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IARC Working Group on the Evaluation of Carcinogenic Risk to Humans. Pharmaceuticals. Lyon (FR): International Agency for Research on Cancer; 2012. (IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, No. 100A.)

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CYCLOPHOSPHAMIDE

Cyclophosphamide was considered by previous IARC Working Groups in 1980 and 1987 (IARC, 1981, 1987a). Since that time, new data have become available, these have been incorporated into the Monograph, and taken into consideration in the present evaluation.

1. Exposure Data

1.1. Identification of the agent

  • Chem. Abstr. Serv. Reg. No.: 50-18-0
  • Chem. Abstr. Name: 2H-1,3,2-Oxazaphosphorin-2-amine, N,N-bis(2-chloroethyl)tetrahydro-, 2-oxide
  • IUPAC Systematic Name: N,N-Bis(2-chloroethyl)-1-oxo-6-oxa-2-aza-1λ5-phosphacyclohexan-1-amine
  • Synonyms: 2-[Bis(2-chloroethyl)amino]tetrahydro-2H-1,3,2-oxazaphosphorin 2-oxide; bis(2-chloroethyl)phosphoramide cyclic propanolamide ester; N,N-bis(β-chloroethyl)-N′,O-trimethylenephosphoric acid ester diamide; N,N-bis(2-chloroethyl)-N′,O-propylenephosphoric acid ester diamide; Cytoxan; Endoxan; Neosar
  • Description: Crystalline solid [anhydrous form] (O’Neil, 2006)

1.1.1 Structural and molecular formulae, and relative molecular mass

Image 978-9283213185-C009-F001.jpg

C7H15Cl2N2O2P

Relative molecular mass: 261.1

1.2. Use of the agent

Cyclophosphamide is an antineoplastic agent metabolized to active alkylating metabolites with properties similar to those of chlormethine. It also possesses marked immunosuppressant properties. It is widely used, often in combination with other agents, in the treatment of several malignant diseases. Information for Section 1.2 is taken from McEvoy, (2007), Royal Pharmaceutical Society of Great Britain (2007), and Sweetman (2008).

1.2.1 Indications

Cyclophosphamide is used in the treatment of chronic lymphocytic leukaemia, lymphomas, soft tissue and osteogenic sarcoma, and solid tumours. It is given orally or intravenously. Cyclophosphamide is inactive until metabolized by the liver.

(a) Hodgkin lymphoma

Cyclophosphamide is used in combination regimens (e.g. bleomycin, etoposide, doxorubicin, cyclophosphamide, vincristine, procarbazine, and prednisone [known as BEACOPP]) for the treatment of Hodgkin lymphoma.

(b) Non-Hodgkin lymphoma

Cyclophosphamide is used in combination therapy for the treatment of non-Hodgkin lymphoma, including high-grade lymphomas, such as Burkitt lymphoma and lymphoblastic lymphoma, as well as intermediate- and low-grade lymphomas. Cyclophosphamide is commonly used with doxorubicin (hydroxydaunorubicin), vincristine (oncovin), and prednisone (known as the CHOP regimen), with or without other agents, in the treatment of various types of intermediate-grade non-Hodgkin lymphoma. Cyclophosphamide has also been used as a single agent in the treatment of low-grade lymphomas.

(c) Multiple myeloma

Cyclophosphamide is used in combination with prednisone, or as a component of combination chemotherapy (i.e. vincristine, carmustine, melphalan, cyclophosphamide, and prednisone [VBMCP]) for the treatment of multiple myeloma.

(d) Leukaemia

Cyclophosphamide is used commonly for the treatment of chronic lymphocytic (lymphoblastic) leukaemia. Cyclophosphamide is used in combination with busulfan as a conditioning regimen before allogeneic haematopoietic progenitor cell transplantation in patients with chronic myelogenous leukaemia.

Cyclophosphamide is used in the treatment of acute lymphoblastic leukaemia, especially in children. In the treatment of acute myeloid (myelogenous, non-lymphocytic) leukaemia, cyclophosphamide has been used as an additional drug for induction or post-induction therapy.

(e) Cutaneous T-cell lymphoma

Cyclophosphamide is used alone or in combination regimens for the treatment of advanced mycosis fungoides, a form of cutaneous T-cell lymphoma.

(f) Neuroblastoma

Cyclophosphamide alone is used in the treatment of disseminated neuroblastoma. Combination chemotherapy that includes cyclophosphamide is also used for this neoplasm.

(g) Cancer of the ovary

Cyclophosphamide is used in combination chemotherapy (vincristine, actinomycin D, and cyclophosphamide [VAC]) as an alternative regimen for the treatment of ovarian germ-cell tumours.

Cyclophosphamide has been used in combination with a platinum-containing agent for the treatment of advanced (Stage III or IV) epithelial ovarian cancer.

(h) Retinoblastoma

Cyclophosphamide is used in combination therapy for the treatment of retinoblastoma.

(i) Cancer of the breast

Cyclophosphamide is used alone and also in combination therapy for the treatment of breast cancer.

Combination chemotherapy with cyclophosphamide is used as an adjunct to surgery in premenopausal and postmenopausal women with node-negative or -positive early (TNM Stage I or II) breast cancer. Adjuvant combination chemotherapy that includes cyclophosphamide, methotrexate, and fluorouracil has been used extensively.

Adjuvant combination chemotherapy (e.g. cyclophosphamide, methotrexate, and fluorouracil; cyclophosphamide, adriamycin, and fluorouracil; cyclophosphamide and adriamycin with or without tamoxifen) is used in patients with node-positive early breast cancer (Stage II) in both premenopausal and postmenopausal patients once treatment to control local disease (surgery, with or without radiation therapy) has been undertaken.

In Stage III (locally advanced) breast cancer, combination chemotherapy (with or without hormonal therapy) is used sequentially following surgery and radiation therapy for operable disease or following biopsy and radiation therapy for inoperable disease; commonly employed effective regimens include cyclophosphamide, methotrexate, and fluorouracil; cyclophosphamide, doxorubicin, and fluorouracil; and cyclophosphamide, methotrexate, fluorouracil, and prednisone. These and other regimens also have been used in the treatment of more advanced (Stage IV) and recurrent disease.

(j) Small cell cancer of the lung

Cyclophosphamide is used in combination chemotherapy regimens (e.g. cyclophosphamide, adriamycin, and vincristine [CAV]; cyclophosphamide, adriamycin, and etoposide [CAE]) for the treatment of extensive-stage small cell lung cancer.

(k) Sarcoma

Cyclophosphamide has been used in combination regimens (usually with dactinomycin and vincristine) and as an adjunct to surgery and radiation therapy in the treatment of rhabdomyosarcoma and Ewing sarcoma.

1.2.2 Dosage

Cyclophosphamide is administered orally or by intravenous injection or infusion. Less frequently, the drug has been administered intramuscularly and by intracavitary (e.g. intrapleural, intraperitoneal) injection and direct perfusion.

In patients with no haematological deficiencies receiving cyclophosphamide monotherapy, induction therapy in adults and children is usually initiated with an intravenous cyclophosphamide loading dose of 40–50 mg/kg administered in divided doses over 2–5 days. Other regimens for intravenous administration include 10–15 mg/kg every 7–10 days or 3–5 mg/kg twice weekly.

When cyclophosphamide is administered orally, the usual dose for induction or maintenance therapy is 1–5 mg/kg daily, depending on the tolerance of the patient.

A daily oral dose of 2–3 mg/kg for 60–90 days has been used in children with nephrotic syndrome, and in whom corticosteroids have been unsuccessful. In patients who are to undergo stem-cell transplantation, very high doses of cyclophosphamide such as 60 mg/kg daily for 2 days may be given as part of the conditioning regimen.

Various cyclophosphamide-containing combination chemotherapy regimens have been used in the treatment of breast cancer. One commonly employed regimen for the treatment of early breast cancer includes a cyclophosphamide dosage of 100 mg/m2 orally on Days 1 through 14 of each cycle combined with intravenous methotrexate at 40 mg/m2 on Days 1 and 8 of each cycle, and intravenous fluorouracil at 600 mg/m2 on Days 1 and 8 of each cycle. In patients older than 60 years of age, the initial intravenous methotrexate dosage is reduced to 30 mg/m2 and the initial intravenous fluorouracil dosage is reduced to 400 mg/m2. Dosage is also reduced if myelosuppression develops. Cycles are generally repeated monthly (i.e. allowing a 2-week rest period between cycles) for a total of 6–12 cycles (i.e. 6–12 months of therapy).

Cyclophosphamide is available as 25 and 50 mg tablets for oral administration, and as 200 mg, 500 mg, 1 g, or 2 g vials of powder for reconstitution for parenteral administration.

1.2.3 Trends in use

No information was available to the Working Group.

2. Cancer in Humans

The carcinogenicity of cyclophosphamide in humans was established initially on the basis of a large number of case reports, as well as several epidemiological studies (IARC 1981, 1987a). The interpretation of the epidemiological studies was limited by the small numbers of cases, the difficulty in separating the role of cyclophosphamide from other agents, or both factors.

The most substantial evidence available to previous Working Groups was a Danish study of 602 patients treated “mainly with cyclophosphamide” for non-Hodgkin lymphoma, in which nine cases of acute myeloid leukaemia were observed compared to 0.12 expected (Pedersen-Bjergaard et al., 1985), and a case–control study of leukaemia following ovarian cancer in the former German Democratic Republic where a strong dose–response relationship was observed (Haas et al., 1987). All other studies reported at most three cases of leukaemia or bladder cancer in people who had received cyclophosphamide as the only potentially carcinogenic agent (IARC, 1981; Kinlen, 1985; Greene et al., 1986).

Subsequently, further studies have been published that have provided more detailed information on the carcinogenicity of cyclophosphamide. This review is restricted to epidemiological studies that have used appropriate comparison groups to investigate the role of cyclophosphamide as the cause of specific types of cancer.

There have been several reported cohort studies in which patients treated with cyclophosphamide were followed up, and the occurrence of second cancers investigated. Valagussa et al. (1994) followed 2465 women who had received treatment with cyclophosphamide, methotrexate and fluorouracil, a combination in which only cyclophosphamide is considered to have carcinogenic potential in humans. There were three cases of acute myeloid leukaemia observed compared to 1.3 expected, and five cases of bladder cancer compared to 2.1 expected. Statistical significance was not reported but was calculated by the Working Group to be greater than 0.05 for both types of cancer. Smith et al. (2003) followed 8563 women who had received cyclophosphamide and doxorubicin as adjuvant therapy for breast cancer and observed 43 cases of acute myeloid leukaemia or myelodysplastic syndromes (AML/MDS). The incidence of AML/MDS overall was seven times higher than expected rates in the general population, and was increased 3-fold in regimens that contained double the cumulative dose of cyclophosphamide.

Several case–control studies have also been reported. For leukaemia, Kaldor et al. (1990) investigated 114 cases of a cohort of ovarian cancer patients. The relative risks were, respectively, 2.2 and 4.1 in two increasing dose categories of cyclophosphamide. Neither increase was reported as statistically significant. Travis et al. (1994) carried out a study involving 35 cases of leukaemia following non-hodgkin lymphoma, and found that prior treatment with cyclophosphamide was associated with a relative risk of 1.8 that was not statistically significant when comparison was made to treatment with radiotherapy alone. In an investigation by Nandakumar et al. (1991) of 97 cases of myeloid leukaemia as second primary cancers, patients receiving cyclophosphamide had a relative risk of 12.6 compared to those treated surgically, and was substantially higher when prednisone was co-administered with cyclophosphamide. Curtis et al. (1992) compared 90 women who developed acute myeloid leukaemia following breast cancer to controls, and found that the risk of leukaemia was 2.6 times greater in those who had received cyclophosphamide, compared to women who had been treated by surgery only.

There have also been two case–control studies of bladder cancer in relation to cyclophosphamide. Kaldor et al. (1995) investigated 63 cases of bladder cancer following ovarian cancer, and found that in comparison to surgery alone, the relative risk associated with chemotherapy containing cyclophosphamide as the only potential bladder-cancer-causing agent was 4.2 (P = 0.025) in the absence of radiotherapy, and 3.2 (P = 0.08) with radiotherapy. Travis et al. (1995) studied 31 cases of bladder cancer and 17 cases of kidney cancer as well as matched controls within a cohort of 2-year survivors of non-Hodgkin lymphoma. The relative risk associated with cyclophosphamide treatment was 4.5 (P < 0.05) for bladder cancer, and 1.3 for kidney cancer.

2.1. Synthesis

The studies summarized above provide a comprehensive epidemiological basis for identifying cyclosphosphamide as an independent cause of acute myeloid leukaemia and bladder cancer, that fully supports the conclusions drawn from earlier case reports, and more limited studies. Several studies have assessed the risk of all second primary cancers following cyclophosphamide treatment, and some have found rates of occurrence that appear to be elevated, but have not provided evidence for risk of other specific cancer types.

3. Cancer in Experimental Animals

Cyclophosphamide has been tested for carcinogenicity by oral administration to mice and rats, by subcutaneous injection to mice, by topical application to mice, by intravenous injection to rats, by intraperitoneal injection to mice and rats, and by perinatal exposure to mice.

Oral administration of cyclophosphamide resulted in skin tumours in transgenic mice (Yamamoto et al., 1996; Eastin et al., 2001), and in urinary bladder carcinoma, leukaemia, and nervous system tumours in rats (Schmähl & Habs, 1979; Habs & Schmähl, 1983). Subcutaneous injection of cyclophosphamide to mice caused a variety of neoplasms, including mammary gland carcinoma and leukaemia (Schmähl & Osswald, 1970; Walker & Bole, 1971, 1973; Walker & Anver, 1979, 1983; Petru et al., 1989).

Intravenous injection of cyclophosphamide to rats caused both benign and malignant neoplasms (Schmähl, 1967, 1974; Schmähl & Osswald, 1970).

Intraperitoneal administration of cyclophosphamide increased the incidences of lung adenoma and adenocarcinoma, bladder papilloma, and leukaemia in mice (Shimkin et al., 1966; Weisburger et al., 1975; Mahgoub et al., 1999), and mammary gland adenoma and carcinoma in rats (Weisburger et al., 1975).

Administration of cyclophosphamide to newborn mice caused lung and liver adenoma and carcinoma, and Harderian gland adenoma (Kelly et al., 1974; McClain et al., 2001).

See Table 3.1.

Table 3.1. Studies of cancer in experimental animals exposed to cyclophosphamide.

Table 3.1

Studies of cancer in experimental animals exposed to cyclophosphamide.

4. Other Relevant Data

4.1. Absorption, distribution, metabolism, and excretion

In most species, cyclophosphamide is rapidly absorbed, metabolized, and excreted. Its metabolic pathway has been studied in several species including mice, rats, hamsters, rabbit, dogs, sheep, and monkeys. Cyclophosphamide is not cytotoxic per se, because it requires metabolic activation before it can act as an alkylating agent. Activation takes place predominantly in the liver, although this may occur in other tissues (IARC, 1981).

Cyclophosphamide undergoes metabolism to several intermediates with alkylating activity. The principal metabolites identified are phosphoramide mustard, and acrolein. Phosphoramide mustard can undergo dephosphoramidation to yield nornitrogen mustard, which also has alkylating activity. Metabolites of cyclophosphamide can interact with DNA and proteins, resulting in the formation of adducts. The metabolism of cyclophosphamide and DNA adducts formation are summarized in Fig. 4.1.

Fig. 4.1

Fig. 4.1

Metabolic pathway of cyclophosphamide

A minor pathway results in dechloroethylation and the formation of 2-dechloroethylcyclophosphamide and another alkylating agent, chloroacetaldehyde (Balu et al., 2002).

The other compounds such as 4-ketocyclophosphamide and propionic acid derivative are relatively non-toxic, and are the major urinary metabolites of cyclophosphamide in several species (IARC, 1981).

4.2. Genetic and related effects

4.2.1 Interaction with DNA

Using 4-hydroperoxycyclophosphamide as an activated form of cyclophosphamide, Mirkes et al. (1992) identified by mass spectrometric analysis the formation of the monofunctional adduct N-(2-chloroethyl)-N-[2-(7-guaninyl)ethyl]amine (nor-G) and the bifunctional adduct N,N-bis[2-(7-guaninyl)ethyl]amine (G-nor-G) in rat embryos in in-vitro culture. The monofunctional adduct N-(2-hydroxyethyl)-N-[2-(7-guaninyl)ethyl]amine (nor-G-OH) was detected in bladder tissue of rats injected with [3H]cyclophosphamide (Benson et al., 1988). Using 32P-postlabelling analysis, a phosphotriester was shown to be formed: (1) when phosphoramide mustard was reacted with deoxyguanosine 5′-monophosphate, (2) when cyclophosphamide was incubated with calf thymus DNA in the presence of reconstituted cytochrome P450 (CYP) metabolizing system, and (3) in liver DNA from mice injected intraperitoneally with cyclophosphamide (Maccubbin et al., 1991).

Nornitrogen mustard reacts with guanosine and with guanine bases in DNA to form nor-G initially, but this is converted to a hydroxylated derivative (nor-G-OH), and to a cross-linked (between guanines) adduct (G-nor-G) (Hemminki, 1987). Both monofunctional adducts, but not the cross-linked adduct, were also detected when phosphoramide mustard was reacted with DNA (Cushnir et al., 1990). Acrolein reacts with DNA to form O6-(n-propanalyl)guanine, and the product of chloroacetaldehyde reaction with DNA is O6-(ethanalyl)guanine (Balu et al., 2002). Acrolein can produce exocyclic adducts in DNA, including 1,N2-hydroxypropanodeoxyguanosine and 1,N6-hydroxypropanodeoxyadenosine (Chung et al., 1984; Foiles et al., 1990; Smith et al., 1990). The former was detected in acrolein-treated human fibroblasts and in peripheral blood lymphocytes of a dog treated with cyclophosphamide (Wilson et al., 1991).

Nornitrogen mustard also reacts covalently with proteins, and a method for the detection of cysteine-34 residue adducts in human serum albumin has been described (Noort et al., 2002).

The single-cell gel comet assay is used to detect single-strand breaks and other alkali-labile lesions in DNA exposed to cyclophosphamide. In vitro studies have demonstrated the comet-forming activity of cyclophosphamide in human hepatoma (Hep G2) cells (Uhl et al., 2000; Yusuf et al., 2000), in primary cultures of rat and human urinary bladder cells (Robbiano et al., 2002), in primary cultures of human leukocytes in the presence of metabolic activation system S9 mix (Hartmann et al., 1995; Hartmann & Speit, 1995; Frenzilli et al., 2000), and in extended-term cultures of human T-lymphocytes, also in the presence of S9 (Andersson et al., 2003). Comet formation was also detected in vivo in the urinary bladder mucosa of rats given cyclophosphamide orally (Robbiano et al., 2002), and in peripheral blood cells of patients administered the drug (Hartmann et al., 1995).

4.2.2 Genotoxic effects in humans

There are few reports of DNA-adduct formation by cyclophosphamide in humans. Acrolein-derived DNA adducts, detected by immunochemical methods, were found in blood leukocytes of cancer patients receiving cyclophosphamide (McDiarmid et al., 1991). In another study, mono-adducts and interstrand cross-links derived from phosphoramide mustard were detected in a single patient administered 1 g/m2 cyclophosphamide (Souliotis et al., 2003). Increased DNA damage (comet formation) was also observed in the lymphocytes of patients administered cyclophosphamide (Hartmann et al., 1995).

Increased frequencies of several biomarkers of genotoxicity have been observed in the lymphocytes of patients treated with cyclophosphamide, relative to control subjects. These include mutations at the hypoxanthine-(guanine) phosphoribosyl transferase (HPRT) locus (Palmer et al., 1986, 1988; Tates et al., 1994; Sanderson et al., 2001), and sister chromatide exchange (Raposa & Várkonyi, 1987; McDiarmid et al., 1990; Sardaş et al., 1994; Mertens et al., 1995; Hartmann et al., 1995).

Other studies reported positive findings for elevated chromosomal aberrations frequencies (Sessink et al., 1994; Rubes et al., 1998; Burgaz et al., 2002), and micronuclei (Yager et al., 1988; Tates et al., 1994; Zúñiga et al., 1996; Burgaz et al., 1999; Rekhadevi et al., 2007) in medical personnel exposed to cyclophosphamide. Increases in frequencies of micronuclei were also detected in buccal cells in some studies (Cavallo et al., 2005; Rekhadevi et al., 2007), but not in another (Burgaz et al., 1999).

4.2.3 Genotoxic effects in experimental systems

(a) Mutagenic effects in vitro

The previous IARC Monograph (IARC, 1987b) states that cyclophosphamide induced chromosomal aberrations, sister chromatid exchange, and DNA damage in human cells in vitro. It also induced morphological transformation, chromosomal aberrations, sister chromatid exchange, mutation, and unscheduled DNA synthesis (UDS) in rodent cells in vitro. It further induced aneuploidy, mutation, recombination, gene conversion, and DNA damage in fungi. It was also reported to act as a mutagen and DNA-damaging agent in bacteria.

The mutagenicity of cyclophosphamide in Salmonella typhimurium was enhanced by increased induction of CYPs in S9 liver fractions by a combination of β-naphthoflavone and sodium phenobarbital (Paolini et al., 1991a). Comparison of S9 from liver and kidney of pregnant mice revealed that liver S9 was more effective in activating cyclophosphamide to mutagenic metabolites in S. typhimurium, and also in inducing sister chromatid exchange in human peripheral lymphocytes, and Chinese hamster ovary (CHO) cells (Winckler et al., 1987).

In Saccharomyces cerevisiae, higher rates of mitotic gene conversion and point mutation by cyclophosphamide were associated with induction of class 2B CYPs in co-cultured epithelial cell lines from fetal mouse liver (Paolini et al., 1991b). A recombinant plasmid containing a full-length cDNA encoding the rat cytochrome CYP2B1 introduced into S. cerevisiae also increased the mutation frequency induced by cyclophosphamide (Black et al., 1989).

CYP2B1 expressed in Chinese hamster V79-derived SD1 cell lines also potentiated cyclophosphamide mutagenesis (6-thioguanine resistance), whereas CYP1A1 expressed in V79-derived XEM2 cell lines did not (Doehmer et al., 1990, 1992).

Cyclophosphamide was weakly mutagenic (detected by induction of resistance to 6-thioguanine) in differentiated Reuber hepatoma cells H4IIEC3/G-, but markedly cytotoxic and clastogenic (micronucleus formation) (Roscher & Wiebel, 1988), and also mutagenic in a Chinese hamster epithelial liver cell line (6-thioguanine resistance) (Turchi et al., 1992), and in Chinese hamster lung (CHL) cells in the presence of S9, as measured at microsatellite loci (Kikuno et al., 1995).

Using 4-hydroperoxycyclophosphamide and phosphoradiamidic mustard, the role of different repair enzymes in defining sensitivity was investigated by Andersson et al. (1996) in CHO cells. Mutations in excision repair cross-complementing ERCC1 and ERCC4 genes caused hypersensitivity to the cyclophosphamide analogues.

Cyclophosphamide induced sister chromatid exchange in mouse primary bone-marrow and spleen cells (Soler-Niedziela et al., 1989), and micronuclei in mouse lymphoma in L5178Y tk+/- cells (Kirsch-Volders et al., 2003), and in parental V79 cells (Kalweit et al., 1999) in the presence of rat liver S9. Of several V79 cell lines engineered to express rat CYPs, increases in micronuclei (Ellard et al., 1991) and sister chromatid exchange (Kulka et al., 1993) were seen in the cells expressing CYP2B1. The rat hepatoma cells lines H4IIEC3/G- and 2sFou were also susceptible to micronuclei induction by cyclophosphamide (Tafazoli et al., 1995).

Human T-lymphocytes were more susceptible than B-lymphocytes to both chromosomal aberrations and sister chromatid exchange induction by cyclophosphamide in the presence of rat liver S9 (Miller 1991a, b). This difference between T- and B-lymphocytes was not found with mouse cells treated with 4-hydroxycyclophosphamide or phosphoramide mustard (Kwanyuen et al., 1990). In another study (Kugler et al., 1987), rat liver microsomal mix was more effective than rat liver S9 in activating cyclophosphamide to induce chromosomal aberrations. Human lymphocytes from women carrying mutations in the breast cancer susceptibility gene BRCA1 were more susceptible to micronuclei induction than cells from non-carriers (Trenz et al., 2003). Hep G2 human hepatoma cells were susceptible to sister chromatid exchange and micronuclei induction by cyclophosphamide (Natarajan & Darroudi, 1991) and, in analogous studies, the S9 microsomal fraction of these cells were shown to be capable in activating cyclophosphamide to induce sister chromatid exchange and micronuclei in CHO cells (Darroudi & Natarajan, 1993). Human dental pulp cells formed chromosomal aberrations when exposed to cyclophosphamide in the presence of rat liver S9 (Tsutsui et al., 2006).

In the presence of rat liver S9, cyclophosphamide induced morphological transformation of BALB/3T3 mouse embryonic fibroblast cells (McCarvill et al., 1990).

(b) Mutagenic effects in vivo

The previous IARC Monograph (IARC, 1987b) states that cyclophosphamide was found to bind to kidney, liver and lung DNA in mice. It also induced dominant lethality, chromosomal aberrations, micronuclei, sister chromatid exchange, mutations, and DNA damage in rodents in vivo. In Drosophila, it induced aneuploidy, heritable translocations, and somatic and sex-linked recessive lethal mutations. In patients administered cyclophosphamide, increased incidences of chromosomal aberrations and sister chromatid exchange in peripheral lymphocytes and bone marrow were observed.

In Drosophila melanogaster, cyclophosphamide tested positive for the somatic white-ivory mutation (Batiste-Alentorn et al., 1994), and produced chromosome breaks in spermatocytes (Zijlstra & Vogel, 1989).

Several studies have examined the mutagenic effects of cyclophosphamide in transgenic mice. In MutaMouse, mutation induction was observed in bone marrow (other tissues not studied) (Hoorn et al., 1993). In Big Blue mice, mutation frequencies were elevated in the liver, but not in the testis or spleen in one study (Hoyes et al., 1998), and in another study, in the lung and urinary bladder, but not in the kidney, bone-marrow or splenic T-cells (Gorelick et al., 1999). Another study compared the lacI locus in Big Blue mice with the Hprt locus in conventional B6C3F1 mice, and cyclophosphamide induced mutations in the endogenous gene in splenic lymphocytes, but not in the transgene (Walker et al., 1999). In rats, cyclophosphamide produced the ‘common deletion’ mutation in liver mitochondrial DNA, and folic acid supplementation was found to be protective against this damage (Branda et al., 2002).

In two related studies investigating oncogene and tumour-suppressor gene expression in mice, cyclophosphamide was found to induce expression of several genes, including c-Myc and Tp53, in the spleen and thymus, but not in other tissues (Ember et al., 1995; Ember & Kiss, 1997).

Many studies have investigated the cytogenicity of cyclophosphamide in newts, rodents, dogs, and non-human primates. Results are invariably positive for this compound, and are summarized in Table 4.1.

Table 4.1. Positive cytogenicity studies of cyclophosphamide in newts, rodents, dogs, and non-human primates.

Table 4.1

Positive cytogenicity studies of cyclophosphamide in newts, rodents, dogs, and non-human primates.

(c) Mutagenic effects in germ cells

Anderson et al. (1995) reviewed the activity of cyclophosphamide in germ cells, and in summary, the germ cell stages that are most sensitive to cyclophosphamide are the postmeiotic stages. Tests for germ-cell damage that examine effects in F1 progeny in which cyclophosphamide gave positive results include dominant lethality, heritable translocations, specific locus mutations, and malformations. Although cyclophosphamide is not an effective aneugen, it causes structural and numerical chromosomal damage in second meiotic metaphases and first cleavage metaphases, and in F1 embryos. It is also positive for inducing sister chromatid exchange in germ cells and causes abnormal sperm-head morphology. Most studies have been carried out in mice, but positive results have also been observed in rats and rabbits, e.g. induction of unscheduled DNA synthesis in the testes (reviewed in Anderson et al., 1995), and also in hamsters (Waters & Nolan, 1995).

More recent studies in mice have demonstrated the dominant lethal effects of cyclosphosphamide (Dobrzyńska et al., 1998) as well as intrachromosomal gene conversion and mutation events primarily in meiotic stage cells (Schimenti et al., 1997). In female rats, administration of cyclophosphamide at 16 days of gestation significantly increased nucleolar and synaptonemal complex fragmentation (Cusidó et al., 1995), and in male rats chronic exposure to cyclophosphamide disrupted meiotic events before pachynema during spermatogenesis (Barton et al., 2003).

(d) Modulation of mutagenicity by other agents

A large number of studies have investigated the effects of agents in modulating the genotoxicity of cyclophosphamide, and are summarized in Table 4.2.

Table 4.2. Studies of modulation of cyclophosphamide genotoxicity in vivo and in vitro.

Table 4.2

Studies of modulation of cyclophosphamide genotoxicity in vivo and in vitro.

4.3. Mechanisms of carcinogenesis

All of the available evidence indicates that cyclophosphamide exerts its carcinogenic activity via a genotoxic mechanism (McCarroll et al., 2008). The metabolite widely thought to be responsible for the antitumour activity of cyclophosphamide is the phosphoramide mustard (Povirk & Shuker, 1994). This metabolite is also generally considered to be the most genotoxic, but the contribution of acrolein, which is highly toxic, to the genotoxic activity of cyclophosphamide is less clear.

It is well established that the treatment of cancer patients with cyclophosphamide results in inflammation of the urinary bladder (haemorrhagic cystitis), which is not seen with other alkylating agents (Forni et al., 1964; Liedberg et al., 1970). In rats, cyclophosphamide treatment resulted in cystitis as well (Crocitto et al., 1996), and in mice, mutagenic activity has been detected in urine following cyclophosphamide treatment (Te et al., 1997). The ultimate alkylating metabolite of cyclophosphamide, phosphoramide mustard, is metabolized but was not shown to cause cytotoxicity and had minimal morphological effects on the mouse bladder, but an intermediate in the formation of the acrolein metabolite, diethylcyclophosphamide administered by intraperitoneal injection, caused severe cystitis in male rats, and less extensive inflammation in female rats (Cox, 1979). Acrolein administered to rats by intraperitoneal injections increased urothelial cell proliferation (Sakata et al., 1989). Acrolein is the only metabolite of cyclophosphamide that is known to be both reactive and cytotoxic (IARC, 1995). Collectively, these data indicate that acrolein is the likely causative agent in cyclophosphamide-induced cystitis. Cystitis is an established condition associated with the development of both squamous cell and urothelial bladder cancers (Michaud, 2007). However, intraperitoneal injections of acrolein by itself only induced bladder hyperplasia, not cancer (Cohen et al., 1992), and oral administration studies in mice and rats did not result in carcinogenic effects (IARC, 1995). Thus it is plausible that acrolein-induced cystitis plays a promoting role in cyclophosphamide bladder tumorigenesis that is initiated by other cyclophosphamide metabolites.

The protective effect of O6-alkylguanine-DNA alkyltransferase (AGT) against cyclophosphamide mutagenicity (Hprt mutations) (Cai et al., 1999), and cytotoxicity (Friedman et al., 1999) in CHO cells implies some involvement of acrolein-derived DNA damage. However, mice deficient in this protein (called O6-methylguanine-DNA methyl transferase [MGMT] in this study) were less susceptible to cyclophosphamide tumorigenesis, not more (Nagasubramanian et al., 2008). Studies of sister chromatid exchange induced in human lymphocytes by acrolein and phosphoramide mustard suggest that phosphoramide mustard is the more potent genotoxic agent (Wilmer et al., 1990). Furthermore, analysis of TP53 mutations in cyclophosphamide-associated human bladder cancers suggests that the mutations detected are characteristic of DNA damage caused by phosphoramide mustard, rather than by acrolein (Khan et al., 1998).

4.4. Synthesis

Cyclophosphamide, after its bioactivation to alkylating metabolites, is carcinogenic via a genotoxic mechanism.

5. Evaluation

There is sufficient evidence in humans for the carcinogenicity of cyclophosphamide. Cyclophosphamide causes cancer of the bladder, and acute myeloid leukaemia.

There is sufficient evidence in experimental animals for the carcinogenicity of cyclophosphamide.

Cyclophosphamide is carcinogenic to humans (Group 1).

References

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