Talc and human cancer: a systematic review of the experimental animal and mechanistic evidence

Figures & data

Table 1. PECOS elements and corresponding inclusion and exclusion criteria for experimental animal carcinogenicity studies.

PECOS element	Inclusion criteria	Exclusion criteria
Population	Any non-human mammal at any life stage Whole animals (in vivo)	Human populations Non-mammalian animals Animal tissues/cells only (in vitro, ex vivo)
Exposure	Repeated exposure to talc alone Any exposure route	A single exposure to talc Exposure to talc only in a mixture with other chemicals
Comparator	Non-human mammals (same species as exposed) concurrently exposed to vehicle-only treatment and/or untreated control	Lack of unexposed or vehicle-exposed group
Outcomes	Tumor incidence	Biochemical or other mechanistic outcomes (e.g. gene or protein expression, clinical chemistry, and genotoxicity) Tumor characteristics after induction (e.g. aggressiveness and metastasis)
Study design	Primary experimental studies Published in English	Reviews, letters, editorials, meta-analyses, commentaries, or conference abstracts Not published in English

PECOS: population, exposure, comparator, outcomes, and study design.

Table 2. PECOS elements and corresponding inclusion and exclusion criteria for mechanistic studies.

PECOS element	Inclusion criteria	Exclusion criteria
Population	Any human, non-human mammalian, or bacterial cells, tissues, or model systems (in vitro, ex vivo) Whole, non-human mammalian animals (in vivo)	Non-mammalian animal species
Exposure	Exposure to talc alone Any exposure route or duration	Exposure to talc only in a mixture with other chemicals
Comparator	Test system (same as exposed) concurrently exposed to vehicle-only treatment and/or untreated control	Lack of unexposed or vehicle-exposed group
Outcomes	Any key biological or chemical events potentially associated with carcinogenesis in general, or for talc specifically (as identified during scoping of the review)	Tumor incidence
Study design	Primary experimental studies Published in English	Reviews, letters, editorials, meta-analyses, commentaries, or conference abstracts Not published in English

PECOS: population, exposure, comparator, outcomes, and study design.

Table 3. PubMed and Scopus search strategies for experimental animal carcinogenicity studies.

Search number	Query
PubMed
1	((("Talc"[Mesh] OR talc OR talcum OR "fibrous dusts" OR "fibrous minerals") NOT (pudrage OR poudrage OR pleurodesis) AND (english[Filter])) AND ((Cancer* OR leukemia OR carcinogen* OR lymphoma OR myeloma OR neoplasm* OR carcinoma OR tumor OR tumors OR tumorous OR tumour OR sarcoma OR "Hodgkin Lymphoma" OR Mesothelioma OR Leiomyoma OR "Neoplasms"[Mesh] OR "Leukemia"[Mesh] OR "Lymphoma, Non-Hodgkin"[Mesh] OR "Carcinogens"[Mesh] OR "Carcinogens, Environmental"[Mesh] OR "Lymphoma"[Mesh] OR "Carcinoma"[Mesh] OR "Sarcoma"[Mesh] OR Mesothelioma OR "Mesothelioma"[Mesh] OR "Mesothelioma, Malignant"[Mesh] OR "Brain Neoplasms"[Mesh] OR "Breast Neoplasms"[Mesh] OR "Uterine Cervical Neoplasms"[Mesh] OR "Colorectal Neoplasms"[Mesh] OR "Esophageal Neoplasms"[Mesh] OR "Stomach Neoplasms"[Mesh] OR "Kidney Neoplasms"[Mesh] OR "Laryngeal Neoplasms"[Mesh] OR "Laryngeal Neoplasms"[Mesh] OR "Lung Neoplasms"[Mesh] OR Melanoma OR "Multiple Myeloma"[Mesh] OR "Ovarian Neoplasms"[Mesh] OR "Pancreatic Neoplasms"[Mesh] OR "Prostatic Neoplasms"[Mesh] OR "Carcinoma, Renal Cell"[Mesh] OR "Skin Neoplasms"[Mesh] OR "Stomach Neoplasms"[Mesh] OR "Thyroid Neoplasms"[Mesh] OR "Leiomyoma"[Mesh] OR toxicity OR toxicolog* OR toxic OR toxicant OR cardiotox* OR Toxicokinetics OR immunotox* OR hepatotox* OR genotox* OR Cytotox* OR PBPK OR Pharmacokinetics OR Mutagen OR "Pharmacokinetics"[Mesh] OR "Threshold of Toxicological Concern"[Title/Abstract] OR "Toxicological Concern"[Title/Abstract] OR "toxicity" [Subheading] OR fibrogenic))) AND english[Filter]
Scopus
1	TITLE-ABS-KEY (talc OR talcum OR "fibrous dusts" OR "fibrous minerals") AND (TITLE-ABS-KEY (cancer* OR leukemia OR carcinogen* OR lymphoma OR myeloma OR neoplasm* OR carcinoma OR tumor OR tumors OR tumorous OR tumor OR sarcoma OR "Hodgkin Lymphoma" OR mesothelioma OR leiomyoma OR toxicity OR toxicolog* OR toxic OR toxicant OR cardiotox* OR toxicokinetics OR immunotox* OR hepatotox* OR genotox* OR cytotox* OR pbpk OR pharmacokinetics OR mutagen OR pharmacokinetics OR "Threshold of Toxicological Concern" OR "Toxicological Concern" OR fibrogenic OR mutagenic*) OR SRCTITLE (toxciol*)) AND (LIMIT-TO (SUBJAREA, "MEDI") OR LIMIT-TO (SUBJAREA, "PHAR") OR LIMIT-TO (SUBJAREA, "BIOC") OR LIMIT-TO (SUBJAREA, "IMMU")) AND (LIMIT-TO (LANGUAGE, "English"))

Table 4. PubMed and Scopus search strategies for mechanistic studies.

Search number	Query
PubMed
1	("Talc"[Mesh] OR talc OR talcum OR "fibrous dusts" OR "fibrous minerals") AND (mechanistic OR mechanism OR "mode of action" OR inflammation OR "oxidative stress" OR "immune response" OR mucin or MUC1 OR mutagenic* OR genotoxic* OR "Inflammation"[Mesh] OR "Oxidative Stress"[Mesh] OR "Immunity"[Mesh] OR "Mucins"[Mesh] OR epithelial OR "oxidative damage" OR macrophage OR immun* OR mucin) AND english[Filter])
Scopus
1	TITLE-ABS (talc OR talcum OR "fibrous dusts" OR "fibrous minerals") AND TITLE-ABS (mechanistic OR mechanism OR "mode of action" OR inflammation OR "oxidative stress" OR "immune response" OR mucin OR muc1 OR mutagenic* OR genotoxic* OR epithelial OR "oxidative damage" OR macrophage OR immun* OR mucin) AND (LIMIT-TO (SUBJAREA, "MEDI") OR LIMIT-TO (SUBJAREA, "PHAR") OR LIMIT-TO (SUBJAREA, "IMMU")) AND (LIMIT-TO (LANGUAGE, "English"))

Table 5. Study quality domains and criteria for experimental animal studies.

Domain*	Criteria for higher quality	Criteria for lower quality
Allocation	Allocation of animals to experimental groups was randomized.	No randomization or no specification that allocation to experimental groups was randomized.
Observational bias/blinding	Measures to reduce observational bias were described, such as blinding to conceal treatment groups during endpoint evaluation or consensus-based evaluations of histopathology.	Measures to reduce observational bias were not described.
Confounding	No indication that there were differences across treatment groups that could bias results (e.g. differences in co-exposures [intended or unintended], vehicle, diet, gavage error, palatability, animal source, husbandry, and animal health status).	Variables not equally balanced across experimental groups, potentially biasing the results.
Selective reporting	Results were reported for all prespecified outcomes, exposure groups, and evaluation time points. If any results were omitted, an explanation was provided, or the omission is not expected to impact interpretation of results.	Results are missing for many prespecified outcomes and omissions were not explained and could impact interpretation of results.
Attrition	Results were reported for all animals, or attrition is explained (e.g. death or unscheduled sacrifice during study) and not expected to impact interpretation of results.	A moderate to high level of attrition occurred that is not explained and could impact interpretation of results.
Chemical administration and characterization	Chemical source, purity, and composition were adequate; chemical purity was analytically verified; and steps were taken to ensure reported exposure levels were accurate (e.g. analytical verification). Any uncertainties are expected to have minimal impact on interpretation of results.	Chemical source, purity, or composition were inadequate; chemical purity was not analytically verified; or no steps were taken to ensure reported exposure levels were accurate.
Endpoint measurement	Methods and animal models were adequately described. Methods are appropriate (e.g. generally accepted and reliable). Sample size is adequate. Appropriate control groups were used and any use of nonconcurrent or historical control data was justified (e.g. for evaluation of rare tumors).	Methods and animal models were not adequately described. Methods are not appropriate or lack specificity for investigating the outcome of interest. Sample size is inadequate. No use of appropriate control groups, and comparisons to nonconcurrent or historical controls are not adequately justified.
Results presentation	Results were quantified or otherwise presented in a manner that allows for independent consideration of the data. There are no concerns with the completeness of the reported results for any specific outcome. Statistical analyses were conducted to detect differences between treated and control groups.	Data were presented in a nonpreferred manner (e.g. data were pooled when responses are expected to differ substantially across sexes or ages). Data presentation is incomplete (e.g. presentation of mean without variance data; concurrent control data not presented). Statistical analyses were not conducted to detect differences between treated and control groups.
Sensitivity	The exposure period, timing (e.g. lifestage), frequency, and duration, as well as the animal model, were appropriate and sensitive for evaluating the outcome. There are not significant concerns with the ability of the experimental design to detect the specific outcome of interest. The observation period was appropriate and sensitive.	The exposure period, timing, frequency, duration, and animal model were not appropriate or sensitive for evaluating the outcome. There are concerns that are expected to decrease the sensitivity of the study to detect a response in the exposed groups. The observation period was not appropriate nor sensitive.

* Risk of bias domains are italicized; sensitivity domains are in regular font.

Table 6. Study quality domains and criteria for in vitro mechanistic studies.

Domain*	Criteria for higher quality	Criteria for lower quality
Observational bias/blinding	Measures to reduce observational bias were described, such as blinding to conceal treatment groups or collection of data from randomly selected fields. Alternatively, observational bias is not a concern because of the use of automated approaches (e.g. microplate readers) or standard laboratory kits.	Measures to reduce observational bias were not described.
Variable control	No indication that there were differences across treatment groups that could bias results (e.g. differences in culture conditions, cell passages, co-exposures, reagent production lots, and mycoplasma infections). No indication that the chemical properties of the test substance could bias the results away from the null (i.e. false positive results). No known variations in cell signaling unique to model system that could influence the detection of effects of interest.	Study variables, chemical properties, or features of the test system that are expected to impact the results were uncontrolled or inconsistent across groups.
Selective reporting	Results were reported for all prespecified outcomes, exposure groups, and evaluation time points. If any results were omitted, an explanation was provided, or the omission is not expected to impact interpretation of results.	Results are missing for many prespecified outcomes and omissions were not explained and could impact interpretation of results.
Chemical administration and characterization	Chemical source, purity, and composition were adequate; chemical purity was analytically verified; and there are no concerns about stability of the chemical in the test system or the specific methods of administration. Any uncertainties are expected to have minimal impact on interpretation of results.	Chemical source, stability, purity, or composition was inadequate; chemical purity was not analytically verified; deficient administration methods were used.
Endpoint measurement	Methods of endpoint measurement and test system were adequately described. Methods are appropriate (e.g. generally accepted and reliable). Sample size or number of replicates is adequate for the assay or protocol. Appropriate negative control groups were used and any use of nonconcurrent or historical control data was justified (e.g. to compare to background levels in negative controls).	Methods and test system were not adequately described. Methods are not appropriate or lack specificity for investigating the outcome of interest. Sample size or number of replicates is inadequate. No use of appropriate negative control groups, and comparisons to nonconcurrent or historical controls are not adequately justified.
Results presentation	Results were quantified or otherwise presented in a manner that allows for independent consideration of the data. There are no concerns with the completeness of the reported results for any specific outcome. Statistical analyses were conducted to detect differences between treated and control groups.	Data were presented in a nonpreferred manner or without quantitative results (e.g. averaging technical replicates rather than biological replicates; presenting western blot images without numerical densitometry values). Data presentation is incomplete (e.g. presentation of mean without variance data; concurrent control data not presented; failure to report or address overt cytotoxicity). Statistical analyses were not conducted to detect differences between treated and control groups.
Sensitivity	The exposure period, timing (e.g. lifestage), frequency, and duration, as well as the test system, were appropriate and sensitive for evaluating the outcome. There are not significant concerns with the ability of the experimental design to detect the specific outcome of interest. Timing of endpoint measurement in relation to chemical exposure was appropriate and sensitive. If deemed necessary, positive controls were used (e.g. if effects of interest may be inhibited or poorly manifested in the test system because of differences from in vivo biology), and the positive control test substance (and dose) was reasonable, such that the intended positive response was induced.	The exposure period, timing, frequency, duration, and test system were not appropriate or sensitive for evaluating the outcome. There are concerns that are expected to decrease the sensitivity of the study to detect a response in the exposed groups. The timing of endpoint measurement in relation to chemical exposure was not appropriate nor sensitive. Positive controls were deemed necessary but not used, or they were used but the positive test substance (and dose) was not reasonable, and the intended positive response was not induced.

* Risk of bias domains are italicized; sensitivity domains are in regular font.

Figure 1. Literature search and study selection flowchart for experimental animal carcinogenicity studies.

Table 7. Characteristics of experimental animal carcinogenicity studies.

Reference	Species/strain	Dose group size/sex	Age at start of talc exposure	Age at examination	Exposure frequency/duration	Talc source/purity/particle size	Exposure levels	Organs/tissues evaluated	Statistical method(s)
Intravaginal or perineal exposure
Keskin et al. (2009)	Rat/Sprague Dawley	7 F	NR	Upon cessation of exposure	Daily for 12 weeks	NR	0 (no treatment or saline) or 100 mg (intravaginal or perineal)	Vulvar, vaginal, uterine, fallopian, and ovarian	Fisher’s exact test
Oral exposure
Wagner et al. (1977)	Rat/Wistar	8–16 M, 8–16 F	21–26 weeks	Upon natural death	Once daily on 101 days during a 5-month (∼150 day) period	Northern Italy mine/92%, no asbestos/mean diameter 25 μm	0 or 100 mg/day	Liver, spleen, kidney, lung, stomach, intestine, peritoneum, uterus, and other unspecified organs	None
Intraperitoneal exposure
Pott et al. (1974)	Rat/Wistar	40–80/sex NR	NR	Upon spontaneous death or sacrifice	Once per week for 4 weeks	NR	0 or 25 mg	All*	None
Intratracheal instillation
Stenback and Rowland (1978)	Hamster/Syrian golden	24 M, 24 F	9 weeks	Upon spontaneous death or when moribund	Once per week for 18 weeks	U.S.P. Fisher Scientific/93–97%/<25 μm for 93.3% of particles	0 (no treatment or saline) or 3 mg	Lung, larynx, trachea, liver, spleen, kidneys, and other tissues showing gross lesions	None
Inhalation exposure
NTP (1993)	Rat/F344/N	50 M, 50 F	6–7 weeks	120–121 weeks (males); 129–130 weeks (females)	6 h/day, 5 days/week for 113 weeks (males) or 122 weeks (females)	MP 10–52 microtalc from Pfizer/∼98%, no asbestos/MMAD = 2.7–3.2 μm	0, 6, or 18 mg/m^3	All^†	Logistic regression; Life table test; Fisher’s exact test; Cochran–Armitage trend test
	Mouse/B6C3F1	50 M, 50 F	7 weeks	112–113 weeks	6 h/day, 5 days/week for 103 or 104 weeks	MP 10–52 microtalc from Pfizer/∼98%, no asbestos/MMAD = 3.3–3.6 μm
Wagner et al. (1977, 1979)	Rat/Wistar	48 M, 48 F	6–8 weeks	One year after cessation of exposure or upon natural death	7.5 h/day, 5 days/week for 3, 6, or 12 months	Northern Italy mine/92%, no asbestos/mean diameter 25 μm	0 or 10.8 mg/m^3	Lung	None
Wehner et al. (1977)	Hamster/Syrian golden	25–50 M, 25–50 F	4 weeks	Upon natural death or when moribund	3, 30, or 150 min/day, 5 days/week for 30 days	Johnson’s Baby Powder (Johnson & Johnson)/≥95%, no asbestos/MMAD = 4–9 μm	0 or 37.1 mg/m^3	Lung, liver, kidney, stomach, ovary, uterus, testis, and other tissues showing gross lesions	None
			7 weeks		30 or 150 min/day, 5 days/week for 300 days		0 or 27.4 mg/m^3

F: female; M: male; MMAD: mass median aerodynamic diameter; NR: not reported; NTP: National Toxicology Program.

* Pott et al. (1974) did not specify which tissues were evaluated, but stated that "all tumors were studied histologically".

^† NTP (1993) conducted histopathological evaluations on 35–40 tissues/organs per animal.

Table 8. Study quality ratings for experimental animal carcinogenicity studies.

Reference	Study quality domains*
	Allocation	Observational bias/blinding	Confounding	Selective reporting	Attrition	Chemical administration/ characterization	Endpoint measurement	Results presentation	Sensitivity
Keskin et al. (2009)	L	L	H	H	H	L	L	H	L
Pott et al. (1974)	L	L	L	L	H	L	L	L	L
Stenback and Rowland (1978)	L	L	H	H	H	L	L	L	L
NTP (1993)	H	H	H	H	H	H	H	H	H
Wehner, Zwicker, et al. (1977)	H	L	H	H	L	L	H	L	L
Wagner et al. (1977, 1979)	H	L	H	H	L	L	L	L	L

H: higher quality; L: lower quality; NTP: National Toxicology Program; US EPA: United States Environmental Protection Agency.

* The study quality criteria for each domain are based on criteria from US EPA (2022), NTP (2015), and Waspe et al. (2021).

Table 9. Results of experimental animal carcinogenicity studies.

Reference	Species/strain	Dose group size/sex	Talc dose	Exposure duration	Tumor type	Tumor incidence (%)	p Value	Ptrend
Intravaginal or perineal exposure
Keskin et al. (2009)	Rat; Sprague-Dawley	7 F	0 mg (no treatment)	12 weeks	Vulvar, vaginal, uterine, fallopian, or ovarian	0	NA	NA
			0 mg (saline)
			100 mg (intravaginal)
			100 mg (perineal)
Oral exposure
Wagner et al. (1977)	Rat; Wistar	16 M + F	0 mg	5 months	Stomach leiomyosarcoma	0	NR	NA
		32 M + F	100 mg			3.1
		16 M + F	0 mg		Uterine sarcoma	0
		32 M + F	100 mg			6.3
		16 M + F	0 mg		Liver, spleen, lung, peritoneum, intestine, or kidney tumors	0
		32 M + F	100 mg			0
Intraperitoneal exposure
Pott et al. (1974)	Rat; Wistar	80	0 mg	4 weeks	Unspecified, but mainly mesothelioma	0	NR	NA
		40	25 mg			2.5
Intratracheal instillation
Stenback and Rowland (1978)	Hamster; Syrian	48 M + F	0 mg (no treatment)	18 weeks	Lymphomas	4.2	NR	NA
			0 mg (saline)			0
			3 mg			0
			0 mg (no treatment)		Lung, larynx, trachea, liver, kidney, or spleen tumors	0
			0 mg (saline)			0
			3 mg			0
Inhalation exposure
NTP (1993)*	Rat; F344/N	49 M	0 mg/m^3	113 weeks	Adrenal pheochromocytoma, benign, malignant, or complex	53	NA	0.007
		48 M	6 mg/m^3			67	.147
		47 M	18 mg/m^3			79	.006
		48 F	0 mg/m^3	122 weeks	Adrenal pheochromocytoma, and malignant	0	NA	<0.001
		47 F	6 mg/m^3			2	.509
		49 F	18 mg/m^3			20	.001
		48 F	0 mg/m^3		Adrenal pheochromocytoma, benign, malignant, or complex	27	NA	0.014
		47 F	6 mg/m^3			30	.541
		49 F	18 mg/m^3			47	.024
		50 F	0 mg/m^3		Lung alveolar/bronchiolar carcinoma	0	NA	0.003
		48 F	6 mg/m^3			0	NR
		50 F	18 mg/m^3			10	.028
		50 F	0 mg/m^3		Lung alveolar/bronchiolar adenoma or carcinoma	2	NA	<0.001
		48 F	6 mg/m^3			0	.503
		50 F	18 mg/m^3			26	<.001
Wagner et al. (1977, 1979)	Rat; Wistar	48 M + F	0 mg/m^3	3 months	Lung adenocarcinoma or mesothelioma	0	NR	NA
		48 M + F	10.8 mg/m^3
		24 M + F	0 mg/m^3	6 months
		24 M + F	10.8 mg/m^3
		24 M + F	0 mg/m^3	12 months
		24 M + F	10.8 mg/m^3
Wehner, Zwicker, et al. (1977)	Hamster; Syrian	25 M, 25 F	0 mg/m^3	30 days	Adrenal pheochromocytoma	0	NR	NA
		50 M, 50 F	37.1 mg/m^3 (3 min/day)			0
			37.1 mg/m^3 (30 min/day)			0
			37.1 mg/m^3 (150 min/day)			1
		25 M, 25 F	0 mg/m^3		Lung carcinoma (metastatic)	0
		50 M, 50 F	37.1 mg/m^3 (3 min/day)			1
			37.1 mg/m^3 (30 min/day)			0
			37.1 mg/m^3 (150 min/day)			0
		25 M, 25 F	0 mg/m^3		Liver cholangiocarcinoma	0
		50 M, 50 F	37.1 mg/m^3 (3 min/day)			1
			37.1 mg/m^3 (30 min/day)			0
			37.1 mg/m^3 (150 min/day)			0
		25 M, 25 F	0 mg/m^3		Rhabdomyosarcoma	2
		50 M, 50 F	37.1 mg/m^3 (3 min/day)			0
			37.1 mg/m^3 (30 min/day)			0
			37.1 mg/m^3 (150 min/day)			0
		25 M, 25 F	0 mg/m^3		Osteosarcoma, liver osteosarcoma, malignant lymphoma, or metastatic lymphoma of the liver	0
		50 M, 50 F	37.1 mg/m^3 (3 min/day)			0
			37.1 mg/m^3 (30 min/day)			0
			37.1 mg/m^3 (150 min/day)			0
		25 M, 25 F	0 mg/m^3	300 days	Lung carcinoma (metastatic), osteosarcoma, malignant lymphoma, lymphoma of the liver (metastatic), and carcinoma of unknown origin^†	2
		50 M, 50 F	27.4 mg/m^3 (30 min/day)			0
			27.4 mg/m^3 (150 min/day)			0
		25 M, 25 F	0 mg/m^3		Liver osteosarcoma	0
		50 M, 50 F	27.4 mg/m^3 (30 min/day)			0
			27.4 mg/m^3 (150 min/day)			1
		25 M, 25 F	0 mg/m^3		Adrenal pheochromocytoma, rhabdomyosarcoma, or liver cholangiosarcoma	0
		50 M, 50 F	27.4 mg/m^3 (30 min/day)			0
			27.4 mg/m^3 (150 min/day)			0

F: female; M: male; NA: not applicable; NR: not reported; NTP: National Toxicology Program.

* NTP (1993) conducted histopathological evaluations on 35–40 tissues/organs per animal; only those tumors with statistically significant increases are shown in this table.

^† Each of these tumor types was found in one animal in the control group for the 300-day exposure duration.

Figure 2. Literature search and study selection flowchart for mechanistic studies.

Table 10. Characteristics of in vivo mechanistic studies.

Reference	Species (sex)	Dose group size	Exposure route	Exposure frequency/duration	Talc source/purity/particle size	Exposure levels	Endpoints evaluated	Statistical method(s)
Litton Bionetics, Inc (1974)	Rat (M)	10 dosed males mated with 2 females/week for 8 weeks	Oral gavage	Single exposure (acute study); once per day for 5 days (subacute study)	FDA 71-43 talc/NR	0, 30, 300, 3000, or 5000 mg/kg-day	Dominant lethal test	Chi-square and Armitage trend test; t-test
	Rat (M)	3–5 per group per time point	Oral gavage	6, 24, or 48 hours (acute study); once per day for 5 days (subacute study)			Chromosomal aberrations in bone marrow cells	NR
Beck et al. (1987)	Hamster (NR)	3–6	Intratracheal instillation	Single exposure	Mill in Vermont/NR, no fibrous material, <1% quartz/MMAD = 12.5 μm	0, 1.5, 7.5, or 37.5 mg/kg-day	PMN and macrophage count in BALF	Student’s two-tailed t-test
Shim et al. (2015)	Rat (M, F)	3 M, 3 F	Inhalation	6 hours/day, 5 days/week for 4 weeks	Ultrafine talc from Rex Material/96.7%, no asbestos/MMAD = 3.88 μm	0, 5, 50, or 100 mg/m^3	Macrophage infiltration in lung; oxidative stress in lung	One-way ANOVA followed by Tukey’s test

ANOVA: analysis of variance; BALF: bronchoalveolar lavage fluid; F: female; FDA: Food and Drug Administration; M: male; MMAD: mass median aerodynamic diameter; NR: not reported; PMN: polymorphonuclear neutrophil.

Table 11. Characteristics of in vitro mechanistic studies.

Reference	Cell line/assay	Number of replicates	Exposure duration	Talc source/purity/particle size	Exposure levels	Endpoints evaluated	Statistical methods
Litton Bionetics, Inc (1974)	Saccharomyces D3, Salmonella TA1530, and Salmonella G-46	NR	24 or 48 hours	FDA 71-43 talc/NR	NR	Mutagenicity	NR
	WI-38 human embryonic lung cells		24–48 hours		0, 2, 20, or 200 µg/mL	Chromosomal aberrations	NR
Endo-Capron et al. (1993)	Rat pleural mesothelial cells	6 replicates in each of 3 experiments	24 hours	Spanish talc (no. 5725), Italian talc (no. 5726), and French talc (no. 7841)/90–95%, no asbestos/diameter 2.6–4.0 μm	0, 50, 100, or 250 µg/mL	Unscheduled DNA synthesis	Student’s t-test
		30 metaphases/group in each of 3 experiments	48 hours		0, 15, 37.5, 75, or 112.5 µg/mL	Sister chromatid exchange	Mann–Whitney’s test
Buz’Zard and Lau (2007)	OSE2a human immortalized normal ovarian epithelial cells; GC1a human immortalized normal ovarian granulosa cells	3	72 hours	Sigma talc/NR	0, 5, 20, or 100 µg/mL	Neoplastic transformation	Student’s t-test
	OSE2a human immortalized normal ovarian epithelial cells; GC1a human immortalized normal ovarian granulosa cells; human polymorphonuclear neutrophils	3	24, 72, or 120 hours (ovarian cells) 24 or 72 hours (neutrophils)		0, 0.5, 5, 20, 50, 100, or 500 µg/mL	ROS generation
Harper et al. (2023)	HPOE human primary normal ovarian epithelial cells; HOSEpiC human ovarian epithelial cells; normal human peritoneal fibroblast cells	3	72 hours	Johnson & Johnson talcum baby powder/NR	0, 100, or 500 µg/mL	Neoplastic transformation	ANOVA with Tukey’s post hoc test
Mandarino et al. (2020)	IC21, J774, and RAW264.7 mouse macrophages	2	4 hours	JP Baker talc, USP grade/NR, no asbestos/diameter <10 μm	0 or 10 µg talc/well	ROS generation	One-way ANOVA
Ghio et al. (2012)	MeT-5A human mesothelial cells; BEAS-2B human bronchial epithelial cells	NR	24 hours	Sclerosol sterile talc powder/NR, no fibers/diameter 10–50 μm	0,50, 100, or 250 µg/mL	IL-8 and IL-6 cytokine release	One-way ANOVA
			1, 2, or 3 hours		0, 50, 100, 200, or 500 µg/mL	ROS generation
Acencio et al. (2007)	Rabbit pleural mesothelial cells	NR	6, 24, or 48 hours	Talc/NR/median diameter 21.2 μm	0 or 125 µg/mL	IL-8, TGF-β1, and VEGF cytokine release	One-way ANOVA with Tukey’s test
Fletcher et al. (2019)	EL-1 human macrophages; human primary normal ovarian epithelial cells; FT33, immortalized human fallopian tube secretory epithelial cells	3	24 hours	Johnson & Johnson talcum baby powder/NR/NR, but filtered in 0.2 μm filter before use	0 or 100 µg/mL	Cell proliferation	Generalized linear models with Tukey-Kramer test
			72 hours			CA-125 release
Wylie et al. (1997)	Hamster tracheal epithelial cells; rat pleural mesothelial cells	3–4 replicates in each of 2 experiments	7 days	FD14, S157, and CPS183 fibrous talc samples from the New York State Gouverneur Mining District/37–60%/surface area of particles 4.9–6.2 mm^2/g	0, 0.025, 0.05, 0.1, 0.5, 1, 5, 10, or 15 µg/cm^2	Cell proliferation/cytotoxicity (colony-forming efficiency)	ANOVA and trend analysis

ANOVA: analysis of variance; FDA: Food and Drug Administration; IL: interleukin; NR: not reported; ROS: reactive oxygen species; TGF: transforming growth factor; VEGF: vascular endothelial growth factor.

Table 12. Study quality ratings for in vivo mechanistic studies.

	Study quality domains*
Reference	Allocation	Observational bias/blinding	Confounding	Selective reporting	Attrition	Chemical administration/ characterization	Endpoint measurement	Results presentation	Sensitivity
Litton Bionetics, Inc (1974)	H	L	H	H	H	L	H	H	H
Beck et al. (1987)	L	L	H	H	H	L	H	H	H
Shim et al. (2015)	L	L	H	H	H	H	H	H	H

H: higher quality; L: lower quality; NTP: National Toxicology Program; US EPA: United States Environmental Protection Agency.

* The study quality criteria for each domain are based on criteria from US EPA (2022), NTP (2015), and Waspe et al. (2021).

Table 13. Study quality ratings for in vitro mechanistic studies.

Reference	Study quality domains*
	Observational bias/blinding	Variable control	Selective reporting	Chemical administration/characterization	Endpoint measurement	Results presentation	Sensitivity
Litton Bionetics, Inc (1974)	L	H	H	L	L	L	H
Endo-Capron et al. (1993)	H	H	H	L	H	H	H
Buz’Zard and Lau (2007)	H	H	H	L	H	H	H
Harper et al. (2023)	H	H	H	H	H	H	H
Mandarino et al. (2020)	H	H	H	L	L	H	H
Ghio et al. (2012)	H	H	H	L	L	H	H
Acencio et al. (2007)	H	H	H	L	L	H	H
Fletcher et al. (2019)	H	H	H	H	H	H	H
Wylie et al. (1997)	H	H	H	L	H	H	H

H: higher quality; L: lower quality; US EPA: United States Environmental Protection Agency.

* The study quality criteria for each domain are based on criteria from US EPA (2022) and Waspe et al. (2021).

Table 14. Results of in vivo mechanistic studies.

Reference	Species (sex)	Endpoint	Exposure parameters/timepoints	Dose/exposure levels	Results	Statistical significance
Litton Bionetics, Inc (1974)	Rat*	Fertility index	Acute or subacute study	30, 300, 3000, or 5000 mg/kg-day	No change	NS
		Average number of implantations/pregnant females	Acute study	30 mg/kg-day	No change	NS
				300 mg/kg-day	Increased	p < .05
				3000 mg/kg-day	No change	NS
				5000 mg/kg-day	No change	NS
			Subacute study	30, 300, or 3000 mg/kg-day	No change	NS
				5000 mg/kg-day	Increased	p < .05
		Average number of corpora lutea/pregnant female	Acute or subacute study	30, 300, 3000, or 5000 mg/kg-day	No change	NS
		Average preimplantation losses/pregnant female	Acute study	30, 300, 3000, or 5000 mg/kg-day	No change	NS
			Subacute study	30, 300, or 3000 mg/kg-day	No change	NS
				5000 mg/kg-day	Decreased	p < .01
		Average resorptions/pregnant female	Acute study	30 mg/kg-day	Decreased	p < .05
				300 mg/kg-day	No change	NS
				3000 mg/kg-day	No change	NS
				5000 mg/kg-day	No change	NS
			Subacute study	30, 300, 3000, or 5000 mg/kg-day	No change	NS
		Proportion of females with one or more dead implantations	Acute or subacute study	30, 300, 3000, or 5000 mg/kg-day	No change	NS
		Proportion of females with two or more dead implantations	Acute or subacute study	30, 300, 3000, or 5000 mg/kg-day	No change	NS
		Dead implants/total implants	Acute study	30 mg/kg-day	Decreased	p < .05
				300 mg/kg-day	No change	NS
				3000 mg/kg-day	No change	NS
				5000 mg/kg-day	No change	NS
			Subacute study	30, 300, 3000, or 5000 mg/kg-day	No change	NS
	Rat (M)	Chromosomal aberrations	Acute study (6, 24, or 48 hours)	30, 300, 3000, or 5000 mg/kg-day	No change	NR
			Subacute study	30, 300, 3000, or 5000 mg/kg-day
Beck et al. (1987)	Hamster (NR)	Macrophage count in BALF	Dose–response study: 1-day post-exposure	1.5, 7.5, or 37.5 mg/kg-day	No change	NS
			Time-course study: 1-day post-exposure	37.5 mg/kg-day	No change	NS
			Time-course study: 4-days post-exposure		No change	NS
			Time-course study: 7-days post-exposure		No change	NS
			Time-course study: 14-days post-exposure		Increased	p < .01
		PMN count in BALF	Dose–response study: 1-day post-exposure	1.5 mg/kg-day	Increased	p < .05
				7.5 mg/kg-day	Increased	p < .01
				37.5 mg/kg-day	Increased	p < .01
			Time-course study: 1-day post-exposure	37.5 mg/kg-day	Increased	p < .01
			Time-course study: 4-days post-exposure
			Time-course study: 7-days post-exposure
			Time-course study: 14-days post-exposure
Shim et al. (2015)	Rat (M)	Macrophage infiltration in lungs	4 weeks	5 mg/m^3	No change	NR
				50 mg/m^3	Increased	NR
				100 mg/m^3	Increased	NR
	Rat (F)			5 mg/m^3	No change	NR
				50 mg/m^3	Increased	NR
				100 mg/m^3	Increased	NR
	Rat (M)	SOD2 expression in lung		5 mg/m^3	Increased	p < .05
				50 mg/m^3	No change	NS
				100 mg/m^3	Increased	p < .05
	Rat (F)			5 mg/m^3	No change	NS
				50 mg/m^3	No change	NS
				100 mg/m^3	Increased	p < .05
	Rat (M)	GPx expression in lung		5 mg/m^3	Increased	p < .05
				50 mg/m^3	No change	NS
				100 mg/m^3	No change	NS
	Rat (F)			5 mg/m^3	No change	NS
				50 mg/m^3	No change	NS
				100 mg/m^3	No change	NS

BALF: bronchoalveolar lavage fluid; F: female; GPx: glutathione peroxidase; M: male; NR: not reported; NS: not significant; PMN: polymorphonuclear neutrophil; SOD2: superoxide dismutase 2.

* For the dominant lethal assay, Litton Bionetics, Inc (1974) mated exposed males to two virgin females each week for 7 or 8 weeks; only the results of the last mating for each study are presented in the table, as the authors did not report any time-trend patterns in the results.

Table 15. Results of in vitro mechanistic studies.

Reference	Cell Line/assay	Endpoint	Exposure parameter/timepoints	Exposure levels	Results	Statistical significance
Litton Bionetics, Inc (1974)	Saccharomyces D3, Salmonella TA1530, and Salmonella G-46	Mutagenicity	24 or 48 hours	NR	Negative	NR
	WI-38 human embryonic lung cells	Chromosomal aberrations	24–48 hours	2 µg/mL	Negative	NR
				20 µg/mL	Negative	NR
				200 µg/mL	Negative	NR
Endo-Capron et al. (1993)	Rat pleural mesothelial cells	Unscheduled DNA synthesis	24 hours (Spanish, Italian, or French talc)	50, 100, or 250 µg/mL	No change	NS
		Sister chromatid exchange	48 hours (Spanish, Italian, or French talc)	15, 37.5, 75, or 112.5 µg/mL	No change	NS
Buz’Zard and Lau (2007)	OSE2a human immortalized normal ovarian epithelial cells	Neoplastic transformation	72 hours	5 µg/mL	Increased	p < .05
				20 µg/mL	Increased	p < .01
				100 µg/mL	Decreased	p < .05
	GC1a human immortalized normal ovarian granulosa cells			5 µg/mL	Increased	p < .01
				20 µg/mL	Increased	p < .001
				100 µg/mL	Increased	p < .001
	Human polymorphonuclear neutrophils	ROS production	24 hours	0.5 µg/mL	Increased	p < .05
				5 µg/mL	Increased	p < .05
				20 µg/mL	Increased	p < .01
				50 µg/mL	Increased	p < .05
				100 µg/mL	Increased	p < .01
				500 µg/mL	Increased	p < .001
			72 hours	0.5 µg/mL	No change	NS
				5 µg/mL	No change	NS
				20 µg/mL	No change	NS
				50 µg/mL	No change	NS
				100 µg/mL	Increased	p < .05
				500 µg/mL	Increased	p < .01
Harper et al. (2023)	HPOE human primary normal ovarian epithelial cells	Neoplastic transformation	72 hours	100 µg/mL	Increased	p < .05
				500 µg/mL	Increased	p < .05
	HOSEpiC human ovarian epithelial cells			100 µg/mL	Increased	p < .05
				500 µg/mL	Increased	p < .05
	Human normal peritoneal fibroblast cells			100 µg/mL	No change	NS
				500 µg/mL	No change	NS
Mandarino et al. (2020)	IC21 macrophages	ROS production	4 hours	10 µg/well	Increased	p < .05
	J774 macrophages				No change	NS
	RAW264.7 macrophages				Increased	p < .01
Ghio et al. (2012)	MeT-5A human mesothelial cells	IL-8 cytokine release	24 hours	50 µg/mL	No change	NS
				100 µg/mL	Increase	NR
				250 µg/mL	Increase
		IL-6 cytokine release	24 hours	50, 100, or 250 µg/mL	No change	NS
		ROS production	60-, 120-, or 180 minutes post-exposure	50 µg/mL	Time- and concentration-dependent increase	NR
				100 µg/mL
				200 µg/mL
				500 µg/mL
	BEAS-2B human bronchial epithelial cells	IL-8 cytokine concentration	24 hours	50 µg/mL	Increase	NR
				100 µg/mL	Increase
				250 µg/mL	Increase
		IL-6 cytokine release	24 hours	50, 100, or 250 µg/mL	No change	NS
		ROS production	60-, 120-, or 180-minutes post-exposure	50 µg/mL	Time- and concentration-dependent increase	NR
				100 µg/mL
				200 µg/mL
				500 µg/mL
Acencio et al. (2007)	Rabbit pleural mesothelial cells	IL-8 cytokine concentration	6, 24, or 48 hours	125 µg/mL	Increased	p < .05
		TGF-β1 cytokine concentration	6, 24, or 48 hours		Increased	p < .05
		VEGF cytokine concentration	6, 24, or 48 hours		Increased	p < .05
Fletcher et al. (2019)	EL-1 human macrophages	Cell proliferation	24 hours	100 µg/mL	Increased	p < .05
	Human primary normal ovarian epithelial cells				Increased	p < .05
	FT33 immortalized human fallopian tube secretory epithelial cells				Increased	p < .05
	EL-1 human macrophages	CA-125 release	72 hours	100 µg/mL	No change	NS
	Human primary normal ovarian epithelial cells				Increased	p < .05
	FT33 immortalized human fallopian tube secretory epithelial cells				Increased	p < .05
Wylie et al. (1997)	Hamster tracheal epithelial cells	Cell proliferation/cytotoxicity (colony-forming efficiency)	FD14 talc	0.025 µg/cm^2	No change	NS
				0.05 µg/cm^2	No change	NS
				0.1 µg/cm^2	Decreased	p < .05
				0.5 µg/cm^2	Increased	p < .05
				1 µg/cm^2	No change	NS
				5 µg/cm^2	No change	NS
				15 µg/cm^2	No change	NS
			S157 talc	0.025 µg/cm^2	No change	NS
				0.05 µg/cm^2	No change	NS
				0.1 µg/cm^2	Decreased	p < .05
				0.5 µg/cm^2	No change	NS
				1 µg/cm^2	Decreased	p < .05
				10 µg/cm^2	Decreased	p < .05
				15 µg/cm^2	No change	NS
			CPS183 talc	0.025 µg/cm^2	No change	NS
				0.05 µg/cm^2	Decreased	p < .05
				0.1 µg/cm^2	No change	NS
				1 µg/cm^2	Decreased	p < .05
				5 µg/cm^2	Decreased	p < .05
				10 µg/cm^2	Decreased	p < .05
	Rat pleural mesothelial cells		FD14 talc	0.025 µg/cm^2	No change	NS
				0.05 µg/cm^2	No change	NS
				0.1 µg/cm^2	Decreased	p < .05
				0.5 µg/cm^2	No change	NS
				1 µg/cm^2	No change	NS
				5 µg/cm^2	No change	NS
				15 µg/cm^2	Decreased	p < .05
			S157 talc	0.025 µg/cm^2	No change	NS
				0.05 µg/cm^2	No change	NS
				0.1 µg/cm^2	No change	NS
				0.5 µg/cm^2	Decreased	p < .05
				1 µg/cm^2	Decreased	p < .05
				10 µg/cm^2	Decreased	p < .05
				15 µg/cm^2	Decreased	p < .05
			CPS183 talc	0.025 µg/cm^2	No change	NS
				0.05 µg/cm^2	No change	NS
				0.1 µg/cm^2	No change	NS
				1 µg/cm^2	Decreased	p < .05
				5 µg/cm^2	Decreased	p < .05
				10 µg/cm^2	Decreased	p < .05

IL: interleukin; NR: not reported; NS: not significant; ROS: reactive oxygen species; TGF: transforming growth factor; VEGF: vascular endothelial growth factor.

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