Weight of evidence evaluation for chemical-induced immunotoxicity for PFOA and PFOS: findings from an independent panel of experts

Figures & data

Table 1. Key epidemiological studies for human immune response to PFAS exposures (as PFAS in serum).

Topic area group	Study	Brief description^a
Vaccine response
Tetanus and diphtheria antibodies	Grandjean et al. (2012)	Serum antibodies for Faroe Islands population, children at ages five and seven years. (® PFOA; ® PFOS)
	Grandjean et al. (2017)	Serum antibodies for Faroe Islands population, children at age 13 years. (® PFOA; – PFOS)
	Granum et al. (2013)	Prospective birth cohort of mother-child pairs and serum antibodies. Tetanus = (– PFOA; – PFOS).
	Grandjean, Heilmann, Weihe, Nielsen, Mogensen Ulla, et al. (2017)	Serum antibodies for Faroe Islands population, children at five years. (® PFAS)
	Budtz-Jørgensen and Grandjean (2018)	Benchmark dose modeling of Faroe Islands serum data for children ages five and seven years.
	Timmermann et al. (2022)	Cross sectional study of Greenland population serum data for children ages 7 through 12 years. (– PFOA; ® PFOS)
	Abraham et al. (2020)	Cross sectional study of serum data for one year old children; also reported a decrease in Haemophilus influenza antibodies. (® PFOA; – PFOS)
Measles mumps rubella (MMR) antibodies	Stein et al. (2016)	Cross sectional study of serum data in NHANES, children ages 12 through 19 years. (– PFOA; ® PFOS)
	Granum et al. (2013)	Prospective birth cohort of mother-child pairs and serum antibodies (rubella only). (® PFOA; ® PFOS). No association with measles vaccine.
	Pilkerton et al. (2018)	Cross sectional analysis of adults and adolescents (>12 years) (® PFOA; – PFOS)
	Timmermann et al. (2020)	Serum antibodies for Guinea-Bissau population, infants and children ages birth to two years. (–PFOA; ® PFOS)
Other antibody endpoints	Zeng et al. (2019)	Serum antibodies for two viruses associated with hand, foot, and mouth disease (HFMD), in infants at birth to three months. (® PFOA; ® PFOS)
	Zeng et al. (2020)	Hepatitis B surface antibodies (HBsAB), cross sectional study of PFAS serum concentrations. (– PFOA; ® PFOS)
Clinical observations of disease
Common cold	Granum et al. (2013)	Prospective birth cohort of mother-child pairs and incidence of the common cold. (↑ PFOA; – PFOS)
	Looker et al. (2014)	Cross sectional study of adults and incidences of cold and/or cold symptoms. (® PFOA; – PFOS)
Hospitalizations	Fei et al. (2010)	Prospective study of maternal plasma concentrations and infectious disease hospitalizations in children ages ∼6–11 years. (– PFOA; – PFOS)
COVID-19	Grandjean et al. (2020)	Severity of COVID-19 and elevated exposure to perfluorinated alkylates. (– PFOA; – PFOS)
Immune hypersensitivity
Asthma/eczema/ wheezing	Granum et al. (2013)	Prospective birth cohort of mother-child pairs and incidence of asthma. (– PFOA; – PFOS)
	Smit et al. (2015)	Cohort of mother-child pairs ages five to nine years. (– PFOA; – PFOS)
	Beck et al. (2019)	Prospective birth cohort of mother-child pairs and asthma in children aged five years. (– PFOA; – PFOS)
	Manzano-Salgado et al. (2019)	Prospective cohort of mother-child pairs from birth to seven years. (® PFOA; – PFOS)
	Jackson-Browne et al. (2020)	Cross sectional analysis of asthma in children ages three to five years. (– PFOA; ® PFOS)
	Timmermann et al. (2017)	Serum antibodies for Faroe Islands population, children ages 5 and 13 years. (↑ PFOA; – PFOS)

^aParentheses following the description provide a general indication of the effect observed/associated with increases in serum concentrations of either PFOA or PFOS as follows: ® = decrease; ↑ = increase; – = no effect observed.

Table 2. Key animal toxicity studies to evaluate immune response to PFOA and/or PFOS since 2016.

Study	Observed changed in immunotoxicity endpoints^a	Brief description
PFOA
DeWitt et al. (2016)	↓ Relative spleen and relative thymus weights ↓ SRBC-specific IgM antibody responses ↓ DNP-specific IgM antibody response Splenic lymphocyte subpopulations	Female mice exposed to varying doses of PFOA in drinking water for up to 15 days.
Lee et al. (2017)	↓ Rectal temperature ↑ Serum histamine ↑ Serum TNF-α ↑ IgG1 and IgE	Male mice sensitized with oral ovalbumin and two treatment doses of PFOA for up to 13 days.
NTP (2020)	↓ Absolute and relative spleen weight Lymphoid follicle atrophy Pigment in spleen	Male and female Sprague Dawley rats exposed to varying doses PFOA in feed for 16 weeks.
PFOS
Zhong et al. (2016)	↑ Absolute spleen and thymus weight ↑ Thymic cellularity Lymphocyte population ↑ Absolute liver weight ↓ Splenic cellularity Changes in splenic lymphocyte populations ↓ Splenic lymphocyte proliferation ↓ Splenic NK cell activity ↓ Splenic PFCs IL-2 and IL-4 levels	Pregnant mice exposed to three doses of PFOS via gavage from gestation day 1–17; pups examined at 4 and eight weeks of age.
Xing et al. (2016)	↓ Absolute spleen weight	Male mice exposed to three doses of PFOS via gavage for 30 days.
Suo et al. (2017)	↓ Pathogen clearance at late stage infection Induction of IL-22 from ILC3 and Th17 cells ↓ Mucin	Mice exposed to one dose of PFOS for 25 days; infected with Citrobacter at day 7.
Han et al. (2018)	↑ Serum TNF-α ↑ Serum IL-6	Male Sprague Dawley rats exposed to two doses PFOS via gavage for four weeks.
Lee et al. (2017)	↓ Rectal temperature ↑ Histamine ↑ TNF-α ↑ IgG and IgE	Male mice sensitized with oral ovalbumin and three treatment doses of PFOA for up to 13 days.
NTP (2019)	↓ White blood cells ↓ Neutrophils; ↓ Eosinophils; ↓ Relative thymus weight	Male and female Sprague Dawley rats exposed to five doses of PFOS for 28 days via gavage.

Notes: Table adapted from OEHHA (2021). DNP: dinitrophenyl-ficoll; Ig: immunoglobulin; IL: interleukin; NK: natural killer cell; PFC: plaque forming cell; SRBC: sheep red blood cell; TNF-α: tumor necrosis factor alpha.

^aArrows indicate direction of change observed; ↑ for increase; ↓ for decrease.

Table 3. Expert panel participants.

Subpanel	Name	Country	Affiliation	Advanced degree	Years experience, post-degree	Number of publications	Expertise
Animal data	Dr. Peter Vogel	United States	St Jude Children’s Research Hospital	DVM; PhD	29	225	Carcinogenesis, hepatic toxicology, immunotoxicology, ocular toxicology, pathology, pulmonary toxicology, renal toxicology, veterinary toxicology
	Dr. Jorge Morales-Montor	Mexico	Universidad Nacional Autonoma De Mexico	PhD	25	215	Carcinogenesis, drug discovery, ecotoxicology, environmental health, immunotoxicology, in vitro toxicology, MoAs, metals toxicology, neurotoxicology, pathology, pulmonary toxicology, veterinary toxicology, neuroimmunoendocrinology
	Dr. Rajeev Tyagi	India	CSIR- IMTECH	PhD	11	49	Biotechnology, carcinogenesis, clinical and translational toxicology, drug discovery, hepatic toxicology, immunotoxicology, in vitro toxicology, medical devices, microbiology, nanotechnology, pathology, stem cells, vaccine and diagnostics, translational biomedical research
Dose-response	Dr. Kenny Crump	United States	Self-employed	PhD	53	181	Dose-response modeling, risk assessment
	Dr. Paul Villeneuve	Canada	Carleton University	PhD	21	200	Environmental health, epidemiology, risk assessment, meta-analysis, biostatistics
	Dr. Tony Cox	United States	Cox Associates, LLC	PhD	35	200	Biological modeling and pharmacokinetics, Carcinogenesis, dose-response modeling, epidemiology, food safety, immunotoxicology, MoAs, occupational and public health, pulmonary toxicology, risk assessment; artificial intelligence, machine learning, statistics, decision analysis
Epidemiology data	Dr. Yang Cao	Sweden	Örebro University	PhD	18	292	Dose-response modeling, environmental health, epidemiology, exposure assessment, occupational and public health, risk assessment
	Dr. Pierluigi Cocco	Italy	University of Manchester	MD	45	364	Carcinogenesis, environmental health, epidemiology, exposure assessment, occupational and public health
	Dr. Wendy Oddy	Australia	University of Tasmania	PhD	22	235	Epidemiology, other; nutrition
Oversight/WOE	Dr. Mahin Khatami	United States	Self-employed	PhD	42	60	Carcinogenesis, clinical and translational toxicology, immunotoxicology, MoAs, metals toxicology, neurotoxicology, ocular toxicology and inflammatory disease; inflammation, drug evaluation, biomarkers, cancer bioenergetics, protein chemistry, mitochondrial and histamine biology
	Dr. Michael Luster	United States	Self-employed	PhD	48	380	Immunotoxicology
	Dr. Jennifer Seed	United States	Self-employed	PhD	35	44	Regulatory toxicology, reproductive toxicology
	Dr. Rita Schoeny	United States	Rita Schoeny LLC	PhD	45	89	Carcinogenesis, dose-response modeling, environmental health, in vitro toxicology, MoAs, metals toxicology, mixtures, risk assessment
	Dr. Douglas Weed	United States	DLW Consulting Service	MD; PhD; MPH	40	145	Environmental health, epidemiology; methodology weight of evidence cancer causality nutrition research on pesticides and cancer, pharmacoepidemiology, environmental chemicals, ethics, cancer etiology, cancer prevention, cancer screening
Totals	N = 14	US: 8	–	PhD: 13	>450	>2600	–
		AU: 1		MD: 2
		CA: 1		DVM: 1
		IN: 1		MPH: 1
		IT: 1
		MX: 1
		SE: 1

Table 4. Key areas of improvement with current animal models for immunotoxicity.

Category	Deficit	Recommended improvement
Test duration	Short term studies only Basic screenings only	Studies should reflect chronic human exposures Consider intelligent design by consortium for future studies
Test species	One strain of rodent used (C57BL/6) Only male rodents used in most studies Different immune responses in animals vs. human (e.g. (IgM mediated vs. IgG-mediated responses)	Multiple strains/humanized mice Non-rodent studies (canine, non-human primate) Both sexes for hormonally influenced fluctuations
Dosing regimens	Not reflective of human exposures Exposure route (gavage) can alter immune function Variations in dosing schedules	Route, dose and duration harmonization Controlling for exposure route influence
Covariates	Not generally controlled in most studies	Identify and control Conduct large meta-analysis or large-scale study with controls
Results/conclusions	Shows association, not causation Differences in metabolites and half-lives when compared to human data Selection of outcome measures	Establish MoA for immune-mediated toxicity

Table 5. Key covariates to consider for establishing fluctuations in vaccine antibody titer (VAT).

Category	Covariate
Vaccine type	Manufacturer Vaccine adjuvants (incipients/excipients) Route of administration (oral, subcutaneous, intramuscular)
Dose and regimen	Dose provided by vaccine Time between inoculations Time post-vaccination Age at vaccine
Individual health	Age Lifestyle Nutrition status Includes diet, particularly seafood Co- morbidities (e.g. diabetes) Sex Gut microbes/flora Exercise/physical activity patterns Previous/concurrent infections Smoking/Alcohol/Caffeine intake Geographic residence Genetics BMI Other factors in blood (e.g. mercury, PCBs) Sleep patterns Ethnicity Income Tap water source Pregnancy/breastfeeding status Th1 phenotype

Table 6. Recommended biomarkers and metrics for establishing potential immunotoxicity in human studies and animal bioassays.

Category	Biomarker/metric
Serum biomarkers	Oxido-redox potential Histamine levels Anti-inflammatory agents Nucleic acids Proteins Free radicals (reactive oxygen/nitrogen species (ROS/RNS)) Blood enzymes (ALT, c-reactive proteins (CRP))
Immunoglobulins	Immunoglobulins (IgE, IgG, IgM) Pre- and post-vaccine levels Metabolites Cytokine profiling (e.g. interleukins, interferons, tumor necrosis factors) Natural killer (NK) cells Mast cells Macrophages Local lymph node assay Cell counts White cells Eosinophils CD cells T cells B cells
Other tests	Proteomic studies Luminex High throughput (HTP) assays

Figure 1. Panelist responses to how the Budtz-Jørgensen and Grandjean (2018) study should be used in a risk assessment for PFAS/PFOA.

Figure 2. Mean confidence ratings by subpanel for each of the modified Bradford Hill criteria for causality.

Figure 3. Ratings for how well evidence for PFOA and PFOS support extrapolation to other PFAAs. 1 = extrapolation is not supported; 5 = extrapolation strongly supported.

Grandjean P, Andersen EW, Budtz-Jørgensen E, Nielsen F, Mølbak K, Weihe P, Heilmann C. 2012. Serum vaccine antibody concentrations in children exposed to perfluorinated compounds. J Am Med Assoc. 307(4):391–397.

 PubMed Web of Science ®Google Scholar

Grandjean P, Heilmann C, Weihe P, Nielsen F, Mogensen Ulla B, Budtz-Jørgensen E. 2017. Serum vaccine antibody concentrations in adolescents exposed to perfluorinated compounds. Environ Health Perspect. 125(7):077018.

 PubMed Web of Science ®Google Scholar

Granum B, Haug LS, Namork E, Stølevik SB, Thomsen C, Aaberge IS, van Loveren H, Løvik M, Nygaard UC. 2013. Pre-natal exposure to perfluoroalkyl substances may be associated with altered vaccine antibody levels and immune-related health outcomes in early childhood. J Immunotoxicol. 10(4):373–379.

 PubMed Web of Science ®Google Scholar

Grandjean P, Heilmann C, Weihe P, Nielsen F, Mogensen Ulla B, Timmermann A, Budtz-Jørgensen E. 2017. Estimated exposures to perfluorinated compounds in infancy predict attenuated vaccine antibody concentrations at age 5-years. J Immunotoxicol. 14(1):188–195.

 PubMed Web of Science ®Google Scholar

Budtz-Jørgensen E, Grandjean P. 2018. Application of benchmark analysis for mixed contaminant exposures: mutual adjustment of perfluoroalkylate substances associated with immunotoxicity. PLoS One. 13(10):e0205388.

 PubMed Web of Science ®Google Scholar

Timmermann CAG, Pedersen HS, Weihe P, Bjerregaard P, Nielsen F, Heilmann C, Grandjean P. 2022. Concentrations of tetanus and diphtheria antibodies in vaccinated Greenlandic children aged 7–12 years exposed to marine pollutants, a cross sectional study. Environ Res. 203:111712.

 PubMed Web of Science ®Google Scholar

Abraham K, Mielke H, Fromme H, Völkel W, Menzel J, Peiser M, Zepp F, Willich SN, Weikert C. 2020. Internal exposure to perfluoroalkyl substances (PFASs) and biological markers in 101 healthy 1-year-old children: associations between levels of perfluorooctanoic acid (PFOA) and vaccine response. Arch Toxicol. 94(6):2131–2147.

 PubMed Web of Science ®Google Scholar

Stein CR, McGovern KJ, Pajak AM, Maglione PJ, Wolff MS. 2016. Perfluoroalkyl and polyfluoroalkyl substances and indicators of immune function in children aged 12-19 y: National Health and Nutrition Examination Survey. Pediatr Res. 79(2):348–357.

 PubMed Web of Science ®Google Scholar

Pilkerton CS, Hobbs GR, Lilly C, Knox SS. 2018. Rubella immunity and serum perfluoroalkyl substances: sex and analytic strategy. PLOS One. 13(9):e0203330.

 PubMed Web of Science ®Google Scholar

Timmermann CAG, Jensen KJ, Nielsen F, Budtz-Jørgensen E, van der Klis F, Benn CS, Grandjean P, Fisker AB. 2020. Serum perfluoroalkyl substances, vaccine responses, and morbidity in a cohort of Guinea-Bissau children. Environ Health Perspect. 128(8):87002.

 PubMed Web of Science ®Google Scholar

Zeng XW, Bloom MS, Dharmage SC, Lodge CJ, Chen D, Li S, Guo Y, Roponen M, Jalava P, Hirvonen MR, et al. 2019. Prenatal exposure to perfluoroalkyl substances is associated with lower hand, foot and mouth disease viruses antibody response in infancy: findings from the Guangzhou Birth Cohort Study. Sci Total Environ. 663:60–67.

 PubMed Web of Science ®Google Scholar

Zeng XW, Li QQ, Chu C, Ye WL, Yu S, Ma H, Zeng XY, Zhou Y, Yu HY, Hu LW, et al. 2020. Apr Alternatives of perfluoroalkyl acids and hepatitis B virus surface antibody in adults: isomers of C8 health project in China. Environ Pollut. 259:113857.

 PubMed Web of Science ®Google Scholar

Looker C, Luster MI, Calafat AM, Johnson VJ, Burleson GR, Burleson FG, Fletcher T. 2014. Influenza vaccine response in adults exposed to perfluorooctanoate and perfluorooctanesulfonate. Toxicol Sci. 138(1):76–88.

 PubMed Web of Science ®Google Scholar

Fei C, McLaughlin JK, Lipworth L, Olsen J. 2010. Prenatal exposure to PFOA and PFOS and risk of hospitalization for infectious diseases in early childhood. Environ Res. 110(8):773–777.

 PubMed Web of Science ®Google Scholar

Grandjean P, Timmermann CAG, Kruse M, Nielsen F, Vinholt PJ, Boding L, Heilmann C, Mølbak K. 2020. Dec 31 Severity of COVID-19 at elevated exposure to perfluorinated alkylates. PLOS One. 15(12):e0244815.

 PubMed Web of Science ®Google Scholar

Smit LA, Lenters V, Høyer BB, Lindh CH, Pedersen HS, Liermontova I, Jönsson BA, Piersma AH, Bonde JP, Toft G, et al. 2015. Prenatal exposure to environmental chemical contaminants and asthma and eczema in school-age children. Allergy. 70(6):653–660.

 PubMed Web of Science ®Google Scholar

Beck IH, Timmermann CAG, Nielsen F, Schoeters G, Jøhnk C, Kyhl HB, Høst A, Jensen TK. 2019. Association between prenatal exposure to perfluoroalkyl substances and asthma in 5-year-old children in the Odense Child Cohort. Environ Health. 18(1):97.

 PubMed Web of Science ®Google Scholar

Manzano-Salgado CB, Granum B, Lopez-Espinosa MJ, Ballester F, Iñiguez C, Gascón M, Martínez D, Guxens M, Basterretxea M, Zabaleta C, et al. 2019. Prenatal exposure to perfluoroalkyl substances, immune-related outcomes, and lung function in children from a Spanish birth cohort study. Int J Hyg Environ Health. 222(6):945–954.

 PubMed Web of Science ®Google Scholar

Jackson-Browne MS, Eliot M, Patti M, Spanier AJ, Braun JM. 2020. PFAS (per- and polyfluoroalkyl substances) and asthma in young children: NHANES 2013–2014. Int J Hyg Environ Health. 229:113565.

 PubMed Web of Science ®Google Scholar

Timmermann CA, Budtz-Jørgensen E, Jensen TK, Osuna CE, Petersen MS, Steuerwald U, Nielsen F, Poulsen LK, Weihe P, Grandjean P. 2017. Association between perfluoroalkyl substance exposure and asthma and allergic disease in children as modified by MMR vaccination. J Immunotoxicol. 14(1):39–49.

 PubMed Web of Science ®Google Scholar

DeWitt JC, Germolec DR, Luebke RW, Johnson VJ. 2016. Associating changes in the immune system with clinical diseases for interpretation in risk assessment. Curr Protoc Toxicol. 67(1):1811–18122.

 Google Scholar

Lee JK, Lee S, Baek MC, Lee BH, Lee HS, Kwon TK, Park PH, Shin TY, Khang D, Kim SH. 2017. Association between perfluorooctanoic acid exposure and degranulation of mast cells in allergic inflammation. J Appl Toxicol. 37(5):554–562.

 PubMed Web of Science ®Google Scholar

[NTP] National Toxicology Program. 2020. NTP technical report on the toxicology and carcinogenesis studies of perfluorooctanoic acid (CASRN 335-67-1) administered in feed to Sprague Dawley (Hsd: Sprague Dawley® SD®) rats [Internet]. [place unknown]: National Institute of Environmental Health Sciences, National Institutes of Health, U.S. Department of Health and Human Services.

 Google Scholar

Zhong S-Q, Chen Z-X, Kong M-L, Xie Y-Q, Zhou Y, Qin X-D, Paul G, Zeng X-W, Dong G-H. 2016. Testosterone-mediated endocrine function and TH1/TH2 cytokine balance after prenatal exposure to perfluorooctane sulfonate: by sex status. Int J Mol Sci. 17(9):1509.

 PubMed Web of Science ®Google Scholar

Xing J, Wang G, Zhao J, Wang E, Yin B, Fang D, Zhao J, Zhang H, Chen YQ, Chen W. 2016. Toxicity assessment of perfluorooctane sulfonate using acute and subchronic male C57BL/6J mouse models. Environ Pollut. 210:388–396.

 PubMed Web of Science ®Google Scholar

Suo C, Fan Z, Zhou L, Qiu J. 2017. Jul 12 Perfluorooctane sulfonate affects intestinal immunity against bacterial infection. Sci Rep. 7(1):5166.

 PubMedGoogle Scholar

Han R, Zhang F, Wan C, Liu L, Zhong Q, Ding W. 2018. Effect of perfluorooctane sulphonate-induced Kupffer cell activation on hepatocyte proliferation through the NF-κB/TNF-α/IL-6-dependent pathway. Chemosphere. 200:283–294.

 PubMed Web of Science ®Google Scholar

[NTP] National Toxicology Program. 2019. NTP technical report on the toxicity studies of perfluoroalkyl carboxylates (perfluorohexanoic acid, perfluorooctanoic acid, perfluorononanoic acid, and perfluorodecanoic acid) administered by gavage to Sprague Dawley (Hsd: Sprague Dawley SD) rats [Internet]. [place unknown]: National Institute of Environmental Health Sciences, National Institutes of Health, U.S. Department of Health and Human Services.

 Google Scholar

[OEHHA] Office of Environmental Health Hazard Assessment. 2021. Public health goals. First public reviewed draft. Perfluorooctanoic acid and perfluorooctane sulfonic acid in drinking water [Internet]. [place unknown]: California Environmental Protection Agency. https://oehha.ca.gov/media/downloads/crnr/pfoapfosphgdraft061021.pdf.

 Google Scholar