Supporting Information

Large-scale assessment of extractables and leachables in single-use

bags for biomanufacturing

Noemí Dorival-García1, Sara Carillo1, Christine Ta1, Dominic Roberts2, Kate Comstock3,
Simon Lofthouse4, Elena Ciceri5, Kyle D'Silva4, Gerald Kierans6, Christian Kaisermayer7,
Ronan Hayes8, and Jonathan Bones1,9*
1
Characterisation and Comparability Laboratory, NIBRT−The Naonal Instute for Bioprocessing
Research and Training, Foster Avenue, Mount Merrion, Blackrock, Co., Dublin, Ireland
2
Thermo Fisher Scientific, Manor Park, Tudor Rd, Cheshire, Runcorn WA7 1TA, UK
3
Thermo Fisher Scientific, 355 River Oaks Pkwy, San Jose, CA 95134, USA
4
Thermo Fisher Scientific, Stafford House, Boundary Park, Hemel Hempstead HP2 7GE, UK
5
Thermo Fisher Scientific, Via Milano, 4, 20090 Rodano MI, Italy
6
Pfizer Ireland Pharmaceuticals. Grange Castle Business Park. Nangor Road, Clondalkin, Dublin 22, D22
V8F8, Ireland.
7
BioMarin International Limited. Shanbally, Ringaskiddy, Co. Cork, P43 R298, Ireland
8
Janssen Sciences Ireland UC. Barnahely, Ringaskiddy, Co Cork, P43 FA46, Ireland
9
School of Chemical and Bioprocess Engineering, University College Dublin, Belfield, Dublin 4, Ireland

Keywords: Extractables and leachables; Single-use bags; Additives; Bioprocessing; Cell culture media;
Extraction solvent systems.

_________________________
* Corresponding author: Tel.: + 353 1215 8100; Fax: + 353 1215 8116, E–mail address:
[email protected]

1
Table of Contents:

Figure S-1: GC chromatograms for IPA:H2O, 1:1 extracts from the blank sample (A), and two different
bag samples (B) and (C).

Figure S-2: Frequency chart of identified compounds per brand in IPA:H2O, 1:1 extracts, using GC-MS.

Figure S-3: LC chromatograms for IPA:H2O, 1:1 extracts from the blank sample (A), and two different bag
samples (B) and (C).

Figure S-4: Distribution of compounds by brands/models in all tested solvents for extractables analysis
by LC-MS.

Figure S-5: (A) Frequency chart for identified leachables, and (B) Distribution of identified leachables by
brands/models, in media extracts and by LC-MS.

Figure S-6: Frequency chart for detected elements in SUBs grouped by extraction solvent. The orange
area includes the most frequent elements, and the blue area, the less frequent. Elements in red showed
the highest concentrations, followed by elements in green, and finally by the ones in black. Elements in
blue reported the lowest concentrations (< 2 ng cm–2).

Figure S-7: Distribution and frequency of detected elements in SUBs by extraction solvent.

Figure S-8: Average concentrations of major elements detected in the different brands/models of SUBs
in 0.1 M H3PO4 extracts.

Figure S-9: Average concentrations of major elements detected in the different brands of SUBs in media
extracts.

Figure S-10: Found organic solvents in SUBs grouped by concentration levels. Solvents in red belong to
Class 1, in black to Class 2, and in red to Class 3, according to USP 467 Guideline.

Table S-1: Summary of identified extractables by GC-MS

Table S-2: Heat maps of relative frequencies per brand/model for the detected compounds in SUBs in all
tested extraction solvents by GC-MS.

2
Table S-3: Summary of identified extractables and leachables by LC-MS.

Table S-4: Heat maps of relative frequencies per brand/model for the detected compounds in SUBs in all
tested extraction solvents by LC-MS.

Table S-5: Heat maps of frequencies per extraction solvent for extractable compounds in SUBs by LC-MS.

Table S-6: Overview of target elements. Sources of their limits and regulatory background.

Table S-7: Lists of extractable metals to be determined according to the specifications of USP 661.1.

Table S-8: (A) Heat map for the concentrations found in SUBs for the analyzed elements by ICP-MS. (B)
Heat map of frequency for the analyzed elements in SUBs by ICP-MS.

Table S-9: Overview of target solvents. Regulatory background and limits.

Table S-10: Heat maps showing the average concentrations found in SUBs for analyzed solvents by HS-
GC-MS.

3
Figure S-1: GC chromatograms for IPA:H2O, 1:1 extracts from the blank sample (A), and two different
bag samples (B) and (C).

4
Figure S-2: Frequency chart of identified compounds per brand in IPA:H2O, 1:1 extracts, using GC-MS.

5
Figure S-3: LC chromatograms for IPA:H2O, 1:1 extracts from the blank sample (A), and two different bag
samples (B) and (C).

6
Figure S-4: Distribution of compounds by brands/models in all tested solvents for extractables analysis
by LC-MS.

7
Figure S-5: (A) Frequency chart for identified leachables, and (B) Distribution of identified leachables by brands/models, in media extracts and by
LC-MS.

8
Figure S-6: Frequency chart for detected elements in SUBs grouped by extraction solvent. The orange area includes the most frequent elements,
and the blue area, the less frequent. Elements in red showed the highest concentrations, followed by elements in green, and finally by the ones
in black. Elements in blue reported the lowest concentrations (< 2 ng cm–2).

9
Figure S-7: Distribution and frequency of detected elements in SUBs by extraction solvent.

10
Figure S-8: Average concentrations of major elements detected in the different brands/models of SUBs
in 0.1 M H3PO4 extracts.

11
Figure S-9: Average concentrations of major elements detected in the different brands of SUBs in media
extracts.

12
Figure S-10: Found organic solvents in SUBs grouped by concentration levels. Solvents in red belong to Class 1, in black to Class 2, and in red to
Class 3, according to USP <467> Guideline.

13
Table S-1: Summary of identified extractables by GC-MS
(A) IPA:H2O, 1:1
Carcinogenicity alerts
Retention time Frequency Base peak Search Cramer
Compound name Formula Annotation Tentative function In silico (1)
(min) (%) m/z index class (1)
A B

Confirmed compounds
16.86 59 219.17431 788 4-tert-Butyl-2,6-diisopropylphenol C16H26O 57354-65-1 Antioxidant/degradation product
C12H36O6Si
12.30 45 341.01799 797 Cyclohexasiloxane, dodecamethyl- 540-97-6 Coating III NO NO
6
Tentatively Antioxidant/degradation
15.94 18 179.14305 660 4-tert-Butylcatechol, dimethyl ether C12H18O2 41280-64-2
product
15.16 14 169.06482 887 o-hydroxybiphenyl C12H10O 90-43-7 Tentatively preservative/desinfectant III NO YES
15.60 14 219.17432 788 Phenol, 2,6-bis(1,1-dimethylethyl)-4-ethyl- C16H26O 4130-42-1 Degradation product of antioxidant II NO NO
C16H48O6Si
17.21 14 221.08442 613 Heptasiloxane, hexadecamethyl- 541-01-5 Coating III NO NO
7
(2)
13.23 5 193.12228 788 Benzene, 1,3-dimethoxy-2-butyl-5-methyl C13H20O2 41395-20-4 NA I NO NO
C14H42O7Si
14.50 5 326.96569 573 Cycloheptasiloxane, tetradecamethyl- 107-50-6 Coating III NO NO
7

16.62 5 205.15870 784 Phenol, 2,4,6-tris(1-methylethyl)- C15H24O 2934-07-8 Antioxidant/degradation product I NO NO

18.43 5 120.02068 780 Salicylic acid C7H6O3 69-72-7 Photostabilizer I NO NO
23.17 5 123.04415 686 Benzoic acid, hexyl ester C13H18O2 6789-88-4 Tentatively Plasticizers I NO NO
23.25 5 178.06245 900 2-Propenoic acid, 3-(4-methoxyphenyl)-, 2-ethylhexyl ester C18H26O3 5466-77-3 Tentatively Plasticizers I NO YES
26.48 5 149.02338 892 Phtalic acid, cyclobutyl tridecyl ester C25H38O4 959274-64-7 Plasticizer I NO YES
26.67 5 149.02338 844 Phthalic acid, nonyl 2-pentyl ester C22H34O4 959267-42-6 Plasticizer I NO YES
26.88 5 149.02338 897 Phthalic acid, 3-methylbut-3-enyl nonyl ester C22H32O4 NA (2) Plasticizer I NO YES
27.08 5 149.02338 796 Phthalic acid, hexyl nonyl ester C23H36O4 88216-56-2 Plasticizer I NO YES
27.10 5 81.06991 762 Squalene C30H50 111-02-4 Polymer fragment I NO NO
27.27 5 149.02338 801 Phthalic acid, 6-methylhept-2-yl nonyl ester C25H40O4 NA (2) Plasticizer I NO YES
27.33 5 149.02338 778 Phthalic acid, cyclobutyl tetradecyl ester C26H40O4 959274-65-8 Plasticizer I NO YES
27.46 5 149.02338 851 Phthalic acid, cyclobutyl decyl ester C22H32O4 959274-63-6 Plasticizer I NO YES

Confident compounds
Benzene, 1,3-bis(1,1-dimethylethyl)- or Benzene, 1,4-bis(1,1-
11.59 77 175.14811 854 C14H22 1014-60-4 or 1012-72-2 Degradation product of antioxidant I/I NO NO
dimethylethyl)

Tentative compounds
17.78 64 207.10148 Unknown (benzene derivative with ester and t-butyl groups) C16H24O3 NA (2) Antioxidant/degradation product NA (2) NA (2) NA (2)
(2) (2) (2) (2)
15.57 55 181.08593 Unknown C12H24O2 NA NA NA NA NA (2)
17.64 45 207.10150 Unknown (benzene derivative with ester and t-butyl groups) C16H24O3 NA (2) Antioxidant/degradation product NA (2) NA (2) NA (2)
18.83 18 221.08443 Unknown (Siloxanes, Silicone oligomers) NA (2) NA (2) Coating NA (2) NA (2) NA (2)
(2) (2) (2) (2)
9.86 14 266.99922 Unknown (Siloxanes, Silicone oligomers) NA NA Coating NA NA NA (2)
No match

(2) (2) (2) (2)

20.30 14 221.08443 Unknown (Siloxanes, Silicone oligomers) NA NA Coating NA NA NA (2)
(2) (2) (2)
12.05 5 125.05979 Unknown (ketone) C8H12O2 NA Degradation product of polymer NA NA NA (2)
(2) (2) (2) (2)
19.74 5 245.15374 Unknown (benzene derivative with ketone groups) C17H24O2 NA NA NA NA NA (2)
(2) (2) (2) (2)
21.65 5 221.08443 Unknown (Siloxanes, Silicone oligomers) NA NA Coating NA NA NA (2)
(2) (2) (2) (2)
22.90 5 221.08443 Unknown (Siloxanes, Silicone oligomers) NA NA Coating NA NA NA (2)
(2) (2) (2) (2)
24.06 5 221.08443 Unknown (Siloxanes, Silicone oligomers) NA NA Coating NA NA NA (2)

14
(B) 0.1M H3PO4
Carcinogenicity alerts
Retention Frequency Base peak Search Cramer
Compound name Formula Annotation Tentative function In silico (1)
time (min) (%) m/z index class (1)
A B
Degradation product of tackifier (abietic
20.07 100 227.17937 741 10,18-Bisnorabieta-8,11,13-triene C18H26 32624-67-2 II NO NO
acid)
(2)
20.14 100 169.10123 688 3-Methyl-7-phenylhepta-1,3,4-triene C14H16 103240-61-5 NA II NO NO
17.38 100 115.05421 832 Benzene, 1,1'-(3-methyl-1-propene-1,3-diyl)bis- C16H16 54140-12-4 Dimer (polymer precursor) II YES NO
(2)
12.95 100 170.07259 915 Diphenyl ether C12H10O 101-84-8 NA III NO NO
17.63 100 183.08044 804 Phenol, 2-(1-phenylethyl)- C14H14O 4237-44-9 Degradation product of UV stabilizer III NO NO
23.17 100 287.14298 723 Phenol, 2,4-bis(1-phenylethyl)- C22H22O 2769-94-0 Degradation product of UV stabilizer III NO NO
(3) (2)
19.86 95 159.11673 788 4b,8-Dimethyl-2-isopropylphenanthrene, 4b,5,6,7,8,8a,9,10-octahydro- C19H28 5323-56-8 NA II NO NO
12.69 95 154.07755 895 Biphenyl C12H10 92-52-4 NA (2) III NO NO
Degradation product of tackifier (abietic
20.59 90 223.14806 752 10,18-Bisnorabieta-5,7,9(10),11,13-pentaene C18H22 6566-19-4 III NO NO
acid)
20.14 75 169.10114 843 1,6-Dimethyl-4-ethylnaphthalene (Norcadalene) C14H16 1451-35-0 Degradant of hydrocarbon fluid III NO NO
2H-2,4a-Methanonaphthalene, 1,3,4,5,6,7-hexahydro-1,1,5,5- (2)
12.92 65 161.13232 830 C15H24 17015-38-2 NA I NO NO
tetramethyl-
Naphthalene, 1,2,3,5,6,7,8,8a-octahydro-1,8a-dimethyl-7-(1-
13.14 65 161.13258 844 C15H24 4630-07-3 Monomer I NO NO
methylethenyl)-, [1R-(1α,7β,8aα)]-
(2)
14.66 60 145.10117 808 Cycloisolongifolene, 9,10-dehydro- C15H22 74842-33-4 NA III NO NO
16.28 55 183.11650 638 2,6-Diisopropylnaphthalene C16H20 24157-81-1 Adhesive III NO NO
21.66 55 219.11688 787 Anthracene, 2-(1,1-dimethylethyl)- C18H18 18801-00-8 Monomer adduct agent III YES NO
10.63 45 141.06075 627 Cyclohexane, isothiocyanato- C7H11NS 1122-82-3 Resin precursor III YES NO
21.67 45 219.11675 794 Phenanthrene, 2,4,5,7-tetramethyl- C18H18 7396-38-5 Coating III YES NO
14.66 40 145.10152 810 aR-Himachalene C15H22 19419-67-1 Slip agent/biofilm inhibitor II NO NO
5,2,1,6,3,4-[2,3]Butanediyl[1,4]diylidenedipentaleno[2,1,6-cde:2',1',6'- (2) (2)
22.96 35 260.15616 632 C20H20 4493-23-6 NA NA NA NA
gha]pentalene, hexadecahydro- (4)
14.48 25 159.11679 885 Naphthalene, 1,2,3,4-tetrahydro-1,6-dimethyl-4-(1-methylethyl)-, (1S-cis)- C15H22 483-77-2 Slip agent/biofilm inhibitor II NO NO
16.27 25 183.11659 702 Naphthalene, 1,6-dimethyl-4-(1-methylethyl)- C15H18 483-78-3 Slip agent/biofilm inhibitor/antioxidant III NO NO
Degradation product from polymerization
20.38 15 167.08545 793 4-Ethylbiphenyl C14H14 5707-44-8 III NO NO
catalyst
16.83 10 135.08057 724 2-Amino-7-methyl-7,8-dihydro-6H-quinazolin-5-one C9H11N3O 850801-35-3 NA (2) III YES NO
20.58 10 223.14832 623 3,4'-Diisopropylbiphenyl C18H22 61434-46-6 Thermoplasticizer III NO NO
20.14 10 169.10121 731 Naphthalene, 2-(1,1-dimethylethyl)- C14H16 2876-35-9 Flame retardant III NO NO
10.56 5 135.01378 884 1,2-Benzisothiazole C7H5NS 272-16-2 Dye III NO NO
20.14 5 169.10123 680 1,6-Dimethyl- 3-ethylnaphthalene C14H16 99486-80-3 NA (2) III NO NO
23.27 5 251.04689 629 4H-1,2,4-triazol-3-amine, 5-[[(4-nitrophenyl)methyl]thio]- C9H9N5O2S 41266-79-9 Thermodye (as silver salt) III YES NO
20.27 5 133.10107 606 Benzene, 1,1'-(1,1,2,2-tetramethyl-1,2-ethanediyl)bis[4-methyl- C20H26 734-17-8 Co-polymer for thermo properties III NO NO
20.15 5 169.10104 706 Naphthalene, 1-(1,1-dimethylethyl)- C14H16 17085-91-5 Filler/stabilizer III NO NO
22.99 5 73.02846 703 Octadecanoic acid, butyl ester C22H44O2 123-95-5 Lubricant I NO NO
22.78 5 192.09334 642 Propyne, 1,3-diphenyl- C15H12 4980-70-5 Coating III NO NO
(1)
From Toxtree using Benigni/Biossa rulebase. A = considering genotoxic effects, B = considering non-genotoxic effects.
(2)
NA = Not available.
(3)
Phenanthrene, 1,2,3,4,4a,9,10,10a-octahydro-1,4a-dimethyl-7-(1-methylethyl)-, or Dehydroabietine.
(4)
[5]Fullerane-C20-Ih or dodecahedrane.

15
Table S-2: Heat maps of relative frequencies per brand/model for the detected compounds in SUBs in all tested extraction solvents by GC-MS.
(A) IPA:H2O, 1:1 (B) Culture media (C) 0.1M H3PO4

Retention Percentages in brand/model Retention Percentages in brand/model Retention Percentages in brand/model

time (min) IA IB IIA IIB III IV VA VB time (min) IA IB IIA IIB III IV VA VB time (min) IA IB IIA IIB III IV VA VB
11.59 100 100 75 100 100 100 17.93 100 100 100 100 100 100 100 100 12.95 100 100 100 100 100 100 100 100
17.78 100 100 100 22.22 33 14 25 100 100 100 33 17.38 100 100 100 100 100 100 100 100
16.86 100 100 75 15.35 14 17.63 100 100 100 100 100 100 100 100
15.57 100 71 100 15.42 33 20.07 100 100 100 100 100 100 100 100
12.30 67 71 75 15.56 100 20,14c 100 100 100 100 100 100 100 100
17.64 67 71 75 16.08 14 23.17 100 100 100 100 100 100 100 100
15.94 67 14 25 16.16 33 12.69 67 100 100 100 100 100 100 100
18.83 100 100 33 16.22 25 19.86 100 100 67 100 100 100 100 100
9.86 33 29 16.31 100 20.59 100 100 100 50 100 100 100 67
15.16 33 29 24.00 25 20,14b 67 100 67 100 100 100 33
15.60 14 100 33 24.04 50 12.92 33 50 67 100 100 100 100 67
17.21 50 100 33 13.14 33 50 67 100 100 100 100 67
20.30 50 100 33 14,66b 100 67 33 50 100 100 33
12.05 14 16.28 100 83 33 100 33
13.23 33 (D) Ammonium buffer, 50 mM, pH 9.5 21.66 33 50 33 100 100 100 67
14.50 14 10.63 67 33 50 100 100 67
16.62 14 21.67 67 50 67 100 33
18.43 100 Percentages in brand/model 14,66a 33 33 100 100 67
Retention
19.74 14 22.96 33 17 67 100 67
time (min)
21.65 100 IA IB IIA IIB III IV VA VB 14.48 33 17 33 50 100 33
22.90 100 16.27 33 100 100 67
9.50 67 17 33 50 100
23.17 100 20.38 17 50 33
23.25 100 14.38 33 16.83 50 33
24.06 100 20,14d 33 33
20.57 17
26.48 33 20.58 50 33
26.67 33 10.56 33
26.88 33 20,14a 100
27.08 33 (E) WFI 20.15 33
27.10 100 20.27 17
27.27 33 22.78 33
27.33 33 22.99 33
Retention Percentages in brand/model
27.46 33 time (min) 23.27 33
IA IB IIA IIB III IV VA VB
22.45 33
16.11 17

16
Table S-3: Summary of identified extractables and leachables by LC-MS.
(A) Extractables.
Confirmed and confident compounds.
Carcinogenicity alerts
Molecular Cramer
Compound name Formula Annotation Tentative function In silico (1)
Weight class (1)
A B
1 2,4,6-trimethylbenzaldehyde C10H12O 148.08890 487-68-3 Polyamide nucleating agent I YES NO
2 2-tert-butyl-1,4-benzoquinone C10H12O2 164.08396 3602-55-9 Polyester radical inhibitor II YES NO
Camphorquinone 10373-78-1 Photoinitiator III NO NO
3 heptane-2,3-dione C10H14O2 166.09940 465-29-2 NA(2) III NO NO
4-tert-butylcatechol 98-29-3 Antioxidant/polimerization inhibitor I NO NO
4 N-phenyldiethanolamine C10H15NO2 181.11066 120-07-0 Light stabilizer III NO NO
4,7,7-trimethylbicyclo[4.1.0]hept-3-ene 13466-78-9 Adhesive/plasticizer I NO NO
1,6-dioxacyclododecane-7,12-dione 777-95-7 Plasticizer I NO NO
5 C10H16O4 200.10465
Dimethyl 1,4-cyclohexanedicarboxylate 94-60-0 Coating I NO NO
1,1-cyclohexanediacetic acid 4355-11-7 Plasticizer I NO NO
6 2-(Diethylamino)ethyl methacrylate C10H19NO2 185.14174 105-16-8 Polymer block degradation product I NO NO
7 PEG n5 C10H22O6 238.14143 25322-68-3 Polymer block degradation product I NO NO
8 Bis[2-(trimethylsilyloxy)ethyl] Ether C10H26O3Si2 250.14205 16654-74-3 Polymerization initiator III NO NO
9 6-methoxyquinoline N-oxide (also leachable) C10H9NO2 175.06337 6563-13-9 Antibacterial III YES NO
10 4-ethoxybenzoic Acid Ethyl Ester C11H14O3 194.09439 23676-09-7 Catalyst degradation product II NO NO
11 N,N-di(2-hydroxyethyl)-p-toluidine C11H17NO2 195.12611 3077-12-1 Photopolymerization/adhesive III NO NO
1,4-dioxacyclotridecane-5,13-dione 4471-27-6 Adhesive for multilayer materials I NO NO
12 C11H18O4 214.12025
3,3-dimethyl-1,5-dioxacycloundecane-6,11-dione 94113-47-0 Contamination formed in polyolefin containers I NO NO
Diethyl Terephthalate 636-09-9 Catalyst degradation product I NO NO
13 C12H14O4 222.08938
Diethyl phthalate 84-66-2 Plasticizer I NO YES
14 2,2-diethoxyacetophenone C12H16O3 208.10996 6175-45-7 Photoinitiator/coating II NO NO
2-cyclohexylidene-cyclohexanone 1011-12-7 Caprolactam degradation product II YES NO
15 C12H18O 178.13575
2-tertbutyl-4-ethylphenol 96-70-8 Stabilizer I NO NO
16 1,3-butanediol dimethacrylate C12H18O4 226.12000 1189-08-8 Adhesive/coating degradation product I NO NO
17 1,4-cyclohexanedimethanol divinyl ether C12H20O2 196.14624 17351-75-6 Photopolymerization initiator/coating III YES NO
18 4-(tert-butyldimethylsilyloxy)phenylboronic Acid C12H21BO2Si 236.14039 159191-56-7 Catalyst degradation product III NO NO
1,8-diazacyclotetradecane-2,7-dione 4266-66-4 Nylon66 building block III NO NO
19 C12H22N2O2 226.16801
1,8-diazacyclotetradecane-2,9-dione 56403-09-9 Caprolactam degradation product III NO NO
Hexanoic acid 2014-58-6 Cu2+ salt: catalyst I NO NO
20 C12H24N2O3 244.17890
4,4'-(oxydi-2,1-ethanediyl)bismorpholine 6425-39-4 Adhesive/coating degradation product III NO NO
21 (3-hydroxy-2,2,4-trimethylpentyl) 2-methylpropanoate C12H24O3 216.17213 25265-77-4 Plasticizer, coating II NO YES
22 1,2-amidododecanoic acid (also leachable) C12H25NO2 215.18841 693-57-2 Residue from production I NO NO
23 PEG n6 C12H26O7 282.16741 25322-68-3 Polymer block degradation product I NO NO
24 N,N-dimethyldecan-1-amine C12H27N 185.21452 1120-24-7 Catalyst degradation product I NO NO
25 tert-butylphenyl glycidylether C13H18O2 206.13068 3101-60-8 Epoxy resin degradation product III YES NO
26 Dibutyl itaconate C13H22O4 242.15199 2155-60-4 Coating I NO NO

17
 Carcinogenicity alerts
Molecular Cramer (1)
Compound name Formula Annotation Tentative function (1) In silico
Weight class
A B
Ethylmalonic acid dibutyl ester 1113-92-4 Propylene copolymer, residue from production I NO YES
27 C13H24O4 244.16699
Diethyl azelate 624-17-9 Plasticizer I NO NO
28 2-(1-hydroxycyclohexyl)-acetophenone C14H18O2 218.13075 57213-26-0 Photoinitiator II NO NO
Dipropyplphthalate 131-16-8 Catalyst degradation product I NO YES
29 C14H18O4 250.12054
Diisopropyl Phthalate 605-45-8 Catalyst degradation product I NO YES
30 2,6-di-tert-butyl-p-benzoquinone C14H20O2 220.14648 719-22-2 Irganox 1010 degradation product II YES NO
31 1,3-adamantanediacetic acid C14H20O4 252.13633 17768-28-4 Polyester monomer III NO NO
32 Isoamyl 4-(dimethylamino)benzoate mixture of isomers C14H21NO2 235.15707 21245-01-2 Photoinitiator III YES NO
33 2-nitrophenyl octyl ether C14H21NO3 251.15206 37682-29-4 Plasticizer III YES NO
2,6-di-tert-butylphenol 128-39-2 Antioxidant/UV stabilizer II NO NO
2,4-di-tert-butylphenol 96-76-4 Antioxidant degradation product I NO NO
34 C14H22O 206.16704
2,5-di-tert-butylphenol 5875-45-6 Antioxidant degradation product I NO NO
4-(2,2,3,3-tetramethylbutyl)phenol 54932-78-4 Emulsifier I NO NO
2, 6-diteritary butyl Phenol 60083-44-5 Antioxidant degradation product II NO NO
35 C14H22O2 222.16198
2,5-di-tert-butylhydroquinone 88-58-4 Antioxidant/inhibitor/stabilizer I NO NO
36 Triethyleneglycol dimethacrylate C14H22O6 286.14130 109-16-0 Monomer I NO NO
37 5-chloro-2-hydroxy-4-methylbenzophenone C14H23NO2 237.17292 68751-90-6 UV stabilizer III NO NO
38 2-propenoic acid 2-methyl- 1,2,2,6,6-pentamethyl-4-piperidinyl ester C14H25NO2 239.18838 68548-08-3 UV stabilizer III NO NO
39 N,N-dimethyllauramide C14H29NO 227.22518 3007-53-2 Plasticizer III NO NO
40 N-(2-hydroxyethyl)-dodecanamide C14H29NO2 243.21957 142-78-9 Antistatic agent III NO NO
41 PEG n7 C14H30O8 326.19407 25322-68-3 Polymer block degradation product I NO NO
42 N,N-dimethyldodecan-1-amine C14H31N 213.24565 112-18-5 Antioxidant I NO NO
43 N,N-dimethyldodecylamine N-oxide (also leachable) C14H31NO 229.24077 1643-20-5 Antistatic agent III NO NO
44 N-phenyl-N'-propan-2-yl-benzene-1,4-diamine (Antioxidant 4010NA) C15H18N2 226.14655 101-72-4 Antioxidant III NO NO
2-ethylhexyl salicylate 118-60-5 UV stabilizer/absorber I NO YES
45 C15H22O3 250.15682
3,5-di-tert-butyl-4-hydroxybenzoic acid 1421-49-4 Irganox 1010 degradation product II NO NO
46 4-(dibutylamino)benzaldehyde C15H23NO 233.17784 90134-10-4 Photoinitiator II NO NO
47 11-maleimidoundecanoic acid C15H23NO4 281.16271 57079-01-3 Coupling agent for fillers III YES YES
48 2,6-di-tert-butyl-4-methoxyphenol C15H24O2 236.17777 489-01-0 Irganox 1010 degradation product II NO NO
49 Tributyrin C15H26O6 302.17294 60-01-5 Plasticizer I NO NO
50 Methyl 3,5-di-tert-butyl-4-hydroxybenzoate C16H24O3 264.17256 2511-22-0 Irganox 1010 degradation product II NO NO
51 2,6-di-tert-butyl-4-ethylphenol (Yoshinox 250) C16H26O 234.19813 4130-42-1 Antioxidant II NO NO
52 hexadecanoic acid amide C16H33NO 255.25616 629-54-9 NA(2) III NO NO
53 2,5-dimethyl-2,5-di(tert-butylperoxy)hexane C16H34O4 290.24535 78-63-7 Polymerization initiator, residue from production III NO NO
54 PEG n8 C16H34O9 370.22028 25322-68-3 Polymer block degradation product I NO NO
55 7,9-di-t-Butyl-1-oxaspiro[4.5] deca-6,9-diene-2,8-dione C17H24O3 276.17254 82304-66-3 NA(2) III YES NO
56 3-(3'5'-di-t-Butyl-1'-hydroxy-4'-oxacyclohexa-2'5'-dienyl) propanoic acid (3) C17H26O4 294.18297 83237-15-4 Irganox 1076 degradation product II YES NO
57 Triton 101 C17H28O2 264.20911 9016-45-9 Surface active agent III NO NO
58 Butylphthalyl butyl glycolate (Morflex 190) C18H24O6 336.15729 85-70-1 Plasticizer I NO YES
59 Octyl 4-methoxycinnamate C18H26O3 290.18817 5466-77-3 NA(2) I NO YES
60 Diamyl Phthalate C18H26O4 306.18316 131-18-0 Plasticizer I NO YES
2,6-di-tert-butyl-4-sec-butylphenol (Isonox 132) 17540-75-9 Antioxidant II NO NO
61 C18H30O 262.22967
2,4,6-tri-tert-butylphenol 732-26-3 Antioxidant II NO NO

18
 Carcinogenicity alerts
Molecular Cramer (1)
Compound name Formula Annotation Tentative function (1) In silico
Weight class
A B
62 PEG n9 C18H38O10 414.24633 25322-68-3 Polymer block degradation product I NO NO
63 PPG n6 C18H38O7 366.26130 25322-69-4 Polymer block degradation product III NO NO
64 2-ethylhexyldiphenyl phosphate (Santicizer) C20H27O4P 362.16400 1241-94-7 Flame retardand, plasticizer III NO YES
65 Triton X-100 C20H34O4 338.24520 9002-93-1 Surfactant for virus inactivation II NO NO
Bis(2-ethylhexyl) Fumarate 141-02-6 Catalyst starting material I NO YES
66 C20H36O4 340.26056
Bis(2-ethylhexyl) maleate 142-16-5 Catalyst starting material I NO YES
67 PEG n10 C20H42O11 458.27249 25322-68-3 Polymer block degradation product I NO NO
68 Triton X-100 C22H38O5 382.27138 9002-93-1 Surfactant for virus inactivation II NO NO
69 Cis-1,3-docosenic acid amide (Kemamide E ultra) C22H43NO 337.33386 112-84-5 Slip agent III NO NO
70 Docosanoic acid amide C22H45NO 339.35026 3061-75-4 Lubricant III NO NO
71 PEG n11 C22H46O12 502.29897 25322-68-3 Polymer block degradation product I NO NO
72 Triton X-100 C24H42O6 426.29761 9002-93-1 Surfactant for virus inactivation II NO NO
73 Triton X-100 reduced C24H48O6 432.34469 92046-34-9 Emulsifier, Plasticizer
74 Bis[4-(vinyloxymethyl)cyclohexylmethyl] glutarate C25H40O6 436.28291 131132-77-9 Crosslinking agent III YES NO
75 Triton X-100 C26H46O7 470.32387 9002-93-1 Surfactant for virus inactivation II NO NO
76 PEG n13 C26H54O14 590.35136 25322-68-3 Polymer block degradation product I NO NO
77 Triton 101 C27H48O7 484.33965 9016-45-9 Surface active agent I NO NO
78 Bis(2,4-di-tert-butylphenyl)phosphate (bDtBPP) (also leachable) C28H43O4P 474.28925 69284-93-1 Antioxidant degradation product III NO NO
n-butyltri-n-hexyl citrate (Citroflex B-6) 82469-79-2 Plasticizer I NO YES
79 C28H50O8 514.35013
Triton X-100 9002-93-1 Surfactant for virus inactivation II NO NO
80 Triton X-100 reduced C28H56O8 520.39748 92046-34-9 Emulsifier, Plasticizer
81 PEG n14 C28H58O15 634.37757 25322-68-3 Polymer block degradation product I NO NO
82 PEG n15 C30H62O16 678.40379 25322-68-3 Polymer block degradation product I NO NO
83 Triton X-100 reduced C32H64O10 608.44995 92046-34-9 Emulsifier, Plasticizer
84 PEG n16 (also leachable) C32H66O17 722.43000 25322-68-3 Polymer block degradation product I NO NO
1,2-benzenedicarboxylic acid ditridecyl ester (Morflex x-1125) 119-06-2 Temperature resistant plasticizer I NO YES
85 C34H58O4 530.43319
Bis(1,1-methyldodecyl) benzene-1,2-dicarboxylate 27253-26-5 Plasticizer I NO YES
86 PEG n17 C34H70O18 766.45621 25322-68-3 Polymer block degradation product I NO NO
87 1,1,3-tris(2-methyl-4-hydroxy-5-tert-butylphenyl)-butane C37H52O3 544.39165 1843-03-4 Antioxidant III NO NO
88 Phenol 4,4'-[[6-(octylthio)-1,3,5-triazine-2,4-diyl]diimino]bis[2,6-bis(11-dimethylethyl)- (Irganox 858) C39H61N5O2S 663.45460 992-53-0 Antioxidant III NO NO
Butyl glycolate 7397-62-8 Plasticizer degradation product I NO NO
1-methoxy-2-propanyl acetate 108-65-6 Solvent III NO NO
89 C6H12O3 132.07859
2-ethoxyethyl acetate 111-15-9 Solvent I NO NO
Di(ethylene glycol) vinyl ether 929-37-3 Solvent I YES NO
2-[2-(2-hydroxyethoxy)ethoxy]ethanol 112-27-6 Solvent I NO NO
90 C6H14O4 150.08923
PEG n3 25322-68-3 Polymer block degradation product I NO NO
91 Hexamethyldisiloxane C6H18OSi2 162.08962 107-46-0 Coating degradation product III NO NO
92 Phenylphosphoric acid C6H7O4P 174.00814 701-64-4 Coating agent III NO NO
2,6-toluenediamine 823-40-5 Polyurethane intermediate III YES NO
93 C7H10N2 122.08440
2,4-toluenediamine 95-80-7 Polyurethane intermediate III YES NO
2-(dimethylamino)ethyl acrylate 2439-35-2 Monomer I NO NO
94 C7H13NO2 143.09468
5-ethyl-1-aza-3,7-dioxabicyclo[3.3.0]octane 7747-35-5 Preservative III NO NO

19
 Carcinogenicity alerts
Molecular Cramer (1)
Compound name Formula Annotation Tentative function (1) In silico
Weight class
A B
95 1,1,1,3,5,5,5-heptamethyltrisiloxane C7H22O2Si3 222.09276 1873-88-7 Slip agent III NO NO
96 Benzamide C7H7NO 121.05313 55-21-0 Photoiniziator, antibacterial I NO NO
97 4-methyl-benzenesulfonamide C7H9NO2 139.06340 70-55-3 Protecting group III NO NO

Vinyl acetate catalyst/olefin epoxidation

5-ethyl-2-methyl-pyridine 104-90-5 III NO NO
98 C8H11N 121.08917 catalyst/solvent for polycarbonate polymerisation

2,4-dimethyl-aniline 95-68-1 Catalyst degradation product III YES NO

1,3-bis(aminomethyl)benzene 1477-55-0 Polyamide modification agent III NO NO
99 C8H12N2 136.10023
Tetramethyl succinonitrile 3333-52-6 Polymerization initiator degradation product III NO NO
100 1-ethenylazepan-2-one C8H13NO 139.09981 2235-00-9 Coating III NO NO
Diethyl succinate 123-25-1 Plasticizer for PVC I NO NO
Octanedioic acid 505-48-6 Lubricant, plasticizer I NO NO
101 3-ethyl-3-methylpentanedioic acid C8H14O4 174.08910 5345-01-7 Catalyst degradation product I NO NO
Dimethyl adipate 627-93-0 Low-viscosity plasticizer for PVC I NO YES
(2)
2,6-dimethyl-13-dioxan-4-ol acetate cis and trans 828-00-2 NA III NO NO
102 n-cyclohexylacetamide C8H15NO 141.11551 1124-53-4 Slip agent III NO NO
3-(dimethylamino)propyl acrylate 18526-07-3 Monomer I NO NO
103 C8H15NO2 157.11029
2-(dimethylamino)ethyl methacrylate 2867-47-2 Monomer I NO NO
[4-(hydroxymethyl)cyclohexyl]methanol 105-08-8 Crosslinking agent, Intermediate from production I NO NO
Butyl butyrate 109-21-7 Residual from production of methacrylate I NO NO
104 C8H16O2 144.11474
Octanoic acid 124-07-2 Lubricant I NO NO
2-ethylhexanoic acid 149-57-5 Lubricant, plasticizer I NO YES
105 1,3-divinyltetramethyldisiloxane C8H18OSi2 186.08962 2627-95-4 Slip agent III NO NO
106 Triethylene glycol dimethyl ether C8H18O4 178.12052 112-49-2 Solvent I NO NO
107 PEG n4 C8H18O5 194.11542 25322-68-3 Polymer block degradation product I NO NO
108 Ethylenedioxybis(trimethylsilane) C8H22O2Si2 206.11583 7381-30-8 Coating agent III NO NO
109 1,3-diethoxy-1,1,3,3-tetramethyldisiloxane C8H22O3Si2 222.11075 18420-09-2 Slip agent, coating III NO NO
110 N-ethyl-2-benzothiazolamine C9H10N2S 178.05652 28291-69-2 NA(2) III YES NO
111 (R)-3-amino-3-phenylpropionic acid (also leachable) C9H11NO2 165.07898 13921-90-9 NA (2) III NO NO
Nonanedioic acid 123-99-9 Plasticizer I NO NO
112 C9H16O4 188.10468
Dimethyl isobutylmalonate 39520-24-6 Catalyst precursor I NO YES
113 2-(diethylamino)ethyl acrylate C9H17NO2 171.12590 2426-54-2 Monomer I NO NO

20
(B) Leachables
Confirmed and confident compounds.
Carcinogenicity alerts
Molecular Cramer (1)
Compound name Formula Annotation Tentative function In silico
Weight class (1)
A B
(2)
1,4-Di-tert-butylbenzene C14H22 1012-72-2 NA I NO NO
1 190.17215
1,3-Di-tert-butyl benzene C14H22 1014-60-4 Antioxidant degradation product I NO NO
2 4-tert-butyl-4'-methoxy-dibenzoylmethane C20H22O3 310.15689 70356-09-1 NA (2) III NO NO
3 3,5-Bis(1,1-dimethylethyl)-4-hydroxybenzenepropanoic acid thiodi-2,1-ethanediyl ester (Tentative) C38H58O6S 642.39541 41484-35-9 Antioxidant II NO NO
(2) (2)
4 Tris(2,4-di-tert-butylphenyl)phosphate C42H63O4P 662.44640 95906-11-9 Antioxidant III NA NA
5 3,9-Bis(octadecyloxy)-2,4,8,1,0-tetroxa-3,9-diphospaspiro[5.5]undecane C41H82O6P2 732.55866 3806-34-6 Color stabilizer III NO NO
6 3-pyridin-1-ium-1-yl propane-1-sulfonate C8H11NO3 169.07389 15471-17-7 NA(2) III NO NO
7 Triton 101 C37H68O12 704.4711 9016-45-9 Surface active agent I NO NO

Tentative compounds.
Carcinogenicity alerts
Molecular Cramer
Compound name Formula Annotation Tentative function In silico (1)
Weight class (1)
A B
8 Irganox 3114 degradation product, one di-tert-butyl-phenol group split off C33H47N3O5 565.35157 NA (2) Antioxidant degradation product NA (2) NA (2) NA (2)
(1)
From Toxtree using Benigni/Biossa rulebase. A = considering genotoxic effects, B = considering non-genotoxic effects.
(2)
NA = Not available
(3)
3,5-bis(1,1-dimethylethyl)-1-hydroxy-4-oxo-2,5-cyclohexadiene-1-propanoic acid

21
Table S-4: Heat maps of relative frequencies per brand/model for the detected compounds in SUBs in all tested extraction solvents by LC-MS.

(A) Extractables
Extracted compounds
Extraction solvent Percentage
Number
(%)
WFI 69 61
0.1M H3PO4 65 58
Ammonium buffer 50mM pH 9.5 62 55
IPA:H2O, 1:1 80 71

C12 H21 B O2 Si
C10 H26 O3 Si2

C12 H22 N2 O2
C12 H24 N2 O3
C10 H15 N O2

C10 H19 N O2

C11 H17 N O2

C12 H25 N O2

C14 H21 N O2
C14 H21 N O3

C14 H23 N O2
C14 H25 N O2
C10 H9 N O2
C10 H12 O2
C10 H14 O2

C10 H16 O4

C10 H22 O6

C11 H14 O3

C11 H18 O4
C12 H14 O4
C12 H16 O3

C12 H18 O4
C12 H20 O2

C12 H24 O3

C12 H26 O7

C13 H18 O2
C13 H22 O4
C13 H24 O4
C14 H18 O2
C14 H18 O4
C14 H20 O2
C14 H20 O4

C14 H22 O2
C14 H22 O6
C10 H12 O

C12 H18 O

C14 H22 O
C12 H27 N
Extraction Brand/
solvent model

IA 29 14 14 29 29 29 29 29 43 14 29 57 43 14 14 29 14 29 29 29 14
IB 29 57 29 57 57 29 71 43 57 29 57 43 57 100 57 57 57 57 57 29 43 29 71 71 57
IIA 25 50 75 50
WFI IIB 29
III 50
IV 100 100 100
VA 100 100
VB 50 25
IA 14 43 86 86 14 14 57 57 43 57 14 86 29 57 14 14 14
IB 71 71 71 71 14 57 14 14 71 57 57 43 14 57 29
IIA 50
0.1M H3PO4 IIB 57
III
IV 100 100
VA 50
VB 25 25
IA 57 57 100 43 100 57 43 100 43 43 29 86 57 57 57 14 71 57 71 29 71 71 71 71 100 71
IB 86 14 71 100 100 100 86 100 100 14 57 100 71 100 43 100 71 100 100 100 86 29 100 100
Ammonium IIA 100 100 50 50
buffer
IIB 86 100 86
50mM
III
pH 9.5
IV 100 100
VA 100 50
VB 100 50
IA 71 71 86 14 100 100 71 14 100 71 14 71 29 100 43 100 29 86 86 14 100 14 100 86 71
IB 71 43 100 86 100 43 71 14 43 14 43 57 29 100 14 57 100 43 86 100 29 100 14 14 100 86 86
IIA 25 75 100
IPA:H2O,
IIB 14 14 43
1:1
III
IV 100 100
VA 100 100 100
VB 100 100 100
22
 C14 H29 N O2

C15 H23 N O4

C20 H27 O4 P
C18 H38 O10

C20 H42 O11

C22 H46 O12

C26 H54 O14

C14 H29 N O

C14 H31 N O

C15 H23 N O

C16 H33 N O

C22 H43 N O
C22 H45 N O
C14 H30 O8

C15 H18 N2
C15 H22 O3

C15 H24 O2
C15 H26 O6
C16 H24 O3

C16 H34 O4
C16 H34 O9
C17 H24 O3
C17 H26 O4
C17 H28 O2
C18 H24 O6
C18 H26 O3
C18 H26 O4

C18 H38 O7

C20 H34 O4
C20 H36 O4

C22 H38 O5

C24 H42 O6
C24 H48 O6
C25 H40 O6
C26 H46 O7
C16 H26 O

C18 H30 O
C14 H31 N
Extraction Brand/
solvent model

IA 29 29 29 14 14 29 29 29 43 29 57 14
IB 57 29 14 57 14 57 57 14 43 86 43 14 14 14 29
IIA 75 50
WFI IIB
III 50
IV 100 100 100
VA 50
VB 100
IA 71 43 71 57 57 43 14 14 71 71 71 57 71 29 57 43 100 57
IB 71 71 71 71 43 29 14 14 71 29 43 57 57 71 71 71 14 29 71 43
IIA 25 25
0.1M H3PO4 IIB 14
III
IV 100 100
VA 50
VB 50
IA 43 43 29 57 29 71 86 71 14 86 71 57 14 14 100 14
IB 57 71 86 100 100 100 100 71 14 14 100 29
Ammonium IIA 25
buffer 50mM IIB
pH 9.5 III
IV 100
VA 50
VB 100
IA 43 100 71 100 100 14 14 86 100 14 14 100 71 100 14 57 14 100 57
IB 14 43 100 57 14 100 100 14 71 100 14 86 14 71 100 14 43 29 100 29
IIA 100 75 25
IPA:H2O, 1:1 IIB 86 14 71 71 71 14 71
III 100 100
IV 100 100 100
VA 100 50 100
VB 100 75 100 50

23
 C39 H61 N5 O2 S

C7 H22 O2 Si3

C8 H22 O2 Si2
C8 H22 O3 Si2
C28 H43 O4 P

C28 H58 O15

C30 H62 O16
C32 H64 O10
C32 H66 O17

C34 H70 O18

C6 H18 O Si2

C8 H18 O Si2
C7 H13 N O2

C8 H15 N O2

C9 H11 N O2

C9 H17 N O2
C9 H10 N2 S
C27 H48 O7

C28 H50 O8
C28 H56 O8

C34 H58 O4

C37 H52 O3

C8 H13 N O

C8 H15 N O
C7 H9 N O2
C6 H7 O4 P
C6 H12 O3
C6 H14 O4

C7 H10 N2

C8 H12 N2

C8 H14 O4

C8 H16 O2

C8 H18 O4
C8 H18 O5

C9 H16 O4
C7 H7 N O

C8 H11 N
Extraction Brand/
solvent model

IA 29 14 29 29 14 14 14 57 14 29 29
IB 14 29 57 14 43 43 14 29 57 29 57 57
IIA 50
WFI IIB
III
IV 100 100 100
VA 50 50 50 50
VB 25 25 25 25
IA 86 43 14 86 14 71 14 14 29 14 71 86 86 14
IB 71 57 71 29 71 71 14 14 71 71 71 14
IIA 75
0.1M H3PO4 IIB
III
IV
VA
VB
IA 14 71 14 86 57 14 71 71 14 71 86 29 86 43 86
IB 100 86 100 14 29 100 29 86 100 86 100 57 100
IIA 50 100
Ammonium
IIB 14 14 14 14
buffer 50mM
III
pH 9.5
IV
VA
VB
IA 86 71 14 14 14 71 14 14 14 100 14 14 14 100 100 100
IB 100 57 14 71 14 100 14 100 100 100
IIA 25 100 100
IPA:H2O, 1:1 IIB 100 71
III 100 100
IV 100 100 100
VA 100
VB 100

24
(B) Leachables
(Extraction solvent: Culture media)

C33 H47 N3 O5

C41 H82 O6 P2
C12 H25 N O2

C28 H43 O4 P

C42 H63 O4 P
C38 H58 O6 S
C32 H66 O17

C37 H68 O12

C14 H31 N O
C10 H9 N O2

C8 H11 N O3
C9 H11 N O2
C20 H22 O3
Brand/ C14 H22
Model

IA 14 43 43 57 71 29
IB 14 29 29 14 14 14 14 14
IIA 25 50 50 50 25
IIB 14 29 14 14 14 14 43
III 50 50 50
IV
VA 50 50 50 50
VB 50 25 50 25 50 50 50 50

25
 (*)
Extracted in 4 solvents Extracted in 3 solvents Extracted in 2 solvents Extracted in 1 solvent
(30 compounds, 27%) (24 compounds, 21%) (25 compounds, 22%) (34 compounds, 30%)

WFI
WFI
WFI
WFI

solvent
solvent
solvent
solvent

Extraction
Extraction
Extraction
Extraction

0.1M H3PO4
0.1M H3PO4
0.1M H3PO4
0.1M H3PO4

IPA:H2O, 1:1
IPA:H2O, 1:1
IPA:H2O, 1:1
IPA:H2O, 1:1

Buffer pH 9.5
Buffer pH 9.5
Buffer pH 9.5
Buffer pH 9.5
C10 H12 O2 C10 H12 O C12 H18 O4 C10 H16 O4

12
6
3
18
C10 H14 O2 C10 H15 N O2 C12 H27 N C10 H19 N O2

21
29
C10 H22 O6 C11 H17 N O2 C14 H20 O2 C10 H9 N O2

29 3 3
3 26
12
C10 H26 O3 Si2 C11 H18 O4 C14 H29 N O C12 H14 O4

6 12 32
3
29
3 26

C11 H14 O3 C12 H16 O3 C15 H18 N2 C13 H24 O4

26 3
6
3
32

C12 H21 B O2 Si C12 H18 O C15 H26 O6 C14 H22 O

3
12

C12 H22 N2 O2 C12 H20 O2 C16 H34 O9 C14 H31 N

29 41 88 29 12 18 38
3 29
C12 H24 N2 O3 C13 H18 O2 C17 H24 O3 C14 H31 N O

24
6 32
15
6 26

Buffer pH 9.5 corresponds to Ammonium buffer, 50 mM, pH 9.5.

C12 H24 O3 C14 H18 O4 C18 H26 O3 C15 H23 N O

24
6

C12 H25 N O2 C14 H21 N O2 C18 H26 O4 C15 H24 O2

6

C12 H26 O7 C14 H21 N O3 C18 H30 O C16 H33 N O

35 3 24 21 38 6 3
6 3 38
29 15
3

C13 H22 O4 C14 H22 O2 C18 H38 O10 C17 H28 O2

12
29

C14 H18 O2 C14 H22 O6 C20 H36 O4 C18 H24 O6

3 41
3
9 26

C14 H20 O4 C14 H25 N O2 C20 H42 O11 C20 H27 O4 P

41 62
6
26

C14 H23 N O2 C15 H23 N O4 C28 H56 O8 C20 H34 O4

15
3
3

C14 H29 N O2 C16 H26 O C30 H62 O16 C22 H38 O5

6 35 32 44 41 6 35 35
3
21
24 15 15

C14 H30 O8 C18 H38 O7 C6 H14 O4 C22 H43 N O

41 62
32
6 21 12 15 3 12 29 21 6 3 18 41
3

C15 H22 O3 3 C22 H45 N O C6 H18 O Si2 C24 H42 O6

3 29
9

C16 H24 O3 C22 H46 O12 C8 H12 N2 C26 H46 O7

24 38 38 41 24 6 9 26 21 18 41 41 35 41 35 35 41 26 41
3 26 41 29 9 26 47 32 6 18 35 26 35 35 35 35 24 29 38
3 9 32 32 3 21 29 3 6 6 32 9 3 6 3 24 29 21 6
15 9 18 6 21 12 26 32 3 9 21 15 12 15 15 18 12 18 18
6
3
6
6 15 15

C16 H34 O4 C28 H58 O15 C8 H13 N O C27 H48 O7

6
3
3
6
9 18 26
3 15 32
3
9

C17 H26 O4 C32 H64 O10 C8 H14 O4 C28 H43 O4 P

3
3
6 12

C24 H48 O6 C32 H66 O17 C8 H15 N O C28 H50 O8

71 12
41 6
21 3
35 6
6 6
18
3 12
6
38

C25 H40 O6 C34 H70 O18 C8 H18 O4 C34 H58 O4

3
6
3
3 94 15 29

C26 H54 O14 C8 H15 N O2 C8 H22 O2 Si2 C37 H52 O3

21
3
3
3 32
6 9
3
Table S-5: Heat maps of frequencies per extraction solvent for extractable compounds in SUBs by LC-MS.

C6 H12 O3 C9 H17 N O2 C39 H61 N5 O2 S

38
3
6

C7 H13 N O2 C6 H7 O4 P

41 18 41 3
41 9 35 35
35 21 32 3
18 3 18 3
6

C8 H16 O2 C7 H10 N2

6
3
3
6
6

C8 H18 O5 C7 H22 O2 Si3

41
32
29
24
9

C8 H22 O3 Si2 C7 H7 N O
32

C9 H16 O4 C7 H9 N O2
53 41
47 21
41 3
24 18
6

C8 H11 N
6

C8 H18 O Si2
6

C9 H10 N2 S
C9 H11 N O2
24 50

Summary chart:

26
Table S-6: Overview of target elements. Sources of their limits and regulatory background.

Regulatory background Limits (ppm)

a
LOD USP USP
Nuclide −1 ICH c d EMEA e
(ng mL ) b EMEA guideline Chapter ICH Q3D Chapter Ph. Eur.
Q3D guideline d
<232> <232>
75 f
As 0.1 1 -
15 - 1.5 1-20
111
Cd 0.006 1 -
5 - 25 1-20
202
Hg 0.02 1 -
30 - 15 1-20
208
Pb 0.06 1 -
5 - 5 1-20
51
V 0.2 2A 1C
100 25 100 1-20
59
Co 0.022 2A - - 50 - - 1-20
78
Se 0.3 2A - - 150 - - -
95
Mo 0.1 2A 1C
3000 25 100 1-20
101 g
Ru 0.011 2B 1B
100 10 100 1-20
103 g
Rh 0.005 2B 1B
100 10 100 1-20
105
Pd 0.006 2B 1A
100 10 100 20
107
Ag 0.010 2B 150 - - -
189 g
Os 0.004 2B 1B
100 10 100 1-20
193 g
Ir 0.04 2B 1B
100 10 100 1-20
195
Pt 0.003 2B 1A
100 10 100 1-20
197
Au 0.02 2B - - 100 - - -
205
Tl 0.02 2B - - 8 - - -
7
Li 0.4 3 - - 550 - - -
52 h
Cr 0.1 3 1C
11000 25 - 1-20
60
Ni 0.1 3 1C
200 25 500 1-20
63
Cu 1.1 3 2
3000 250 1000 1-20
118
Sn 0.127 3 - - 6000 - - 1-20
121
Sb 0.019 3 - - 1200 - - 1-20
137
Ba 0.06 3 - - 1400 - - -
27
Al 4.2 4 - - - - - -
55
Mn 0.1 4 2 - - 250 - 1-20
56
Fe 0.4 4 3
- - 1300 -
66
Zn 0.5 4 3
- - 1300 -
88
Sr 0.048 - - - - - - -
209
Bi 0.02 - - - - - - -
a
LOD = limits of detection of the analytical method used in this study.
b
Class 1 are human toxicants that have limited or no use in the manufacture of pharmaceuticals. Class 2 are elements generally considered as route-
dependent human toxicants. They are divided in sub-classes 2A and 2B based on their high or reduced probability of occurrence in the drug product.
respectively. Class 3 are other classes are elements with low toxicities.
c
Class 1 are metals of significant safety concern. Class 2 are metals of low safety concern. Class 3 are metals of minimal safety concern.
d
Oral permitted daily doses (PDE). µg day–1.
e
Limits in sum (adapted from monographs of substances . if applicable; some substances are not attended by heavy metal limits).
f

= element included in the Guideline USP <232>.
g
Combination not to exceed.
h
Not a safety concern.

27
Table S-7: Lists of extractable metals to be determined according to the specifications of USP 661.1.

Type of plastica
Nuclide
PE CO PP PET, PETG PVC
75
As





111
Cd





202
Hg





208
Pb





52
Cr


60
Ni





118
Sn

121
Sb

137
Ba


27
Al




55
Mn

66
Zn





51
V





59
Co





b
Ti




b
Zr

b
Ge

b
Ca

a
PE = Polyethylene, CO = Cyclic Olefins, PP = Polypropylene, PET = Polyethylene
Terephthalate, PETG = Polyethylene Terephthalate G, PVC = Polyvinyl Chloride.
b
Not analysed in this study.

28
 Table S-8: (A) Heat map for the concentrations found in SUBs for the analyzed elements by ICP-MS. (B) Heat map of frequency for the analyzed elements in SUBs by ICP-MS.

(A) Heat map for the concentrations found in SUBs for the analysed elements by ICP-MS.

Average concentrations (ng cm−2)

Extraction solvents
7 27 51 52 55 56 59 60 63 66 88 95 107 111 118 121 137 193 197 202 208 209
Li Al V Cr Mn Fe Co Ni Cu Zn Sr Mo Ag Cd Sn Sb Ba Ir Au Hg Pb Bi
a a a a a a a a a a a
0.1 M H3PO4  230.82  3.93 9.17 200.85  2.97 8.40 53.54 5.86      5.39    10.83 a
Media 1.32 a 2.53 a a a a 8.77 44.06 a a 0.35 0.33 0.50 3.77 a 1.31 a a a 1.74 a
Ammonium buffer 50mM
0.84 a a 9.77 0.88 a 1.86 24.04 a 11.95 2.09 0.44 a a a a 0.74 a a 0.79 1.07 a
pH 9.5
WFI a 149.13 a 1.88 3.54 11.51 0.48 3.38 8.60 56.69 1.06 a 0.41 0.21 0.95 0.48 3.43 0.32 0.68 0.33 0.68 0.59
 a Not present in the sample in reportable quantities.

(B) Heat map of frequency for the analysed elements in SUBs by ICP-MS.

Frequency (%)
Extraction solvents
7 27 51 52 55 56 59 60 63 66 88 95 107 111 118 121 137 193 197 202 208 209
Li Al V Cr Mn Fe Co Ni Cu Zn Sr Mo Ag Cd Sn Sb Ba Ir Au Hg Pb Bi

0.1 M H3PO4 88 50 100 100 100 38 100 100 100 6

Media 32 9 15 71 12 53 9 29 9 88
Ammonium buffer 50mM
38 74 32 15 6 9 68 21 32 82 9
pH 9.5
WFI 53 100 100 88 9 53 50 38 82 15 32 47 88 41 18 15 32 88 38

29
Table S-9: Overview of target solvents. Regulatory background and limits.

LODa Class USP PDE Concentration

Solvent (µg) CAS number
<467> (mg day-1) limit (ppm)f
d
1,1,1-trichloroethane 0.01 1 71-55-6 1500
d
1,1-dichloroethene 0.01 1 75-35-4 8
d
1,2-dichloroethane 0.01 1 107-06-2 5
d
Benzene 0.02 1 71-43-2 2
d
Carbon tetrachloride 0.003 1 56-23-5 4
1.2-dichloroethylene cisb 0.09 2A 156-59-2 18.7 1870
1.2-dichloroethylene transb 0.02 2A 156-60-5 18.7 1870
1.4-Dioxane 2.0 2A 123-91-1 3.8 380
Ethylbenzene 0.05 2A 100-41-4 3.8 380
Methyl Alcohol 5.0 2A 67-56-1 30.0 3000
Methylcyclohexane 0.09 2A 108-87-2 11.8 1180
Methylene chloride 1.0 2A 75-09-2 6.0 600
m-Xylene/ p-Xylenec 0.5 2A 108-38-3/106-42-3 21.7 2170
o-Xylenec 0.1 2A 95-47-6 21.7 2170
Tetrahydrofuran 0.1 2A 109-99-9 7.2 720
Toluene 0.003 2A 108-88-3 8.9 890
1.2-dimethoxyethane 0.003 2B 110-71-4 1.0 100
2-Hexanone 0.1 2B 591-78-6 0.5 50
Chloroform 0.01 2B 67-66-3 0.6 60
n-Hexane 0.2 2B 110-54-3 2.9 290
Nitromethane 0.01 2B 75-52-5 0.5 50
Pyridine 0.002 2B 110-86-1 2.0 200
Tetralin 0.02 2B 119-64-2 1.0 100
Trichloroethylene 0.002 2B 79-01-6 0.8 80
e e
Acetone 1.50 3 67-64-1
e e
Isopropyl Alcohol 2.00 3 67-63-0
a
LOD = limits of detection of the analytical method used in this study.
b
PDE and concentration limits specified only for 1.2-dichloroethylene.
c
Usually 60% m-xylene. 14% p-xylene. 9% o-xylene with 17% ethylbenzene.
d
No data provided. because they should be avoided.
e
Limited by GMP or other quality-based requirements in drug substances . excipients and drug products.
f –1
ppm units correspond to µg g , as it is indicated in USP 467, the source of this data.

30
Table S-10: Heat maps showing the average concentrations found in SUBs for analyzed solvents by HS-GC-MS.
2
Average concentrations (pg cm− )

1,1,1-trichloroethane

Carbon tetrachloride
1,2-dichloroethylene

1,2-dichloroethylene
Methylene chloride
1,1-dichloroethene

Isopropyl Alcohol

Tetrahydrofuran
Methyl Alcohol

Nitromethane

Chloroform
n-Hexane
Acetone
Brand/Model

trans

cis
I-A 262.8 0.36 39.5 56.9 43.0 0.88 5.3 2.1 0.34 2.4 0.70 2.1 0.33
I-B 353.4 0.35 45.3 63.5 43.3 0.64 7.7 6.0  a 2.4 0.72 2.3 0.45
II-A 159.7 0.67 49.3 1928.8 26.0 2.2 10.6 29.6 1.5 3.1 0.36 1.2 0.18
II-B 958.3 5.7 291.0 9379.2 28.4 6.7 59.0 2.5 1.2 7.3 2.2 0.56 0.11
III 1135.3 0.27 103.6 111.4 37.6 9.2 7380.5 35.6 3.3 5.7 1.1 2.4 0.36
IV 323.7 2.2 76.5 3905.9 26.5 1.2 5.4 6.2  a 25.7 0.18 0.30 0.08
V-A 149.0 0.59 47.1 77.5 58.5 0.57 8.0 15.2  a 3.1 0.82 1.5 0.58
V-B 286.2 0.59 67.6 81.5 60.1 0.64 10.5 10.5  a 3.0 1.2 2.9 0.48

2
Average concentrations (pg cm− )
1,2-dimethoxyethane

Methylcyclohexane
1,2-dichloroethane

Trichloroethylene

Ethylbenzene
1,4-Dioxane

m-p-Xylene

o-Xylene
Benzene

Pyridine

Toluene

Tetralin
Brand/Model

I-A 0.55 0.15 0.86 0.54 2.1 117.1 0.94 0.79 1.6 14.0 2.6 1.6
I-B 2.4 0.16 1.2 0.64 2.3 133.1 1.2 0.69 2.4 17.8 4.3 1.9
II-A 6.8 0.07 0.74 0.30 14.3 69.7 0.41 8.9 12.5 20.8 3.6 0.57
II-B 4.6 0.18 0.39 0.12 26.8 90.3 0.20 9.8 44.9 23.7 5.9 0.36
III 10.8 0.09 1.0 0.57 51.8 214.2 1.0 0.73 9.9 19.1 4.0 2.1
IV 1.8 0.00 0.15 0.05 3.8 59.0 0.05 0.08 13.7 28.3 3.1 0.39
V-A 2.9 0.10 0.84 0.70 3.6 98.2 0.81 8.5 9.0 45.7 14.5 1.4
V-B 2.5 0.27 1.4 0.80 2.8 212.9 1.6 18.8 6.4 26.7 7.9 2.3
 a Not present in the sample in reportable quantities.

31