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## Propylene oxide (PO)

by Paul A. Schulte PhD, Kari Hemmi nki PhD, and Nancy B. Hopf PhD

Citation for most recent IARC review
IARC Monographs 60, 1994

Current evaluation:
Conclusion from the previous Monograph

Propylene oxide is possibly carcinogenic to humans (Group 2B)

.

There is inadequate evidence in humans for the car cinogenicity of propylene oxide.

There is sufficient evidence in experimental animals for the carcinogenicity of propylene

oxide.

Exposure and biomonitoring
Biomarkers of exposure or effect

There are three biomarkers of effect curren tly being applied to measure PO exposure in humans: DNA adducts, hemoglobin adducts, and si ster chromatid exchanges (SCE). These effect biomarkers integrate e xposure for different time interval s, and correlation between all three end points is not expected.

DNA adducts of propylene oxide are formed in various organs of mice, rats and dogs. PO binding in mouse liver DNA was about one-twentie th that of ethylene oxide (EO). The major

adduct formed in DNA after in vitro reaction of PO is 7-HP-guanine, followed by the adducts 3-HP-, 1-HP-adenine, and 3-HP-cytosine. Th ese adducts are chemically unstable: 7-HPguanine and 3-HP-adenine will depurinate, form ing apurinic sites, and 1-HP-adenine and 3- HPcytosine will spontaneously convert to N 6-HP-adenine and 3-HP-uracil, respectively. The level of 1-HP-adenine corresponded to 2% of 7-HP-guanine. 1-HP-adenine is chemically more stable ( t 1/2 = 9.2 days for rearrangement to an N 6 adduct compared with t 1/2 = 5 days for depurination of 7-HP-guanine). Therefore, th is minor adduct could be an alternative for monitoring PO exposures (Czene et al., 2002 and references ther ein). DNA adducts are subject to DNA repair and necrosis of the ce ll, which will alter the cumulative dose estimate depending if the exposure is intermittent (allowi ng for DNA repair time) or constant (no DNA repair time).

PO forms adducts with albumi n and hemoglobin (Hb) in man, dog, rat and mouse. Adducts to Hb are especially suitable because small mol ecules such as PO form chemically stable

adducts with the N-terminal valine of Hb, N-(3-hydroxypropyl)v aline (HOPrVal). Concentration of HOPrVal (Hb) is linearly re lated to air concentrations of PO (see below Boogaard et al., 1999). These adducts do not appear to affect the average life span of the erythrocytes. The exposure dose as measured by determination of Hb-adduct concentrations is integrated over the average life span of human erythrocytes, which is approximately 120 days.80 Hb-adducts are not subject to re pairs and therefore a cumulative dose over the past 120 days

is achieved.

PO may cause SCE, which is a nonmutati onal genotoxic event (do not alter genetic information); however, when they arise in vivo

, they do indicate that a putative mutagen/carcinogen has reached the critical cellu lar target for direct genotoxicity. SCE is not specific to PO exposures, and may be caused by other chemicals.

For dose estimates, monitoring Hb adducts is preferred to DNA adducts for several reasons: availability of large amounts (erythrocytes in whole blood), availability of methods for chemical identification (specific and sensitive), and the well-defined life span due to absence of DNA repair (Ogawa et al., 2006). Also, the hemogl obin adduct is strongly correlated with measured concentrations of PO (8h TWA) (B oogaard et al., 1999). Limitation of Hb adducts is that they cannot be used for monitori ng of acute or short term exposures.

Polymorphisms: Hypoxanthine phosphoribosyl transferase ( HPRT ) gene has been used for studies of the mutagenic effect of EO but not for PO. High exposures to EO in workers showed a significant increase in HPRT mutant frequency compared to controls, while at low exposures

no such association was found.

Occupational exposure

Occupational exposure occurs during the produc tion of propylene oxide and its derivatives and during production of hydroxypro pyl starch ethers. (IARC, 1994). Since the IARC review, three new occupational biomonitoring studies have been conducted: one in a chemical plant manufacturing glycols and glycol ethers from PO and two in PO producing plants. Included also is an exposure to polyethylene (P E) study because PE metabolizes to PO.

An occupational exposure study was performed to establish a relationship between ambient exposure to EO and PO in air and the formati on of their respective adducts to Hb (Boogaard et al., 1999*). The personal air monitoring (PAM) data were measured by gas-diffusion monitors in the breathing zone in three groups of workers: (1) In 1990: male operators (N=20) and controls (N=36 operators from a different di vision of the same plant with no exposure to EO or PO) in a chemical plant manufacturing gl ycols and glycol ethers from EO and PO; (2) In 1997 follow-up: male operators (N=18) from the same plant maintaining and inspecting a shutdown of the plant; (3) In 1997: male workers (N=28) maintaining a styrene-PO plant during shutdown. No correlations between random PAMs and Hb-adducts were found in the 1990 survey of male operators and controls, but correlations were found between PO air concentrations spanning four m onths (the life span of the hum an erythrocyte) and the Hbadducts of male operators in the 1997 follo w-up. However, in the follow-up study, 89 of 112 PAMs (79.5%) were below the limit of detection (0.2 mg m

− 3 = 0.08 ppm 8-h TWA), but did

allow calculations of increments for the 4-mo interval from which daily (8-h) increments and steady-state concentrations we re derived. Background levels of HOPrVal were found in some controls; however, the publica tion does not reveal how many nor what level of HOPrVal81 levels were found in controls and exposed operators. In the 19 97 study of maintenance

workers the median HOPrVal concentrations in creased 21.3 pmol/g globin, a 2-fold increase from pre-maintenance activities (24.4 pmol/g globin) to end of shutdown period (45.7 pmol/g globin). A PO air concentra tion of 10 mg m-3 is equal to 5.3 nmol/g globin, and the operators’ blood levels were measured to 0.005 to 0.161 nmol/g globin in 1997.

Hemoglobin adducts were assessed as a measure of PO exposure in a chemical plant using PO, EO, acrylamide and acrylonitrile in the production of surfactants for the textil e industry in exposed (N=62) and controls selected from the laboratory (N=10) (Schettgen 2002). In addition to PO globin adduct (HOPrVal), othe r globin adducts (N-2-carbamoylethylvaline, N2-cyanoethylvaline, HOEtVal) were also measured for workers at this plant. HOPrVal were not found in either exposed workers or controls. The authors contribute this fact to either the limit of the analytical detection (LOD, 80 pmol/g globin), or due to the low adduct formation of PO, which is five times lower than for EO. This study does not report PO air concentrations or describe what type of j obs the workers were performing.

A small study in China was conducted to de termine DNA adducts (1-hydroxypropyl-adenine or 1HPAdenine), hemoglobin adducts (HOPrV al), and SCE in blood of workers (N=8) occupationally exposed to PO at a PO-producin g plant and control subjects (N=8) at an institute of occupational health (Czene et al., 2002). PO con centrations were determined based on area air samples collected in areas workers occupied the day before blood samples were drawn. PO concentrations ranged from 0.9 to 6.9 ppm, and workers were reported to be present in the areas of highest PO concentration for 1 to 1.5 h per day

. HOPrVal adduct concentrations in the exposed wo rkers ranged from 0.13 to 4.92 pmol mg

− 1 globin for a mean of 2.69 ± 1.52 pmol mg

− 1 globin, which is in the same ra nge as found in the glycol and glycol ether from PO produci ng plants (Boogard et al., 1999)

. For comparison, values for controls ranged from 0.005 to 0.008 pmol mg

− 1 globin. The mean difference between exposed and controls was highly significan t. In this study, Hb- and DNA-adducts were measured in the same worker (Czene et al., 2002). DNA adducts were detected in 7 of the 8 exposed workers with a mean value of 0.66 ± 0.34 mol per 109 mol normal nucleotides. DNA

adducts were not detected in controls. Th e LOD was 0.1 mol per 109 mol normal nucleotides.

1HPAadenine rather than the N7HPG adducts were measured in this study because they are more stable. The group mean difference for DNA adduct concentrations between exposed workers and controls was highly significant, as was the correlation between the individual OHPrVal hemoglobin and 1HPAdenine DNA adduct concentrations when all subjects were included in the analysis (including the cont rols with no detectab le adducts) (r = 0.887, P <0.0001). However, limiting the correlation to only the exposed workers resulted in a correlation with marginal statistical significan ce (r= 0.713, p = .047). For the third biomarker, the difference in mean SCE frequency was significant (p ≥ 0.011) with mean SCE frequency was 3.7 ± 2.11% for the exposed workers and 2.0 ±0.52% for the control group (Czene et al., 2002). Strong and significant correlations were found between levels of N7HPG and SCE frequencies (r =0.792; p = 0.00026) and between OHPrVal and SCEs (r =0.766; p = 0.0014). Excluding smokers gave signifi cant associations between SC E frequency and 1HPAdenine adducts r = 0.851 (p = .00044), and betw een SCE and OHPrVal r=0.757 (p = 0.011).82 Exposures to PO at three manufacturing sites in France and the Netherlands were assessed by determining Hb-adducts (n>800 samples) over a 2-year period from operators, maintenance fitters and office staff (Jones et al., 2005***). The geometric means range from the three plants was 2.9-12.6 pmol g-1 globin, which is four to 16 times lower than the other two plants mentioned previously (Boogard et al., 1999, Czen e et al., 2002). To evaluate smoking as a confounder, workers at one of the plants we re separated into smokers (N=51) and nonsmokers (N=177) and their GMs were 3.8 a nd 3.0 pmol g-1 globin, respectively. Smoking does not seem to be a major confounder when measuring Hb-adduct in blood of very low exposed workers.

Polyethylene (PE) is metabolized to PO. PO concentrations in blood from healthy male nonsmoking volunteers (N=4) resulting from inhalati on of PE (mean concentrations of 9.82 and 23.4 ppm for 180 min) was measured to 0.44 and 0.92 nmol/l for the two PE air levels (Filser et al., 2008**). The authors found a distinct species difference be tween rats and human in the activities of PO metabolizing enzymes of liver and lung cell fractions. Human microsomal epoxide hydrolase activities towa rd PO 2-4x (liver) and 6-8x (lung) higher in humans than

those of rats.

Environmental exposures Household and industrial detergents, paints, adhesi ves, textiles, defoamers, oil field chemicals, cosmetics, functional fluids and lubricants, heat transfer fluids, and automotive brake fluids

contain PO. PO is also an addi tive in food. Levels of PO expo sures from using these articles have not been measured (or reported).

PO is also found in tobacco smoke and is an environmental pollutant.

In a 2-year cancer bio-assay study in rats, the authors compared the results obtained from rats being exposed to a constant PO concentra tion with an unexposed human. The unexposed human level of PO Hb-adduct level was 0.006 pmo l/mg globin (compared to Boogaard et al., 1999 for occupational exposure of 5.32 pmol/m g globin) (Rios-Blan co et al., 2002*****).

Human carcinogenicity:
From the last Monograph (vol. 60, 1994): One ca se-control study provides information about cancer risk in relation to expos ure to propylene oxide specifica lly but does not allow any firm conclusion regarding carcinogenicity. Since th en only one epidemiological study has been

conducted.

Mortality study of workers formerly employe d in a ethylene chlorohydrin and propylene chlorohydrin process plant (Olsen et al., 1997 ). The objective was to compare the SMRs at this plant with pr evious excess mortality from pancre atic cancer and lymphopoietic and hematopoietic cancer found among workers in a nother chlorohydrin unit. All male workers (N=1361) who had worked in the ethylene or propylene production area for a month and worked at either manufacturing site for a year were included in the study. These workers were identified using work histories. Vital st atus was determined from 1940 to 1992. SMR was non-significantly elevated for lymphopoietic and hematopoietic cancers (SMR=129; 95%CI 62-238, observed 10 cases, expected 7.7), and not elevated for malignant neoplasms83 (SMR=94) and pancreatic cancer (SMR=25),. In cluding a latency of 25 years gave increased the SMR to 144 for lymphopoietic and hematopoie tic cancers, but did not reach statistical significance (95%CI 52-312). Compar ing the SMRs across plants ga ve similar results except for lymphopoietic and hematopoietic cancer deaths at one plant SMR=181 (95%CI 66-393, observed 6 cases and expected 3.3). Compar ing SMRs across process gave similar nonsignificant results; however, including a late ncy of 25 years gave a SMR= 194 (95%CI 71- 423, 6 observed, 3.1 expected,) for lymphopoiet ic and hematopoietic cancer among those employees with exposure only to the ethylen e chlorohydrin process. At 10-20 years of employment in the chlorohydrin plants, ther e was a significant Mantel-Haenszel RR (RR 3.56, 95% CI 1.23-10.29) for lymphopoietic and hemat opoietic cancer based on three observed deaths. This effect disappeared with different categories were used in a Poisson regression. Limitation of this study is that following expos ures are not considered : cigarette smoking, the ratio of exposure to chlorohydrin versus oxide, exposure in enclosed versus outdoor production facility, earlier versus later exposur e periods (usually i ndustrial hygiene improves over the years). The mean follow-up time was 25 years and additional follow-up time might change the outcome.

Animal cancer data (excluding mechanisms):
From the last Monograph (vol. 60, 1994): Propylen e oxide was tested by oral gavage in one study in rats, by inhalation in one study in mice a nd in three adequate studies in rats and by subcutaneous administration in one study in mice and in one study in rats.

Propylene oxide administered by oral gavage to rats produced tumours of the forestomach, which were mainly squamous-cell carcinom as. In mice exposed by inhalation, propylene oxide produced hemangiomas and hemangiosarcoma s of the nasal cavity and a few malignant nasal epithelial tumours. In a study in rats of each sex exposed by inhalation, papillary adenomas of the nasal cavity were observed in males and females and thyroid adenomas and carcinomas were found in females; in the second study, in males, papillary adenomas of the nasal cavity and an increased incidence of adrenal phaeochromocytomas were observed; in the third study, in females, increased in cidences of mammary fibroadenomas and

adenocarcinomas were observed. Subcutaneous administration of propylene oxide to mice produced local sarcomas; the study in ra ts was inadequate for evaluation.

Male Fischer 344/N rats in closed exposure chambers were exposed to constant PE concentrations, between 20.1 and 3000 ppm (7 h at least) and the PO concentrations were measured in the blood of these rats (Filser et al., 2008** ). The PO blood concentrations ranged from 53 nmol/l at 20.1 ppm PE to 1750 nmol/l at 3000 ppm PE. The PO blood concentrations measured in this study are too low for resulting in PO treatment–related tumors seen in a long-term study (Renne et al., 1986; U.S. National Toxicology Program, 1985b).

Mechanisms of carcinogenicity:
From the last Monograph (vol 60, 1994): In rats exposed by inhalation, there is strong uptake of propylene oxide, which is then metaboli zed extensively and eliminated rapidly. Metabolism occurs predominantly by conjugation with glutathione. Propylene oxide can also84 be hydrolyzed by epoxide hydrolase to 1,2-propaned iol, which is subsequently metabolized to lactic and pyruvic acids.

Dominant lethal mutations were not induced in rats or mice, and sperm abnormalities were not observed in mice exposed to propylene oxide in vivo. Micronuclei a nd, in single studies,

chromosomal aberrations and sister chromatid exchange were induced in mouse bone marrow after intraperitoneal injection of propylene oxide. Neither sister chromatid exchange nor

chromosomal aberrations were induced in monkeys exposed by inhalation to 300 ppm. Propylene oxide induced chromosomal aberrations and sister chromatid exchange in human lymphocytes and DNA damage, gene mutation, chro mosomal aberrations and sister chromatid exchange in mammalian cells in vitro. It cause d dominant lethal mutation in Drosophila and was mutagenic to yeast, fungi and bacteria.

PO binds covalently to the cyclic ring nitrogens in DNA producing hydroxypropyl (HP) adducts. HP adducts can be non-promutagenic lesions being eliminated spontaneously, leaving behind the depurinated site s that can result in mutations only if not repaired before DNA replication occurs, or the adduct may be at the coding site resu lting in potentially promutagenic lesions (Albertini et al., 2007). These adducts may cause mutation by

transversions. PO can also react with the phosphate groups in the DNA backbone.

PO is a less potent mutagen than the week EO mutagen.

In a 2-year cancer bioassay with rats exposed to constant PO concentration the pattern for

DNA and Hb-adduct accumulation did not correlate with the incidence curve for nasal tumors (Rios-Blanco et al., 2002*****). Tumor forma tion was only seen at >300 ppm exposure (highest levels tested 300 and 500 ppm). Neith er adduct accumulation in tissues can explain

the threshold in nasal tumor formation. The auth ors suggest an increased cell proliferation in the nose occurs at high PO concentration to be a cr itical factor for tumorigenesis in this tissue.

Research needs and recommendations:

The animal data consist of oral, inhalation and subcutaneous studies in three strains of rats and two strains of mice. Propylene oxide caused tumo rs at or near the site of administration in rodents, causing nasal tumors af ter inhalation exposure (NTP, 1985).

One of the major limitations in cancer epidemiol ogical studies is that records of exposure are often incomplete or lacking (Kolman et al., 2002). Measuring hemoglobin adducts, an extremely sensitive effect biomarker, whic h does not undergo repair as DNA-adducts, may overcome this problem. In addition, low background level of OHPrVal shows that contributions from non-occupational sources are minor. The levels of OHPrVal of the

smokers were similar to those of the nonsmoke rs, indicating that tobacco smoking is not the major contributing factor to the found backgroun d levels. Therefore, a future prospective study exploring cytogenetic eff ect would be feasible even though PO exposures are low

because:

1) the great difference in means between expos ed and controls seen in the pilot study will require few participants in the two groups (exposed and unexposed workers) and85 2) the OHPrVal is a sensitive and specific biomarker to PO exposures (even detected in controls with unknown exposures).

The OHPrVal adduct represents cumulative ex posures over a 3-month time period. The number of workers recruited to such a study would only be about 11 a ssuming equal variances in the two groups; however, othe r possible confounders should be included in the study such as gender, age, ethnicity, y ears of education, smoking, and possibly polymorphisms in the GST metabolizing pathway of PO. In additi on, loss of follow-up and errors in power calculations would benefit from a larger study (~ 100 exposed and 100 controls in a balanced design). Various exposure-selective cross sec tional epidemiological studies that look at OHPrVal adducts and cytogeneti c effects would be useful.

Possible sources of future cohorts for such a st udy might be the already established cohorts in PO manufacturing workers (USA ) (Olsen et al., 1997), which did not show an increased mortality rate due to cancer by duration with or without latency or cancer risk by process (PO versus EO); or the manufacturing cohorts in Fr ance and the Netherlands (Jones et al., 2005), and China (Czene et al., 2008). PO production is being expanded, and workers at these new PO manufacturing sites should be recruited for a future st udy. Other possible cohorts for propylene oxide biomarker epidemiological studie s are: processing workers where PO is used as a starting material in polyurethane polyols (NTP 11 th RoC), surfactants for textiles (Schettgen et al., 2002) and glycol/glycol et her manufacturing (Boogaard et al., 1999), and manufacturing of polyethylene (PE), which meta bolizes to PO. Women should be included in the study as PO might be mammary carcinogen (Rudel et al., 2007). Workers’ exposures to PO in paint and automotive fluids have not b een adequately characterized. If these workers have exposures to PO then these could poten tially be included in a future biomarker epidemiological study. Therefore recruiting sufficient workers to a future prospective study

should be feasible.

Selected relevant publications since IARC review:
Albertini RJ, Sweeney LM. Propylene-oxide-G enotoxicity profile of a rodent nasal carcinogen. Crit Rev Toxicol 2007; 37: 489–520.

Boogaard PJ, Rocchi PSJ, van Sittert NJ. Biom onitoring of exposure to ethylene oxide and propylene oxide by determination of hemoglobi n adducts Correlations between airborne exposure and adduct levels. Int Arch Occup Environ Health 1999; 72: 142-150.

Czene K, Osterman-Golkar S, Xiao Y, et al . Analysis of DNA and hemoglobin adducts and sister chromatid exchanges in a human populat ion occupationally exposed to propylene oxide A pilot study. Cancer Epidemiol Biomark Prev 2002; 11: 315–318.

Filser J, Hutzler C, Rampf F, et al. Concen trations of the propylene metabolite propylene oxide in blood of propylene exposed rats a nd humans A basis for risk assessment. Toxicol

Sci 2008; 102: 219–231.86 Jones AL, van der Woord M, Bourrillon F. Use of a whole blood competitive immunoassay for the assessment of worker exposed to pr opylene oxide at three manufacturing facilities. Ann Occup Hyg 2005; 49: 241–243.

Kolman A, Chovanec M, Osterman-Golkar S. Genotoxic effects of ethylene oxide, propylene oxide and epichlorohydrin in hum ans Update review (1990-2001). Mutat Res 2002; 512: 173–

194.

Ogawa M, Oyama T, Isse T, et al. Hemoglobin adducts as a marker of exposure to chemical substances, especially PRTR class I designated chemical substances. J Occup Health 2006;

48: 314-328.

Olsen GW, Lacy SE, Bodner KM, et al. Mortality from pancreatic and lymphopoietic cancer among workers in ethylene and pr opylene chlorohydr in production. Occup Environ Med 1997; 54: 592-598.

Ríos-Blanco MN, Ranasinghe A, Upton P, et al. Exposure dependent accumulation of N-(2- hydroxypropyl)valine in hemogl obin of F344 rats exposed to propylene oxide by the

inhalation route. J Chrom B 2002; 778: 383–391.

Rudel RA, Attfield KR, Schifano JN, Brody JG . Chemicals causing mammary gland tumors in animals signal new directions for epidemio logy, chemicals testing, and risk assessment for breast cancer prevention. Cancer 2007; 109: 2635-2666.

Schettgen T, Broding HC, Angerer J, Drexler H. Hemoglobin adducts of ethylene oxide, propylene oxide, acrylonitrile and acrylamide biomarkers in occupational and environmental

medicine. Toxicol Lett 2002; 134: 65–70.

* Authors from: Shell Research and Technology Ce nter, Amsterdam Shell International Chemicals ** Study paid for by CEFIC – an organization representing the European chemical industry *** Authors from: Lyondell Chemical Europe – a chemical manufacturing company   Authors from: Dow Chemical Company, Health and Environmental Sciences ** *** Study supported in part by ACC PO/PGP and Olefin panel, CEFIC PO/PG sector group, NIH grants



## Formaldehyde

by Bernard D. Goldstein PhD and Martyn T. Smith PhD

Citation for most recent IARC review
IARC Monograph 88, 2006

Current evaluation
Conclusion from the previous Monograph: