 ± 0.04 6.4 ± 0.5

0.1 5 1.05 ± 0.12 8.3 ± 1.4

0.3 4 1.10 ± 0.17 8.1 ± 0.7

1 5 1.06 ± 0.13 8.8 ± 1.1

3 5 0.85 ± 0.10 6.5 ± 1.2

10 5 0.77 ± 0.13 6.4 ± 0.9

30 5 0.77 ± 0.08 7.0 ± 1.6

100 5 0.63 ± 0.07 5.3 ± 0.6



### dose respons e + +



## male rats

standard 5 0.50 ± 0.05 ** 7.3 ±0.6

0 4 0.70 ± 0.05 ** 8.1 ± 0.6

0.1 4 0.70 ± 0.09 8.7 ± 1.2

0.3 5 0.73 ± 0.07 9.2 ± 0.4

1 5 0.74 ± 0.12 10.7 ± 2.0

3 5 0.67 ± 0.04 7.8 ± 1.5

10 4 0.63 ± 0.13 7.7 ± 2.0

30 5 0.63 ± 0.05 7.8 ± 0.8

10 0 5 0.55 ± 0.08 6.6 ± 1.0



### dose response + +

Figures are average ± standard deviation of n animals per dose group ; a , absence of

effect concluded from average values, dose

- response analysis not performed.

Standard, standard fat diet, included as a refer ence for the effect of high oil in the

exposure diets. +, Significant dose response (for results see Table 1 in the text ).

** P<0.001, standard versus high fat diet (T

- test).

1 

### Consisting mainly of retinylesters and retinol

.

# Endocrine effects of hexabromocyclododecane (HBCD) in a one

- generation reproduction study in Wistar rats

Leo TM van der Ven, Ton va n de Kuil, Wout Slob, Pim EG Leonards, Hellmuth Lilienthal, Theo J Visser, Rocío F Cantón, Martin van den Berg, Sabina Litens , Helen Håkansson, Henk van Loveren, Josephus G Vos, Aldert H Piersma



## Supplementary Tables



# Table 12

– Bone parameters ( tibi a)

HBCD dose

(mg/kg bw) n length of tibia

(mm) trabecular BMD

(mg/cm 3 ) cortical BMD

(mg/cm 3 ) total BMD

(mg/cm 3 ) total min eral cont ent

(mg/mm 3 ) total area

(mm 2 ) cortical area

(mm 2 ) cortical thickness

(mm)



### f emale rats

standard 5 37.0 ± 1.9 * 245 ± 27 * 1352 ± 7 1008 ± 31 4.7 ± 0.4 4.7 ± 0.4 3.1 ± 0.1 0.52 ± 0.01

0 5 34.0 ± 0.3 * 294 ± 19 * 1345 ± 16 1008 ± 37 4.4 ± 0.4 4.4 ± 0.3 2.9 ± 0.3 0.50 ± 0.03

0.1 5 34.9 ± 0.8 268 ± 27 1356 ± 11 1018 ± 30 4.7 ± 0.4 4.6 ± 0.3 3.2 ± 0.3 0.53 ± 0.03

0.3 5 34 .6 ± 0.5 253 ± 30 1350 ± 17 1022 ± 41 4.6 ± 0.4 4.5 ± 0.5 3.1 ± 0.3 0.53 ± 0.03

1 5 35.1 ± 0.6 231 ± 35 1350 ± 11 984 ± 10 4.7 ± 0.3 4.7 ± 0.3 3.1 ± 0.2 0.51 ± 0.02

3 5 34.0 ± 0.5 245 ± 31 1358 ± 6 994 ± 17 4.3 ± 0.2 4.3 ± 0.2 2.9 ± 0.1 0.49 ± 0.02

10 5 35.3 ± 0.3 227 ± 28 1350 ± 11 1002 ± 25 4.6 ± 0.3 4.6 ± 0.2 3.1 ± 0.2 0.52 ± 0.03

30 5 34.5 ± 1.0 200 ± 31 1355 ± 7 1016 ± 23 4.7 ± 0.4 4.6 ± 0.4 3.2 ± 0.5 0.53 ± 0.03

100 5 33.6 ± 0.9 234 ± 29 1350 ± 13 1016 ± 24 4.3 ± 0.3 4.3 ± 0.3 2.9 ± 0.2 0.50 ± 0. 02



### dose response + +

- a

- - - -

male rats

standard 5 39.6 ± 0.97 147 ± 9 1319 ± 6 972 ± 17 6.2 ± 0.6 6.4 ± 0.7 4.2 ± 0.4 0.60 ± 0.03

0 5 38.1 ± 1.2 145 ± 25 1326 ± 9 1000 ± 34 5.6 ± 0.6 5.6 ± 0.6 3.8 ± 0.4 0.58 ± 0.04

0.1 5 38.4 ± 0.8 143 ± 20 1332 ± 18 1017 ± 23 5.7 ± 0.4 5.6 ± 0.4 3.9 ± 0.2 0.59 ± 0.02

0.3 5 39.0 ± 0.2 154 ± 23 1321 ± 18 1006 ± 29 5.9 ± 0.4 5.9 ± 0.3 4.1 ± 0.2 0.61 ± 0.02

1 5 39.0 ± 0.9 167 ± 16 1318 ± 12 978 ± 13 6.4 ± 0.5 6.5 ± 0.5 4.4 ± 0.3 0.61 ± 0.02

3 4 37.7 ± 1.1 134 ± 36 1326 ± 7 994 ± 13 5.7 ± 0.6 5.7 ± 0.5 3.9 ± 0.4 0.58 ± 0.03

10 4 37.7 ± 0.7 146 ± 25 1317 ± 14 993 ± 21 5.8 ± 0.4 5.8 ± 0.5 4.0 ± 0.3 0.59 ± 0.02

30 5 38.4 ± 0.8 156 ± 20 1328 ± 27 1024 ± 55 6.2 ± 0.5 6.1 ± 0.6 4.2 ± 0.3 0.63 ± 0.03

100 5 36.5 ± 0.6 16 7 ± 11 1313 ± 12 991 ± 20 5.0 ± 0.2 5.1 ± 0.2 3.4 ± 0.1 0.55 ± 0.01



### dose response +

- - a + + + +

T rabecular parameter is measured at the metaphysic, total and cortical parameters at the diaphysis; BMD, bone mineral content. Figures are average ± standa rd

deviation of n replicates per dose group. +, Significant dose response (for results see Table 1 in the text );

- , no significant dose response; a , absence of effect concluded

from average values, dose

- response analysis not performed. * P<0.01 for differe nce between carrier (oil) control and standard feed control.

# Endocrine effects of hexabromocyclododecane (HBCD) in a one

- generation reproduction study in Wistar rats

Leo TM van der Ven, Ton va n de Kuil, Wout Slob, Pim EG Leonards, Hellmuth Lilienthal, Theo J Visser, Rocío F Cantón, Martin van den Berg, Sabina Litens , Helen Håkansson, Henk van Loveren, Josephus G Vos, Aldert H Piersma



## Supplementary Tables



# Table 1 3

– Antibody titers after immunization against

sheep red blood cells

, male F1 animals

HBCD dose

mg/kg bw n IgM day 7

extincition IgG day 21

extincition

standard 4 0.254 ± 0.148 0.4 34 ± 0.405

0 4 0.146 ± 0.065 0.182 ± 0.128

0.1 4 0.178 ± 0.076 0.362 ± 0.333

0.3 4 0.096 ± 0.083 0.174 ± 0.143

1 2 0.292 ± 0.05 0 0.233 ± 0.169

3 4 0.155 ± 0.052 0.152 ± 0.18 0

10 4 0.17 0 ± 0.061 0.444 ± 0.143

30 4 0.197 ± 0.046 0.856 ± 0.231

100 4 0 .137 ± 0.132 0.469 ± 0.205

dose response

- +

Figures are average ± standard deviation of n replicates per dose group. Figures are average ± standard

deviation of n animals per dose groups. Standard, standard fat diet, included as a reference for the eff ect

of high oil in the exposure diets. +, Significant dose response (for results see Table 1 in the text );

- , no

significant dose response.

This test was not performed in female rats.

# Endocrine effects of hexabromocyclododecane (HBCD) in a one

- generation reproduction study in Wistar rats

Leo TM van der Ven, Ton va n de Kuil, Wout Slob, Pim EG Leonards, Hellmuth Lilienthal, Theo J Visser, Rocío F Cantón, Martin van den Berg, Sabina Litens , Helen Håkansson, Henk van Loveren, Josephus G Vos, Aldert H Piersma



## Supplementary Tables



# Table 1 4

– Spleen immune cells

- FACS analysis of subpopulations a nd NK activity in male F1 rats after

HBCD

subpopulation fractions (%) absolute cell numbers per spleen (x10 7) 

### NK

- activity

HBCD dose

mg/kg bw n CD3

(Tall) CD4

(Th) CD8

(Tc) CD161a

(NK) CD45ra

(B) T/B CD4/CD8 n per spleen CD3

(Tall) CD4

(Th) C D8

(Tc) CD161a

(NK) CD45ra

(B) per

culture `b 

### per spleen c

standard 4 45.9 ± 5.4 26.7 ± 3.2 11.5 ± 1.2 * 10.5 ± 2.1 * 35.9 ± 2.8 1.29 ± 0.22 2.33 ± 0.10 4 49.0 ± 8.7 22.2 ± 2.6 12.9 ± 1.1 5.5 ± 0.5 5.3 ± 1.9 17.8 ± 4.1 110.5 ± 26.3 131.7 ± 75.8

0 5 47.9 ± 3.5 27.7 ± 2.5 13.7 ± 0.9 * 7.9 ± 0.4 * 37.0 ± 2.9 1.30 ± 0.12 2.03 ± 0.20 5 49.6 ± 8.3 2 3.8 ± 4.5 13.7 ± 2.5 6.8 ± 1.2 3.9 ± 0.6 18.4 ± 3.4 100.0 ± 14.8 100.0 ± 12.2

0.1 4 45.2 ± 3.4 26.2 ± 3.0 12.9 ± 1.5 8.8 ± 0.8 36.4 ± 0.9 1.24 ± 0.11 2.03 ± 0.24 4 55.3 ± 12.8 25.0 ± 5.9 14.5 ± 3.9 7.2 ± 2.2 4.9 ± 1.4 20.2 ± 5.1 107.4 ± 15.1 128.6 ± 59.5

0.3 4 45.3 ± 6.0 24.9 ± 2.8 13.6 ± 2.1 8.6 ± 1.4 35.7 ± 4.1 1.27 ± 0.08 1.85 ± 0.29 4 46.2 ± 8.6 21.3 ± 6.2 11.7 ± 3.2 6.4 ± 1.9 3.9 ± 0.8 16.7 ± 4.5 107.6 ± 19.3 9 6.2 ± 25.7

1 3 45.9 ± 1.9 26.9 ± 2.9 13.2 ± 1.2 8.9 ± 1.3 35.6 ± 1.8 1.29 ± 0.08 2.06 ± 0.29 3 47.8 ± 4.8 21.9 ± 1.5 12.8 ± 1.4 6.3 ± 1.2 4.3 ± 0.8 17.0 ± 1.3 110.0 ± 30.6 115.4 ± 66.3

3 4 46.4 ± 3.6 26.9 ± 2.8 13.6 ± 1.1 9.6 ± 0.6 34.2 ± 2.7 1.37 ± 0.20 1.98 ± 0.21 4 45.4 ± 7.4 21.0 ± 3.4 12.2 ± 2.5 6.1 ± 0.7 4.4 ± 0.9 15.5 ± 2.6 115.2 ± 20.5 107.8 ± 27.7

10 4 47.1 ± 3.1 26.6 ± 2.9 13.6 ± 1.4 8.9 ± 0.8 36.9 ± 2.2 1.28 ± 0.13 1.98 ± 0.32 4 49.8 ± 4.9 23.5 ± 3.1 13.1 ± 0.8 6.8 ± 1.2 4.4 ± 0.5 18.4 ± 2.5 1 05.4 ± 11.1 105.4 ± 32.7

30 4 47.2 ± 2.6 27.4 ± 3.0 13.7 ± 0.5 9.0 ± 1.5 35.7 ± 3.0 1.33 ± 0.18 1.99 ± 0.16 4 59.0 ± 17.9 27.6 ± 7.5 15.9 ± 4.1 8.0 ± 2.3 5.2 ± 1.2 21.4 ± 7.7 113.1 ± 20.6 122.4 ± 43.4

100 4 43.0 ± 4.2 23.9 ± 4.5 12.9 ± 0.9 11.3 ± 1.3 36. 5 ± 4.4 1.20 ± 0.25 1.86 ± 0.34 3 43.9 ± 7.2 17.6 ± 0.3 10.3 ± 0.3 5.2 ± 0.3 4.5 ± 0.8 14.9 ± 4.1 124.9 ± 19.2 94.7 ± 21.8



### dose response a

- a + a

- a

- 

### a a a a a

- -



### Figures are average ± standard deviation of n replicates per dose group. Cell numbers of subpopulations were based on total spleen cell count, of which some samples were missing. CD, cluster

differentiation markers; Tall, total T

- lymphocyte population; Th, Thelper lymphocytes; Tc, cytotoxic T

- lymphocytes; NK, natural killer cells; B, B

- lymp hocytes. Standard, standard fat diet, included as a

reference for the effect of high oil in the exposure diets.

+, Significant dose response (for results of total spleen cells and B

- cells see Table 1 in the text );

- , no significant dose response; a , absenc e of effect concluded from average values, dose

- response

analysis not performed. This analysis was not performed in female rats. * P<0.05 for difference between carrier (oil) control and standard diet (T

- test).

This analysis was not performed in female rat s.

# Endocrine effects of hexabromocyclododecane (HBCD) in a one

- generation reproduction study in Wistar rats

Leo TM van der Ven, Ton va n de Kuil, Wout Slob, Pim EG Leonards, Hellmuth Lilienthal, Theo J Visser, Rocío F Cantón, Martin van den Berg, Sabina Litens , Helen Håkansson, Henk van Loveren, Josephus G Vos, Aldert H Piersma



## Supplementary Tables



# Table 1 5

– White blood cells in blood and bone marrow of F1 male rats after HBCD

cell counts (x 10 9 /L) blood cell fractions (%) blood cell count

(x 10 9 /L)

bone marrow



### HBCD dose

mg/kg bw n WBC neutro lympho mono eosino baso LUC neutr o lympho mono eosino Baso LUC WBC

standard 4 4.87 ± 1.26 0

. 41 ± 0

. 08 4.27 ± 1.15 0.061 ± 0.023 0.071 ± 0.038 0.044 ± 0.011 0.008 ± 0.005 8.66 ± 1.46 87.60 ± 0.43 1.25 ± 0.35 1.41 ± 1.13 0.90 ± 0.04 0.15 ± 0.07 18.1 ± 5.7 *

0 4 5.10 ± 1.01 0.38 ± 0.01 4.5 8 ± 1.00 0.036 ± 0.009 0.048 ± 0.007 0.040 ± 0.004 0.005 ± 0.004 7.69 ± 1.40 89.64 ± 0.29 0.74 ± 0.24 1.00 ± 1.87 0.79 ± 0.12 0.13 ± 0.06 9.3 ± 3.4 *

0.1 3 7.18 ± 1.44 0

. 51 ± 0

. 14 6.46 ± 1.37 0.060 ± 0.005 0.063 ± 0.032 0.072 ± 0.016 0.012 ± 0.003 7.28 ± 2.16 89.87 ± 0.26 0.85 ± 0.15 0.85 ± 1.91 0.98 ± 0.10 0.15 ± 0.00 15.0 ± 9.3

0.3 4 5.72 ± 1.79 0

. 40 ± 0

. 09 5.15 ± 1.70 0.053 ± 0.010 0.055 ± 0.018 0.063 ± 0.026 0.006 ± 0.005 7.41 ± 2.43 89.45 ± 0.29 0.96 ± 0.27 1.03 ± 3.03 1.06 ± 0.26 0.10 ± 0.07 17.4 ± 8.5

1 3 6.53 ± 0.72 0

. 45 ± 0

. 09 5.87 ± 0.78 0.055 ± 0.013 0.085 ± 0.013 0.057 ± 0.016 0.010 ± 0.005 7.10 ± 1.88 89.72 ± 0.18 0.90 ± 0.30 1.30 ± 2.14 0.85 ± 0.18 0.15 ± 0.05 13.0 ± 3.0

3 4 4.90 ± 1.71 0

. 41 ± 0

. 15 4.35 ± 1.53 0.041 ± 0.009 0.048 ± 0.010 0. 045 ± 0.016 0.008 ± 0.006 8.33 ± 1.01 88.61 ± 0.40 0.93 ± 0.26 1.05 ± 1.19 0.95 ± 0.25 0.14 ± 0.09 17.9 ± 4.2

10 4 5.92 ± 2.27 0

. 45 ± 0

. 12 5.33 ± 2.13 0.037 ± 0.016 0.051 ± 0.017 0.048 ± 0.028 0.009 ± 0.004 7.93 ± 1.69 89.61 ± 0.25 0.65 ± 0.25 0.88 ± 1.98 0.78 ± 0.18 0.15 ± 0.04 20.2 ± 4.1

30 3 6.55 ± 0.14 0

. 56 ± 0

. 09 5.80 ± 0.08 0.053 ± 0.003 0.050 ± 0.010 0.068 ± 0.008 0.010 ± 0.000 8.57 ± 1.16 88.65 ± 0.15 0.85 ± 0.05 0.75 ± 1.30 1.02 ± 0.10 0.17 ± 0.03 16.3 ± 5.0

100 4 4.05 ± 1.50 0

. 44 ± 0

. 19 3.47 ± 1.28 0.036 ± 0.015 0.051 ± 0.013 0.035 ± 0.030 0.006 ± 0.005 10.86 ± 2.25 85.90 ± 0.23 0.95 ± 0.15 1.33 ± 2.29 0.81 ± 0.32 0.13 ± 0.10 17.6 ± 4.8

dose response + a +

- - +

- + +

- - a a +



### Figures are average ± standard deviation of n replicates per dose group. WBC, white blood cells; neutro, neutrophilic granulocytes; lympho, lymphocytes; mono, monocytes; eosino, eosinophilic

granulocytes; baso, basophilic granulocytes; LUC, large unstained cells

. Standard, standard fat diet, included as a reference for t he effect of high oil in the exposure diets. +, Significant dose response

(for results see Table 1 in the text );

- , No significant dose response; a , absence of effect concluded from average values, dose

- response analysis not performed. * P<0.05 for differe nce between carrier

(oil) control and standard diet (T

- test).

This analysis was not performed in female rats.

# Endocrine effects of hexabromocyclododecane (HBCD) in a one

- generation reproduction study in Wistar rats

Leo TM van der Ven, Ton va n de Kuil, Wout Slob, Pim EG Leonards, Hellmuth Lilienthal, Theo J Visser, Rocío F Cantón, Martin van den Berg, Sabina Litens , Helen Håkansson, Henk van Loveren, Josephus G Vos, Aldert H Piersma



## Supplementary Tables



# Table 16

– Red blood cell parameters in blood of F1 male rats after HBCD

HBCD dose

mg/kg bw n RBC

x 10 12 

### /L HGB

mmoL/L HCT

L/L MCV

fL MCH

fmoL MCHC

mmoL/L RDW

% HDW

mmoL/L PLT

x 10 9 

### /L MPV

fL retic

%

standard 4 7.83 ± 0.25 8.43 ± 0.10 0.400 ± 0.008 51.2 ± 1.3 1.07 ± 0.02 21.0 ± 0.3 10.99 ± 0.18 1.78 ± 0.03 1043 ± 64 6.94 ± 0.13 1.71 ± 0.28

0 4 8.01 ± 0.51 8.58 ± 0.31 0.411 ± 0.0 15 51.4 ± 2.1 1.08 ± 0.04 20.9 ± 0.0 11.20 ± 0.35 1.71 ± 0.07 1160 ± 150 6.56 ± 0.59 1.65 ± 0.24

0.1 3 8.00 ± 0.15 8.72 ± 0.03 0.423 ± 0.008 52.9 ± 0.9 1.09 ± 0.02 20.6 ± 0.3 10.75 ± 0.09 1.62 ± 0.03 1113 ± 58 6.60 ± 0.10 1.73 ± 0.16

0.3 4 7.82 ± 0.15 8. 58 ± 0.13 0.412 ± 0.009 52.8 ± 1.1 1.10 ± 0.02 20.8 ± 0.2 10.88 ± 0.19 1.65 ± 0.05 1168 ± 92 6.78 ± 0.16 1.80 ± 0.11

1 3 7.96 ± 0.34 8.72 ± 0.18 0.411 ± 0.010 51.7 ± 1.2 1.10 ± 0.03 21.2 ± 0.4 10.98 ± 0.08 1.74 ± 0.10 910 ± 54 6.62 ± 0.52 1.67 ± 0.13

3 4 7.83 ± 0.20 8.41 ± 0.19 0.403 ± 0.008 51.5 ± 0.8 1.08 ± 0.01 20.8 ± 0.2 10.96 ± 0.45 1.65 ± 0.18 1188 ± 95 6.79 ± 0.45 1.70 ± 0.12

10 4 7.82 ± 0.27 8.46 ± 0.20 0.412 ± 0.008 52.8 ± 1.1 1.08 ± 0.02 20.5 ± 0.0 10.89 ± 0.06 1.65 ± 0.04 1133 ± 73 6.59 ± 0.20 1.77 ± 0.15

30 3 7.62 ± 0.34 8.27 ± 0.20 0.400 ± 0.009 52.6 ± 1.3 1.09 ± 0.03 20.7 ± 0.1 10.93 ± 0.28 1.60 ± 0.03 1078 ± 149 6.58 ± 0.68 1.82 ± 0.08

100 4 7.80 ± 0.27 8.39 ± 0.09 0.408 ± 0.013 52.4 ± 2.2 1.08 ± 0.05 20.6 ± 0.6 11.08 ± 0.28 1.50 ± 0.16 1 124 ± 60 6.70 ± 0.50 1.89 ± 0.23



### dose response a a a a a a a +

- a +



### Figures are average ± standard deviation of n replicates per dose group. RBC, red blood cells (erythrocytes); HGB, hemoglobin; HCT, hematocrite; MCV, mean cell volume

(erythrocytes); MC H, mean cell hemoglobin mass (erythrocytes); MCHC, mean cell hgb concentration (erythrocytes); RDW, relative distribution wid th (erythrocytes, volume

variation); HDW, relative distribution width of erythrocyte hgb; PLT, platelets; MPV, mean platelet volume ; retic, reticulocytes. Standard, standard fat diet, included as a

reference for the effect of high oil in the exposure diets. +, Significant dose response (for results see Table 1 in the text );

- , No significant dose response; a , absence of effect

conclud ed from average values, dose

- response analysis not performed.

This analysis was not performed in female rats.