Inter-Laboratory Reproducibility and Interchangeability of 3R4F and 1R6F Reference Cigarettes in Mainstream Smoke Chemical Analysis and In Vitro Toxicity Assays

Sakai, Yuka; Mori, Sakura; Yanagimachi, Miyuki; Takahashi, Tomohiro; Shibuya, Kaori; Kumagai, Asami; Ishikawa, Shinkichi; Ito, Shigeaki; Fukushima, Toshiro

doi:10.2478/cttr-2020-0011

Figures & Tables

Relative differences in the chemical constituents of mainstream cigarette smoke between 3R4F and 1R6F under the ISO standard smoking regimen. Circles and triangles indicate the relative differences in the chemical yields of 1R6F compared with 3R4F in the current and Jaccard studies (12), respectively. The boxes show the critical difference ranges of 3R4F. “Empty” or “filled” symbols indicate whether the relative difference was within or out of the critical difference range, respectively. LOQ indicates the limit of quantitation.

Relative differences in the chemical constituents of mainstream cigarette smoke between 3R4F and 1R6F under the ISO intense smoking regimen. Circles and triangles indicate the relative differences in the chemical yields of 1R6F compared with 3R4F in the current and Jaccard studies (12), respectively. The boxes show the critical difference ranges of 3R4F. “Empty” or “filled” symbols indicate whether the relative difference was within or out of the critical difference range, respectively. LOQ indicates the limit of quantitation.

Relative differences between 3R4F and 1R6F in the Ames assay. (A) ISO standard smoking regimen; (B) ISO intense smoking regimen. Circles and triangles indicate the relative differences in the mutagenic slope value of 1R6F compared with 3R4F in the current and Jaccard studies (12), respectively. The boxes show the critical difference ranges of 3R4F. “Empty” symbols indicate the relative difference were within the critical difference ranges. Equivocal indicates there was no reproducible significant increase in revertants over three replicates. Non-mutagenic means there was no significant increase in revertants over three replicates.

Relative differences between 3R4F and 1R6F in the MN assay. (A) ISO standard smoking regimen; (B) ISO intense smoking regimen. Circles and triangles indicate the relative difference in the genotoxic logit slope values of 1R6F compared with 3R4F in the current and Jaccard studies (12), respectively. The boxes show the critical difference ranges of 3R4F. “Empty” or “filled” symbols indicate whether the relative difference was within or out of the critical difference range, respectively.

Relative differences between 3R4F and 1R6F in the NRU assay. (A) ISO standard smoking regimen; (B) ISO intense smoking regimen. Circles and triangles indicate the relative differences in the IC50 values of 1R6F compared with 3R4F in the current and Jaccard studies (12), respectively. The boxes show the critical difference ranges of 3R4F. “Empty” or “filled” symbols indicate whether the relative difference was within or out of the critical difference range, respectively.

Dose-response curves in the cell viability assay. Cell viability was measured after 24 h exposure to TPM. The values for the solvent control were set at 100 (%). “Empty” and “filled” symbols represent the means of the viability for 1R6F and 3R4F, respectively. The error bars indicate standard error (n = 3). The solid and dotted lines indicate the dose-response curves for the ISO standard and intense smoking regimens, respectively.

Dose-response curves in the GSH/GSSG assay. The intracellular GSH/GSSG ratio was determined after exposure for 2 h to the TPM. “Empty” and “filled” symbols represent the means of the GSH/GSSG ratios of 1R6F and 3R4F, respectively. The error bars indicate standard error (n = 3). The solid and dotted lines indicate the dose-response curve under the ISO standard and intense smoking regimens, respectively.

Dose-response curves in the ARE-luciferase reporter assay. Luciferase activity was measured after exposure to the TPM for 24 h to determine ARE gene activity. “Empty” and “filled” symbols represent the means of ARE-luciferase gene activity for 1R6F and 3R4F, respectively. The error bars indicate standard error (n = 3). The solid and dotted lines indicate the dose-response curve under the ISO standard and intense smoking regimens, respectively.

Correlation of the absolute values of the relative differences between 1R6F and 3R4F in chemical analysis. (A) ISO standard smoking regimen; (B) ISO intense smoking regimen. X-axis: relative differences in current study (%), Y-axis: relative differences in Jaccard study (%). The coefficients of determination under the ISO standard and intense smoking regimens were R2 = 0.73 and R2 = 0. 74, respectively.

Analysis methods used for specific analytes in mainstream cigarette smoke_

Analytes	Fraction (smoking machine)	Trap	Chromatograph, detection	Notes	Ref.
TSNAs
NNK, NNN, NAB, NAT	TPM (RM20H^a)	A glass-fiber filter	HPLC; Agilent 1290 infinity system ^c, Triple Quad 4500 MS system ^d	The extract with ammonium acetate solution was syringe filtered.	26
Carbonyls
Formaldehyde, acetaldehyde, acrolein, crotonaldehyde, acetone, propionaldehyde, n-butylaldehyde, MEK	WS (SM450RH^b)	Two impingers with 2, 4-dinitrophenyl-hydrazine solution	HPLC; Agilent 1290 Infinity system ^c, Diode array detection	Deribatized solution was stabilized with Tris base.	27
VOCs
1,3-Butadiene, benzene, isoprene, acrylonitrile, toluene	GVP (SM450RH^b)	Two cryogenic impingers with methanol	GC/MS; 5975C GC/MSD ^c, Electron impact ionization	–	28
Cyanic compound
HCN	GVP TPM (SM450RH^b)	A glass-fiber filter and an impinger with NaOH solution	Continuous flow analyzer; STAT-2000 ^e	The extract with NaOH was syringe filtered.	29
PAH
B[a]P	TPM (SM450RH^b)	A glass-fiber filter	HPLC; HPLC-1100 system ^c, Fluorescence detection	The extract with cyclohexane was syringe filtered and purified using SPE by passing through a silica cartridge and an NH₂ cartridge, followed by the elution with hexane.	30
Phenols
Hydroquinone, resorcinol, catechol, phenol, o-cresol, m-cresol, p-cresol Ammonia	TPM (SM450RH^b)	A glass-fiber filter	HPLC; Agilent 1290 Infinity system ^c, Fluorescence detection	The extract with acetic acid was syringe filtered.	31
Ammonia	GVPTPM (RM20H^a)	A glass-fiber filter and two impingers with sulfuric acid	Cation exchange chromatograph; ICS-3000 ^f, Suppressed ion conductivity detection	The extract with ultrapure water was syringe filtered.	32
NO_x
NO, NO_x	GVP (RM20H^a)	–	Online gas phase chemiluminescence analyzer; CLD822Mh ^g	–	33
SVOCs
Pyridine, quinoline, styrene	GVP TPM (RM20H^a)	A glass-fiber filter and two cyrogenic impingers with methanol	GC/MS; 5975C GC/MSD ^c, Electron impact ionization	TPM was extracted with the impinger solution.	34
Aromatic amines
1-Aminonaphthalene, 2-aminonaphthalene, 3-aminobiphenyl, 4-aminobiphenyl	TPM (RM20H^a)	A glass-fiber filter	GC/MS; 5975C GC/MSD ^c, Chemical ionization source (selected ion monitoring)	The extract with hydrochloric acid solution was syringe filtered and loaded to the conditioned SPE-I with ammonia solution and methanol, and then loaded to the SPE-II with toluene. The derivatized solution with heptafluorobutyric acid anhydride solution was loaded to SPE-III.	35
Heavy metals
Mercury	GVP (RM20H^a)	An inpinger with acidified potassium permanganate	AAS; FIMS 400 ^h, cold vapor atomic absorption spectrometry	Hydrogen peroxide and ultrapure water were added for microwave digestion. Hydroxlyamine hydrochloride was added. Mercury ions in the sample were reduced by stannous chloride.	36
Arsenic, cadmium, chromium, nickel, lead, selenium, beryllium, cobalt	TPM (RM20H^a)	Glass electrostatic precipitate tube	ICP/MS; Agilent 7500cxc	Collected metals with methanol were mixed with Nitric acid solution, TritonX-100 solution by ultrosonication.	–

Chemical constituents in the mainstream smoke of 1R6F and 3R4F_

Analyte	Unit	ISO standard smoking regimen						ISO intense smoking regimen

		1R6F		3R4F		Ratio (1R6F/3R4F)		1R6F		3R4F		Ratio (1R6F/3R4F)

		mean	S.E.	mean	S.E.			mean	S.E.	mean	S.E.
TPM	mg/cig	10.5	0.148	9.9	0.389	106	(–)	42.4	1.19	43.3	0.304	97.9	(–)
Nicotine	mg/cig	0.720	0.0116	0.700	0.0152	103	(111)	1.92	0.0326	1.97	0.0492	97.5	(95.5)
Carbon monoxide	mg/cig	10.0	0.143	10.6	0.371	94.3	(102)	27.5	0.38	30.1	0.622	91.4	(85.9)
TSNAs
NNN	ng/cig	100	3.58	116	4.02	86.2	(76.3)	221	5.37	279	4.92	79.2	(68.2)
NAT	ng/cig	107	0.894	118	2.68	90.7	(80.9)	253	5.37	291	3.13	86.9	(77.5)
NAB	ng/cig	12.9	0.224	15.0	0.224	86.0	(82.7)	29.6	0.626	35.6	0.358	83.1	(76.7)
NNK	ng/cig	77.8	2.33	103	3.13	75.5	(74.8)	177	3.58	243	4.92	72.8	(65.1)
Carbonyls
Formaldehyde	μg/cig	37.6	1.21	27.3	1.07	138	(136)	134	4.02	106	3.58	126.4	(119)
Acetaldehyde	μg/cig	548	9.39	594	11.18	92.3	(89.1)	1570	17.89	1697	14.31	92.5	(87.1)
Acetone	μg/cig	179	2.68	202	3.13	88.6	(86.7)	503	3.13	574	5.81	87.6	(84.8)
Acrolein	μg/cig	55.9	1.21	50.2	1.12	111	(97.4)	177	1.34	166	0.447	106.6	(96.1)
Propionaldehyde	μg/cig	40.3	0.626	42.9	0.805	93.9	(90.9)	115	0.894	124	1.34	92.7	(88.1)
Crotonaldehyde	μg/cig	12.2	0.358	11.1	0.358	110	(101)	54.8	0.402	54.9	0.537	99.8	(92.4)
MEK	μg/cig	41.8	0.581	48.3	0.894	86.5	(85.3)	119	0.447	143	1.79	83.2	(82.4)
n-Butylaldehyde	μg/cig	25.0	0.492	27.5	0.76	90.9	(91.2)	68.1	0.939	77.1	1.3	88.3	(86.3)
VOCs
1,3-Butadiene	μg/cig	40.9	0.358	41.5	1.12	98.6	(104)	96.1	1.21	95.2	1.7	100.9	(102)
Isoprene	μg/cig	314	3.58	341	8.05	92.1	(99.3)	766	15.21	816	16.1	93.9	(94.1)
Acrylonitrile	μg/cig	8.19	0.116	9.39	0.264	87.2	(100)	26.1	0.224	28.9	0.179	90.3	(93.7)
Benzene	μg/cig	42.2	0.358	46.1	1.16	91.5	(100)	91.2	0.671	99.4	1.03	91.8	(92.7)
Toluene	μg/cig	64.7	0.76	78.6	2.1	82.3	(91.8)	160	1.34	191	1.34	83.8	(86.3)
Cyanic compounds
HCN	μg/cig	98.4	1.52	99.2	2.37	99.2	(107)	404	10.29	470	8.94	86.0	(903)
PAH
B[a]P	ng/cig	7.51	0.121	6.49	0.17	116	(110)	16.9	0.268	16.9	0.716	100.0	(91.4)
Phenols
Hydroquinone	μg/cig	35	0.402	30.4	0.313	115	(113)	87.1	0.984	84.1	0.447	103.6	(108)
Resorcinol	μg/cig	0.718	0.0237	0.494	0.0152	145	–	2.15	0.0492	1.6	0.0492	134.4	–
Catechol	μg/cig	40	0.313	36.7	0.313	109	(111)	89.3	0.984	88.1	0.537	101.4	(106)
Phenol	μg/cig	8.79	0.215	6.69	0.085	131	(131)	12.4	0.224	11	0.179	112.7	(105)
p-Cresol	μg/cig	5.09	0.107	4.31	0.0537	118	(115)	8.22	0.157	7.81	0.13	105.2	(100)
m-Cresol	μg/cig	2.01	0.0537	1.78	0.0224	113	(115)	3.08	0.085	2.99	0.0894	103.0	(94.6)
o-Cresol	μg/cig	2.36	0.0537	2.14	0.0268	110	(111)	3.47	0.112	3.72	0.157	93.3	(85.7)
Other amines
Ammonia	μg/cig	8.72	0.13	8.04	0.0581	108	(130)	30.6	0.76	30.4	0.76	100.7	(106)
NO_x
Nitric oxide	μg/cig	148	5.63	194	7.06	76.2	(67.3)	358.86	15.07	529	20.89	67.9	(69.9)
Nitric oxides	μg/cig	163	6.46	217	7.92	75.1	(67.8)	399.08	16.94	589	23.11	67.7	(70.4)
SVOCs
Pyridine	μg/cig	7.33	0.112	7.41	0.148	98.9	(91.6)	36.8	0.581	42.1	0.268	87.4	(103)
Quinoline	μg/cig	0.273	0.00178	0.228	0.00358	120	(152)	0.546	0.0139	0.53	0.00492	103.0	(105)
Styrene	μg/cig	5.68	0.0671	6.79	0.0626	83.7	(76)	23	0.224	27.3	0.134	84.2	(94)
Aromatic amines
1-Aminonaftalene	ng/cig	13	0.358	12.9	0.134	101	(97.2)	24.5	0.358	26.9	0.179	91.1	(91.8)
2-Aminonaftalene	ng/cig	7.69	0.367	7.82	0.0581	98.3	(122)	15.5	0.581	16.7	0.134	92.8	(89.5)
3-Aminobiphenyl	ng/cig	2.04	0.085	2.2	0.0179	92.7	(103)	4.71	0.183	5.21	0.0224	90.4	(82.5)
4-Aminobiphenyl	ng/cig	1.41	0.0626	1.54	0.0134	91.6	(96.1)	3.35	0.125	3.71	0.0313	90.3	(80.1)
Heavy metals
Mercury	ng/cig	2.33	0.0179	2.58	0.0313	90.3	(93.8)	5.26	0.0268	6.01	0.0537	87.5	(95.1)
Lead	ng/cig	11.7	0.268	10.6	0.179	110	(–)	31.1	0.581	33.8	0.626	92.0	(–)
Cadmium	ng/cig	36.6	0.671	34.3	0.179	107	(107)	95.3	0.984	114	1.79	83.6	(81.7)
Chromium	ng/cig	<LOD	–	<LOD	–	–	(–)	<LOD		<LOD	–	–	(–)
Nickel	ng/cig	<LOQ	–	<LOQ	–	–	(–)	<LOQ		<LOQ	–	–	(–)
Beryllium	ng/cig	<LOD	–	<LOD	–	–	(–)	<LOD		<LOD	–	–	(–)
Cobalt	ng/cig	<LOQ	–	<LOQ	–	–	(–)	<LOQ		<LOQ	–	–	(–)
Selenium	ng/cig	<LOQ	–	<LOQ	–	–	(–)	<LOQ		<LOQ	–	–	(–)
Arsenic	ng/cig	2.41	0.0313	2.56	0.0268	94.1	(–)	6.72	0.152	7.92	0.179	84.8	(–)

IC50 values of 1R6F and 3R4F TPM in the GSH/GSSG assay_ IC50 values in the GSH/GSSG assay were calculated by inverse estimation of the effective concentration at which the normalized luminescence was reduced by 50%_ S_E_ stands for standard error_ p values were calculated based on Student's t-test (n = 3)_

Smoking regimen	1R6F IC₅₀ (μg/mL)		3R4F IC₅₀ (μg/mL)		Relative difference (1R6F/3R4F)		p value (t-test)

	Mean	S.E.	Mean	S.E.	Mean	S.E.
Standard	35.0	7.74	40.0	9.36	0.877	0.0128	0.70
Intense	66.5	8.25	73.4	18.9	0.970	0.139	0.76

Characteristics of 1R6F and 3R4F reference cigarettes_ The data were obtained from literature (12, 19, 20)_

Parameter	3R4F	1R6F
Physical data
Cigarette length (mm)	84	83
Tobacco rod length (mm)	57	56
Filter length (mm)	27	27
Tobacco rod circumference (mm)	24.8	24.6
Cigarette weight (g)	1.1	0.89
Filter ventilation (%)	29	33
Paper permeability (CU)	24	45
Resistance to draw (mm H₂O)	128	107
Blend composition
Flue cured (%)	35	34
Burley (%)	22	24
Maryland (%)	1.4	–
Oriental (%)	12	12
Reconstituted (%)	29.6	20
Expanded flue cured (%)	–	7
Expanded Burley (%)	–	3
Humectants
Glycerol (%)	2.7	1.7
Propylene glycol (%)	–	1
Isosweet (%)	6.4	6.3
Yield data from supplier (ISO standard smoking regimen)
Puff count	9.0	7.5
TPM (mg/cig)	11	10
“Tar” (mg/cig)	9.4	8.6
Nicotine (mg/cig)	0.73	0.72
Carbon monoxide (mg/cig)	12.0	10.1
Yield data from supplier (ISO intense smoking regimen)
Puff count	^a	8.7
TPM (mg/cig)	^a	46.8
“Tar” (mg/cig)	^a	29.1
Nicotine (mg/cig)	^a	1.90
Carbon monoxide (mg/cig)	^a	28.0

IC50 values of 1R6F and 3R4F TPM in the cell viability assay_ The IC50 values were calculated by inverse estimation of the effective concentration at which the normalized fluorescence was reduced by 50%_ S_E_ stands for standard error_ p values were calculated based on Student's t-test (n = 3)_

Smoking regimen	1R6F IC₅₀ (μg/mL)		3R4F IC₅₀ (μg/mL)		Relative difference (1R6F/3R4F)		p value (t-test)

	Mean	S.E.	Mean	S.E.	Mean	S.E.
Standard	75.0	2.44	73.3	2.19	1.02	0.0281	0.62
Intense	79.5	3.98	82.2	4.06	0.975	0.0836	0.67

Genotoxic slope parameters of the logit function of 1R6F and 3R4F TPM in the MN assay_

Smoking regimen	Term and S9 metabolic activation	1R6F slope parameter (mg/mL)		3R4F slope parameter (mg/mL)		Ratio (1R6F/3R4F)

		Mean	S.E.	Mean	S.E.	Mean		S.E.
Standard	“Short” without S9	10.2	1.26	8.11	1.22	1.27	(1.18)	0.0836
Standard	“Short” with S9	6.36	1.37	5.67	1.09	1.11	(1.01)	0.520
Standard	“Long” without S9	17.8	3.22	15.2	2.22	1.17	(1.05)	0.100
Intense	“Short” without S9	7.36	1.10	6.37	1.14	1.17	(1.12)	0.0650
Intense	“Short” with S9	4.29	0.850	3.87	0.487	1.09	(1.05)	0.0750
Intense	“Long” without S9	13.8	2.11	11.6	2.02	1.21	(0.894)	0.0550

IC50 values of the TPM and GVP of 1R6F and 3R4F in the NRU assay_

Smoking regimen	Fraction	1R6F IC₅₀ (μg/mL)		3R4F IC₅₀ (μg/mL)		Ratio (1R6F/3R4F)

		Mean	S.E.	Mean	S.E.	Mean		S.E.
Standard	TPM	68.6	1.32	70.9	2.67	0.969	(0.979)	0.0221
Standard	GVP	101	6.89	95.9	7.11	1.05	(1.18)	0.00707
Intense	TPM	94.2	4.84	105	1.97	0.901	(1.05)	0.0429
Intense	GVP	104	6.36	107	4.61	0.971	(1.15)	0.0334

Slope values of 1R6F and 3R4F TPM in the ARE-luciferase reporter assay_ The slope values in the ARE-luciferase reporter assay were calculated by linear regression analysis over the dose range where cell viability was higher than 50%_ S_E_ stands for standard error_ p values were calculated based on Student's t-test (n = 3)_

Smoking regimen	1R6F slope value		3R4F slope value		Relative difference (1R6F/3R4F)		p value (t-test)

	Mean	S.E.	Mean	S.E.	Mean	S.E.
Standard	0.394	0.0441	0.305	0.0456	1.32	0.133	0.23
Intense	0.377	0.0682	0.266	0.0376	1.46	0.292	0.23

Mutagenic slope values of 1R6F and 3R4F TPM in the Ames assay_

Smoking regimen	Strain and S9 metabolic activation	1R6F slope value (Revetants/μg)		3R4F slope value (Revetants/μg)		Ratio (1R6F/3R4F)

		Mean	S.E.	Mean	S.E.	Mean		S.E.
Standard	TA98 with S9	2.31	0.0748	2.40	0.145	0.966	(0.841)	0.0532
Standard	TA98 without S9	equivocal	–	equivocal	–	–	(1.51)	–
Standard	TA100 with S9	0.849	0.0430	0.886	0.0509	0.959	(0.902)	0.00907
Standard	TA100 without S9	0.354	0.0213	0.298	0.0365	1.23	(–)	0.171
Standard	TA1537 with S9	0.300	0.0301	0.305	0.0455	1.00	(1.05)	0.0786
Standard	TA1537 without S9	equivocal	–	equivocal	–	–	(–)	–
Standard	TA1535 with S9	non-mutagenic	–	non-mutagenic	–	–	(–)	–
Standard	TA1535 without S9	non-mutagenic	–	non-mutagenic	–	–	(–)	–
Standard	TA102 with S9	non-mutagenic	–	non-mutagenic	–	–	(–)	–
Standard	TA102 without S9	non-mutagenic	–	non-mutagenic	–	–	(–)	–
Intense	TA98 with S9	1.10	0.0342	1.02	0.0368	1.072	(0.971)	0.0259
Intense	TA98 without S9	non-mutagenic		euivocal	–	–	(1.18)	–
Intense	TA100 with S9	0.475	0.0213	0.426	0.0119	1.12	(1.07)	0.0756
Intense	TA100 without S9	0.233	0.0178	0.190	0.0115	1.24	(–)	0.136
Intense	TA1537 with S9	0.162	0.0258	0.187	9.50E-3	0.864	(0.939)	0.116
Intense	TA1537 without S9	equivocal	–	equivocal	–	–	(–)	–
Intense	TA1535 with S9	non-mutagenic	–	non-mutagenic	–	–	(–)	–
Intense	TA1535 without S9	non-mutagenic	–	non-mutagenic	–	–	(–)	–
Intense	TA102 with S9	non-mutagenic	–	non-mutagenic	–	–	(–)	–
Intense	TA102 without S9	non-mutagenic	–	non-mutagenic	–	–	(–)	–

Inter-Laboratory Reproducibility and Interchangeability of 3R4F and 1R6F Reference Cigarettes in Mainstream Smoke Chemical Analysis and In Vitro Toxicity Assays

Figures & Tables

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Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure A1

Analysis methods used for specific analytes in mainstream cigarette smoke_

Chemical constituents in the mainstream smoke of 1R6F and 3R4F_

Characteristics of 1R6F and 3R4F reference cigarettes_ The data were obtained from literature (12, 19, 20)_

IC50 values of 1R6F and 3R4F TPM in the cell viability assay_ The IC50 values were calculated by inverse estimation of the effective concentration at which the normalized fluorescence was reduced by 50%_ S_E_ stands for standard error_ p values were calculated based on Student's t-test (n = 3)_

Genotoxic slope parameters of the logit function of 1R6F and 3R4F TPM in the MN assay_

IC50 values of the TPM and GVP of 1R6F and 3R4F in the NRU assay_

Mutagenic slope values of 1R6F and 3R4F TPM in the Ames assay_

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