Comprehensive review of epidemiological and animal studies on the potential carcinogenic effects of nicotine per se

Figures & data

Table 1. Plasma and urinary concentrations of nicotine and cotinine in users of nicotine delivery systems (with comparison to conventional cigarettes).

	Blood concentration range (ng/ml)		Urinary concentration range (ng/ml)
Nicotine-containing product	Nicotine	Cotinine	Nicotine	Cotinine
NRT
Dermal patch	6–28*,†,‡,¶	161–170‡	–	–
	7–25¶	80–300¶		300–1300¶
Sublingual tablet	7–30*,†	–	–	–
Nasal spray	3–20*,†	170 ± 95§	–	–
Gum	7–43*,†	–	–	900∥
Inhaler	2–40*,†	–	–	–
Lozenge	35†	–	–	900∥
ENDS	9–24#,**,††,‡‡	99–156¶¶	604–1250§§	1420–2480§§
Conventional cigarette	10–50*	184 ± 106∥∥	1191 ± 1145∥∥	1149 ± 736∥∥

*From Schneider et al. (2001) (review article).

†From Shiffman et al. (2005) (review article; maximum concentrations).

‡From Gourlay and Benowitz (1996) (after 4–6 h of patch application).

¶From Lawson et al. (1998a, 1998b) (approximate values for 11-mg to 44-mg patch users).

§From Benowitz et al. (1997) (mean ± SD).

∥From Kotlyar et al. (2011) (approximate values, 4-mg lozenges or gums).

#From Ramoa et al. (2015).

**From Lopez et al. (2015).

††From D'Ruiz et al. (2015) (after 90 min of ad libitum use).

‡‡From Dawkins and Corcoran (2014) (ad libitum).

¶¶From Pacifici et al. (2015) (switched to e-cigarette for 1–8 months).

§§From Hecht et al. (2015) (95% confidence interval).

∥∥From Roethig et al. (2009) (population estimates; urine data previously unpublished; mean ± SD).

Table 2. Statements by authoritative bodies on the potential carcinogenic effect of nicotine per se.

Authoritative body	Authoritative statement	Attributing statement
WHO FCTC*	–	• Not a carcinogen, but may function as a tumor promoter
IARC†	(No full assessment)	• Not generally considered to be a carcinogen • Biological effects through receptor binding • Likely enhances carcinogenicity
FDA‡	(HPHC list)	• Not listed as a carcinogen
US Surgeon General¶	“The evidence is inadequate to infer the presence or absence of a causal relationship between exposure to nicotine and risk for cancer.”	• Insufficient data to conclude that nicotine causes or contributes to cancer in humans • No support from animal studies for the hypothesis that nicotine is a complete carcinogen • Tumor promoter in some experimental models
US Surgeon General§	–	• Mechanistic data supporting a role of nicotine in stimulating carcinogenesis
UK RCP∥	–	• No direct evidence for carcinogenicity of NRTs

HPHC: harmful and potentially harmful constituents; IARC: International Agency for Research on Cancer; NRT: nicotine replacement therapy product; US FDA: US Food and Drug Administration; UK RCP: Royal College of Physicians; WHO FCTC: World Health Organization Framework Convention on Tobacco Control.

*From WHO Framework Convention on Tobacco Control (2014).

†From International Agency for Research on Cancer (2012).

‡From US Food and Drug Administration (2012).

¶From US Department of Health and Human Services (2014).

§From US Department of Health and Human Services (2010).

∥From UK Royal College of Physicians (2007).

Figure 1. Overview of findings from literature searches and criteria used for the inclusion and exclusion of publications for critical evaluation. *The total number is only 74 because several publications include both complete and modulating cancer studies.

Table 3. Adequacy evaluation criteria for individual animal studies on the potential carcinogenicity of nicotine.

Category	Adequate study parameters
Route of administration	Inhalation, oral, dermal
Group size	50 per sex (100 of single sex)
Dose–response	Three (or two) dose levels
Daily dose (average)	Eliciting signs of toxicity, or
	≥1 mg/(kg × d)
Duration of exposure	≥18 months (complete carcinogen studies)
	Any duration (modulating carcinogenesis studies)
Quality (subjective)	e.g., Nicotine biomonitoring, body weight data

Notes:

(1) Adequacy criteria for group size, dose–response, and study duration were derived from the carcinogenicity study design guidance provided by the Organisation for Economic Co-operation and Development (2009). Other adequacy criteria were subjectively set by the authors.

(2) Adequacy was scored with plus (+) or minus (−). If information was lacking, minus (−) was also applied.

(3) Plus (+) scores of a study were totaled for an overall adequacy score. The maximum plus (+) score is 6 (studies on complete carcinogenesis) or 5 (modulating studies), with >2(+) representing a high-adequacy score and ≤2(+) representing a low-adequacy score.

Figure 2. Overview of nicotine biomonitoring data in mouse and rat studies (various shaped arrows). Data referring to human exposure and blood nicotine levels are presented as a black bar indicating the range of data presented in Table 1 (users of nicotine delivery systems). For species comparison, mouse data are shown from studies with oral administration via the drinking water (red color: Pietilä et al. 1995; Sparks & Pauly 1999; Grabus et al. 2005; Arany et al. 2011; AlSharari et al. 2013), s.c. osmotic minipump (brown color: AlSharari et al. 2013) and feeding (violet color: Theophilus et al. 2012). Rat data are shown from nicotine inhalation (black color: Werley et al. 2014), continuous s.c. administration via minipump (dark blue color: Cheng et al. 2005), and feeding (blue color: Theophilus et al. 2012).

Table 4. Reported findings of relevant studies for evaluating the potential of nicotine to act as a complete carcinogen.

Route/adequacy scoring	Number of (sub-)studies	Positive*	Negative*	Labs†
Inhalation
Total	1	0	1	1
High score	1	0	1	1
Low score	0	0	0	0
Oral
Total	7	0	7	5
High score	5	0	5	5
Low score	2	0	2	2
Dermal
Total	1	0	1	1
High score	0	0	0	0
Low score	1	0	1	1
Other‡
Total	14	2	3	12
High score	1	0	1	1
Low score	13	2	2	11

To facilitate comparisons, the (sub-)studies were grouped into high and low-adequacy scoring studies with overall scores subjectively set to >2 and ≤2, respectively.

*As judged by the authors of the respective studies, where “positive” indicates stimulating carcinogenesis and “negative” indicates a lack of carcinogenic effect.

†Number of different laboratories contributing studies.

‡The two positive studies (both s.c.) were those with the longest exposure period. Many shorter but still chronic studies in this category were not planned as carcinogenicity studies and were, therefore, often not judged by the respective authors as positive or negative for carcinogenesis. This resulted in a total number of studies that was higher than the sum of studies reported as positive or negative by the authors.

Figure 3. Overview of nicotine biomonitoring data in mouse and rat studies investigating potential nicotine-mediated carcinogenesis. Data (indicated by arrows) were generated in a rat inhalation study (Waldum et al. 1996) and in mouse studies with exposure to nicotine via drinking water (Wong et al. 2007; Maier et al. 2011; Murphy et al. 2011; Hermann et al. 2014; Li et al. 2015), via continuous s.c. administration (Hao et al. 2013), via i.v. administration (Li et al. 2015) and via patch and i.p. administration (Davis et al. 2009). Nicotine doses and nicotine and cotinine levels in blood and urine from users of nicotine delivery systems such as NRT products or ENDS are illustrated as black bars (blood) or diamonds and bars (urine) indicating the range of concentrations (from data presented in Table 1). Positive (cancer stimulating) and negative studies are characterized by red and green colors, respectively.

Table 5. Reported findings of relevant studies for evaluating the potential cancer modulating activity of nicotine in studies induced by physical, chemical, or transgenic means.

Route/type/adequacy scoring	Number of (sub-)studies	Positive*	Negative*	Labs†
Inhalation
Total	0	0	0	0
Oral
Total	11	4	7	8
High score	8	4	4	6
Low score	3	0	3	3
Dermal
Total	8	4	4	1
High score	0	0	0	0
Low score	8	4	4	1
Other
Total	16	9	7	10
High score	0	0	0	0
Low score	16	9	7	10

To facilitate comparisons, the (sub-)studies were grouped into high and low-adequacy scoring studies with overall scores subjectively set to >2 and ≤2, respectively.

*As judged by the authors of the respective studies, where “positive” indicates stimulating carcinogenesis and “negative” indicates a lack of carcinogenic effect.

†Number of different laboratories contributing studies.

Table 6. Reported findings of relevant studies for evaluating the potential cancer modulating activity of nicotine in xenograft studies.

Route/type/adequacy scoring	Number of (sub-)studies	Positive*	Negative*	Labs†
Inhalation
Total	0	0	0	0
Oral
Total	20	15	5	11
High score	7	4	3	4
Low score	13	11	2	7
Dermal
Total	1	1	0	1
High score	0	0	0	0
Low score	1	1	0	1
Other
Total	14	8	6	11
High score	4	2	2	3
Low score	10	6	4	8

Note: To facilitate comparisons, the (sub-)studies were grouped into high and low-adequacy scoring studies with overall scores subjectively set to >2 and ≤2, respectively.

*As judged by the authors of the respective studies, where “positive” indicates stimulating carcinogenesis and “negative” indicates a lack of carcinogenic effect.

†Number of different laboratories contributing studies.

Figure 4. Cancer modulating activity as judged positive or negative by respective authors for mouse studies using nicotine administration via drinking water (stratified by average estimated daily nicotine dose). Positive modulating activity refers to stimulating carcinogenesis. Negative activity is the lack of a stimulating effect. Symbols characterize chemical/physical/transgene-based (circles) and cancer cell-based (triangles) studies with high (full symbols) and low (open symbols) adequacy scores.

Table 7. Reported findings of relevant studies for evaluating the potential of nicotine metabolites to act as complete carcinogens.

Route	Number of (sub-)studies	Positive*	Negative*	Labs†
Inhalation
Total	0	0	0	0
Oral
Total	5	1	4	3
High score	4	1	3	2
Low score	1	0	1	1
Dermal
Total	1	0	1	1
High score	0	0	0	0
Low score	1	0	1	1
Other
Total	3	1	2	1
High score	0	0	0	0
Low score	3	1	2	1

To facilitate comparisons, the (sub-)studies were grouped into high- and low- adequacy scoring studies with overall scores subjectively set to >2 and ≤2, respectively.

*As judged by the authors of the respective studies, where “positive” indicates stimulating carcinogenesis and “negative” indicates a lack of carcinogenic effect.

†Number of different laboratories contributing studies.

Table 8. Reported findings of relevant studies for evaluating the potential cancer modulating activity of nicotine metabolites.

Route	Number of (sub-)studies	Positive*	Negative*	Labs†
Inhalation
Total	0	0	0	0
Oral
Total	7	4	3	3
High score	5	3	2	3
Low score	2	1	1	2
Dermal
Total	2	1	1	1
High score	2	1	1	1
Low score	0	0	0	0
Other
Total	0	0	0	0

To facilitate comparisons, the (sub-)studies were grouped into high and low-adequacy scoring studies with overall scores subjectively set to >2 and ≤2, respectively.

*As judged by the authors of the respective studies, where “positive” indicates stimulating carcinogenesis and “negative” indicates a lack of carcinogenic effect.

†Number of different laboratories contributing studies.

Table 9. Strength of evidence classification for nicotine to act as a carcinogen or carcinogenesis modulator in human and animal studies.

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