Phase 1 Dose Escalation of Single‐Agent Mechlorethamine in Dogs With Lymphoma

ABSTRACT

Mechlorethamine is commonly prescribed to dogs at 3 mg/m². The minimal toxicity observed indicates that higher doses of mechlorethamine are likely tolerable. The primary objective of this study was to determine the maximally tolerated dose (MTD) of mechlorethamine in dogs with lymphoma. The secondary objectives were to describe the toxicity associated with increased mechlorethamine dose and to evaluate the response in treatment‐naive dogs treated at the MTD. Dogs with histologically or cytologically confirmed intermediate to large cell lymphoma were enrolled using a 3 + 3 dose escalation model, starting at 3.5 mg/m² mechlorethamine IV, with planned dose increments of 10%–15% between cohorts. Adverse events were monitored per VCOG‐CTCAE guidelines. Dose‐limiting toxicity was defined as any grade 3 or 4 adverse event. Thirty dogs were enrolled across nine cohorts. Two dogs treated at 12.3 mg/m² developed asymptomatic grade 4 neutropenia 7 days after mechlorethamine administration, leading to a MTD of 10.7 mg/m². Low‐grade vomiting, diarrhoea, and inappetence were recorded amongst dogs at several dose levels and were managed with supportive medications. Six of 10 chemotherapy‐naïve dogs treated at the MTD, representing a separate cohort, showed partial responses (PR) 7 days post‐administration; however, PR was also observed at dosages ranging from 3.5 to 12.3 mg/m² in pre‐treated patients. A higher dose of mechlorethamine than previously reported can be safely administered as a single agent to dogs. Increasing the dose of mechlorethamine in combination therapies might offer greater therapeutic benefits.

1. Introduction

Chemotherapeutic drugs have a narrow therapeutic index. Accurate dosing is crucial to ensure optimal outcomes while simultaneously avoiding severe toxicity. The maximally tolerated doses (MTDs) of the most common frontline and rescue chemotherapy drugs used to treat canine lymphoma are well established. Mechlorethamine is a chemotherapeutic agent used in multi‐agent protocols to treat lymphoma in dogs and humans. The MTD of mechlorethamine in dogs is currently unknown.

The first clinical veterinary study examining the efficacy of mechlorethamine in dogs with lymphoma utilized a dosage of 3 mg/m² [1]. The authors empirically selected this dosage based on unpublished data from 17 dogs treated with mechlorethamine at a dosage of 6 mg/m². Of those receiving 6 mg/m², five (29%) died from sepsis. In the published mechlorethamine study, 13% of dogs developed moderate gastrointestinal (GI) toxicity, 4% of dogs developed febrile neutropenia, and 5% of dogs died as a result of treatment‐related toxicity [1]. However, in both the published and unpublished dogs, mechlorethamine was administered in combination with vincristine, prednisone, and procarbazine (MOPP), making it impossible to determine which toxicities were specifically attributable to mechlorethamine versus the other agents.

Additional clinical data on mechlorethamine‐associated toxicity are available. When vincristine was omitted from the MOPP protocol (MPP), 2% of dogs developed moderate GI toxicity [2]. Seventeen percent of dogs developed clinically insignificant thrombocytopenia, and no dogs died. When L‐asparaginase was included in the MOPP protocol, 12% of dogs developed moderate GI toxicity, 38% developed grade 3 or 4 neutropenia, 18% developed grade 3 or 4 thrombocytopenia, and one dog died as a result of toxicity [3]. When vinblastine was substituted for vincristine in the MOPP protocols (MVPP), there were no grade 4 or 5 toxicities and only one grade 3 toxicity (anorexia) [4]. The median starting dosage of mechlorethamine in this study ranged from 2.52 to 3.94 mg/m² [4]. In a retrospective evaluation of chemotherapy overdosages in dogs and cats, one dog received mechlorethamine at 8.6 mg/m² (2.9 times greater than the current conventional dose) [5]. This dog developed grade 3 neutropenia with no other toxicity reported. No studies are available that describe the clinical toxicity of mechlorethamine as a single agent in dogs with cancer.

Given the low rate of AEs events noted in multi‐agent protocol studies, dogs can likely safely tolerate a higher dose of mechlorethamine, both as a single agent and in combination with other chemotherapeutics. As dose correlates to efficacy for several chemotherapeutics, response rates and response duration could improve with an increasing dose of mechlorethamine [6, 7]. We hypothesized that the MTD of mechlorethamine in dogs is higher than the current conventional dosage of 3 mg/m².

The primary purpose of this study was to determine the MTD of mechlorethamine in dogs with lymphoma. The secondary objectives were to describe the toxicities associated with administering increased doses of mechlorethamine and the response rate in a second cohort of chemotherapy‐naïve dogs with lymphoma treated with single‐agent mechlorethamine at the MTD.

2. Methods

This phase I open–label dose escalation trial was performed in client‐owned dogs diagnosed with multicentric nodal lymphoma. This study was conducted at North Carolina State University Veterinary Hospital, with approval from the Animal Care and Use Committee and informed consent. Dogs weighing > 5 kg with a histological or cytological diagnosis of intermediate to large cell multicentric lymphoma, a neutrophil count of > 2500 cells/μL, a platelet count of > 100,000/μL, and serum bilirubin < 0.2 mg/dL on the day of treatment were eligible for treatment. Prior chemotherapy (other than mechlorethamine) was permitted, with a 2‐week washout period. Dogs with lymphoma of the skin, GI tract, or central nervous system were excluded. Dogs were excluded from the study if they exhibited any adverse GI signs before treatment.

Dogs were clinically staged (III–V) and substaged (a = without systemic signs, b = with systemic signs, including hypercalcemia, fever, and/or uveitis) at diagnosis using the World Health Organization criteria for canine lymphoma [8]. All owners were offered an initial staging workup, including a physical examination, complete blood count (CBC) with manual differential, serum biochemical profile, urinalysis, thoracic radiographs, abdominal ultrasonography, and bone marrow aspiration cytology. We also recorded signalment, body weight, phenotype when available, and previous treatment, including corticosteroids if prescribed within 1 month of starting therapy with mechlorethamine.

On the day of treatment with mechlorethamine (mechlorethamine hydrochloride 5 mg, The Compounding Pharmacy, Hickory NC), all dogs underwent a baseline physical examination, calliper‐based lymph node measurement, CBC with differential, chemistry panel, and urinalysis. Dogs were administered a dose of mechlorethamine (intravenously (IV) over 3 to 5 min as a slow bolus, within 45 min of reconstitution of the powdered drug). Each dog received 0.5 mg/kg IV of ondansetron immediately following mechlorethamine administration, as was standard practice for all dogs receiving injectable chemotherapy at the time of trial enrollment. Dog owners were provided standard anti‐emetic (maropitant 2 mg/kg PO q24h) and anti‐diarrheal (metronidazole 10 mg/kg PO q12h) medications to use as needed. Marbofloxacin (3–5 mg/kg PO q24h) was administered prophylactically starting 4 days after treatment for a total of 10 days. On day seven, dogs returned to the North Carolina State Veterinary Hospital for a physical examination, calliper‐based lymph node measurement, and CBC with differential. Toxicities were graded per VCOG‐CTCAE guidelines [9].

Dogs were enrolled using a standard Phase I dose‐escalation 3 + 3 model. A dose‐limiting toxicity (DLT) was defined as any grade 3 or above AE during the treatment period. The MTD was defined as the highest dose that resulted in a DLT in no more than one of six dogs. The first cohort received mechlorethamine at 3.5 mg/m². This dosage was chosen because it was previously well tolerated in other dogs at the investigators' institution. Doses were increased based on cohorts of three dogs. If no dogs developed DLT at day seven, the dose of mechlorethamine was increased by10%. If one dog developed DLT, three additional dogs were enrolled at the same dose. If two or more dogs developed severe toxicity, enrollment in the cohort and dose escalation were discontinued. The dose escalation resumed if no additional dogs experienced severe toxicity at that dosage.

After determining the maximum tolerated dose (MTD), 10 treatment‐naïve dogs (including not having previously received corticosteroids) with histologically or cytologically confirmed intermediate‐ to large‐cell lymphoma were enrolled in a second cohort. Each dog received a single dose of mechlorethamine at the established MTD to further assess toxicity and treatment response. Responses were evaluated 7 days post‐administration using the VCOG‐revised response evaluation criteria for peripheral nodal lymphoma in dogs [10]. After response evaluation to mechlorethamine administration, all dogs were permitted to receive additional chemotherapy protocols as part of their continued lymphoma treatment based on owner preference.

3. Results

Thirty dogs were enrolled in the dose‐escalation cohort of this trial between January 2023 and May 2024. Breeds included mixed breed (n = 12), Beagle (n = 5), German Shepherd (n = 3), Golden Retriever (n = 3), Labrador Retriever (n = 2), Australian Shepherd (n = 2), and one each of the following: Basset Hound, Rottweiler, and West Highland White Terrier. The median weight was 27.5 kg (range 9.3–47 kg), and the median age was 6 years (range 2–13 years). Nineteen dogs were male castrated, and 11 were female spayed. Twenty‐eight dogs were diagnosed with cytology, and two were diagnosed via histopathology. Immunophenotype was available for 22 dogs (21 B‐cell, 1 T‐cell). Nine dogs were at least stage 3, four dogs were suspected to be stage 4 based on imaging findings, and 17 dogs were stage 5 (all due to circulating neoplastic lymphocytes observed on CBC). Not all owners elected for complete staging. Based on the entrance criteria, all dogs were classified as substage a (showing no clinical signs). Nineteen were chemotherapy‐naïve, and 11 had received previous chemotherapy. Of the dogs that had undergone prior chemotherapy, seven were considered resistant to cyclophosphamide, doxorubicin, vincristine, and prednisone, +/− L‐asparaginase (L/CHOP). Seven dogs had received corticosteroids (prednisone) at the time of mechlorethamine administration, while 23 had not.

Three dogs were enrolled in the initial cohort and received a single dosage of mechlorethamine at 3.5 mg/m². This dose was well tolerated, with minimal GI and hematologic toxicities observed. Dose escalation proceeded as planned. No grade 3 or higher AEs were observed in any of the next 10 dogs enrolled in five cohorts up to a dosage of 5.11 mg/m². At that time, the PI of this study (JI) became aware of a contemporaneously enrolling clinical trial evaluating escalating doses of mechlorethamine, which suggested that the MTD was likely higher than initially anticipated. Given this information, to optimise available funding and minimise the number of dogs required, the protocol was amended to revise the dose escalation strategy. The mechlorethamine dosage was immediately increased to 8.5 mg/m², and dose escalations for subsequent cohorts were planned in 15% increments while maintaining the 3 + 3 design. One dog that received mechlorethamine at 9.3 mg/m² developed asymptomatic grade 3 neutropenia 7 days post‐administration (692 cells/μL), necessitating expansion of this cohort to six dogs, with no further neutropenia noted. Two dogs developed asymptomatic grade 4 neutropenia 7 days post‐treatment at 12.3 mg/m² (30 and 92 cells/μL, respectively); therefore, the MTD of mechlorethamine was determined to be 10.7 mg/m². (See Table 1).

TABLE 1.

Haematological toxicity following a single dose of mechlorethamine in dogs.

Mechlorethamine dose mg/m2	# of dogs	Neutropenia grade					Thrombocytopenia grade
		0	1	2	3	4	0	1	2	3	4
3.5	3	3	0	0	0	0	2	1	0	0	0
3.85	3	3	0	0	0	0	1	2	0	0	0
4.23	3	3	0	0	0	0	2	1	0	0	0
4.65	3	3	0	0	0	0	0	2	1	0	0
5.11	1	1	0	0	0	0	0	1	0	0	0
8.1	3	3	0	0	0	0	1	2	0	0	0
9.3	6	5	0	0	1	0	2	4	0	0	0
10.7	3	3	0	0	0	0	0	2	1	0	0
12.3	5	3	0	0	0	2	1	3	1	0	0

Note: No clinically relevant adverse events were observed in 13 dogs at doses up to 5.11 mg/m². Following external correspondence regarding a similar study conducted at another institution, the dose was escalated to 8.1 mg/m², with subsequent cohorts receiving incremental dose increases of 15%. Asymptomatic grade 4 neutropenia was observed in 2 dogs at 12.3 mg/m². The MTD of mechlorethamine was therefore determined to be 10.7 mg/m².

Of the 30 dogs treated over nine cohorts, GI toxicity occurred in 19 dogs (63%), with most experiencing more than one sign. Vomiting occurred in 12 dogs (10 grade 1, 2 grade 2). Diarrhoea occurred in 10 dogs (5 grade 1, 5 grade 2). Fourteen dogs were hyporexic (all grade 1). Three dogs were anorectic (2 grade 1, 1 grade 2). Grade 1 lethargy was observed in 14 (47%) of dogs. (See Table 2).

TABLE 2.

Gastrointestinal toxicity following a single dose of mechlorethamine in dogs.

Mechlorethamine dose mg/m2	# of dogs	Vomiting grade					Diarrhoea grade					Anorexia grade
		0	1	2	3	4	0	1	2	3	4	0	1	2	3	4
3.5	3	3	0	0	0	0	2	0	1	0	0	3	0	0	0	0
3.85	3	1	1	1	0	0	1	1	1	0	0	3	0	0	0	0
4.23	3	3	0	0	0	0	2	1	0	0	0	3	0	0	0	0
4.65	3	2	1	0	0	0	3	0	0	0	0	3	0	0	0	0
5.11	1	1	0	0	0	0	1	0	0	0	0	1	0	0	0	0
8.1	3	1	2	0	0	0	1	2	0	0	0	3	0	0	0	0
9.3	6	3	2	1	0	0	5	0	1	0	0	5	1	0	0	0
10.7	3	2	1	0	0	0	2	0	1	0	0	2	1	0	0	0
12.3	5	2	3	0	0	0	3	1	1	0	0	4	0	1	0	0

Note: No clinically relevant adverse events were observed in 13 dogs at doses up to 5.11 mg/m². Following external correspondence regarding a similar study conducted at another institution, the dose was escalated to 8.1 mg/m², with subsequent cohorts receiving incremental dose increases of 15%. GI toxicity was frequent, low‐grade, and managed with supportive care.

Although not a primary endpoint of the dose escalation cohort, 10 dogs (33%) achieved a response (PR) to mechlorethamine at doses ranging from 3.5 to 12.5 mg/m². Seventeen dogs (57%) had stable disease (SD), and three (10%) had progressive disease (PD). None of the seven dogs identified as resistant to L/CHOP chemotherapy responded to single‐agent mechlorethamine. Of the 10 dogs with PR, nine were naïve to chemotherapy, and two received previous corticosteroids. Of the 17 dogs with SD, eight were naïve to chemotherapy, and five received previous corticosteroids. Of the three dogs with PD, two were naïve to chemotherapy, and none received prior corticosteroids (see Table 3).

TABLE 3.

Response to mechlorethamine treatment in dogs diagnosed with lymphoma.

Mechlorethamine dose mg/m2	# of dogs	Complete response	Partial response	Stable disease	Progressive disease
3.5	3	0	1	2	0
3.85	3	0	1	2	0
4.23	3	0	1	2	0
4.65	3	0	0	1	2
5.11a	1	0	0	1	0
8.1	3	0	3	0	0
9.3	6	0	2	3	1
10.7b	13	0	7	6	0
12.3	5	0	1	4	0
Total	40	0 (0%)	16 (40%)	21 (52.5%)	3 (7.5%)

Note: Overall, 16/40 (40%) dogs treated with single‐agent mechlorethamine had a PR. This consisted of 10/30 (33%) chemotherapy‐pre‐treated dogs and 6/10 (60%) chemotherapy‐naïve dogs. All chemotherapy naïve dogs were treated at the MTD. Response was assessed 7 days post‐treatment.

^a No clinically relevant adverse events were observed in 13 dogs at doses up to 5.11 mg/m². Following external correspondence regarding a similar study conducted at another institution, the dose was escalated to 8.1 mg/m², with subsequent cohorts receiving incremental dose increases of 15%.

^b Indicates MTD of mechlorethamine.

After determining the MTD, an additional 10 dogs with multicentric intermediate to large cell lymphoma who had not received any prior chemotherapy or corticosteroids were enrolled in a second cohort treated with mechlorethamine at a dosage of 10.7 mg/m² between May 2024 and May 2025. Breeds included mixed breed (n = 5), German shepherd (n = 2), and one each of the following: Shiba Inu, Boxer, and Rottweiler. The median weight was 25.3 kg (range 9.9–58.5 kg), and the median age was 8.5 years (range 3–14 years). Five dogs were male (3 intact), and 5 were female spayed. Eight dogs were diagnosed with cytology, and two were diagnosed via histopathology. Immunophenotype was available for 6 dogs (5 B‐cell, 1 T‐cell). One dog was at least stage 2, five dogs were at least stage 3, two dogs were suspected to be stage 4 based on imaging findings, and two dogs were stage 5 (all due to circulating neoplastic lymphocytes observed on CBC). Not all owners elected for complete staging. Based on the entrance criteria, all dogs were classified as substage a (showing no clinical signs). Six of these dogs (60%) showed a PR, while four (40%) had stable disease (SD). (See Table 3) The duration of the response beyond 1 week could not be measured because dogs continued receiving additional chemotherapy. In this group, eight dogs experienced haematological toxicity, including neutropenia (n = 4; 3 grade 1, 1 grade 2) and thrombocytopenia (n = 7; 4 grade 1, 3 grade 2). Six dogs experienced GI toxicity (all grade 1) with some experiencing more than one sign: vomiting occurred in four dogs, diarrhoea occurred in two dogs, hyporexia occurred in four dogs, anorexia occurred in two dogs, and lethargy occurred in four dogs.

Of all 13 dogs treated at the MTD, four developed neutropenia (3 grade 1, 1 grade 2), 10 developed thrombocytopenia (6 grade 1, 4 grade 2). Five developed grade 1 vomiting, three developed diarrhoea (1 grade 1, 2 grade 2), six developed grade 1 hyporexia, three developed grade 1 anorexia, and eight developed grade 1 lethargy.

4. Discussion

The DLT of mechlorethamine in dogs is neutropenia, which corresponds with data from humans where the DLT of mechlorethamine is myelotoxicity, manifested primarily as neutropenia and thrombocytopenia [11]. In people, acute nausea and vomiting are also common following treatment with mechlorethamine and can be severe for some patients [11]. Similarly, 23 of 24 research dogs receiving mechlorethamine between 0.5 and 10 mg/kg IV vomited within 2.5 h of administration [12]. In our study, vomiting was common and typically occurred 24–48 h after treatment, mirroring the pattern seen in humans and dogs receiving higher doses of this drug. Overall, in our study population, GI toxicity was dose‐dependent: three of 13 dogs (23%) treated with mechlorethamine at dosages up to 5.11 mg/m² experienced vomiting, compared to nine of 17 dogs (53%) receiving higher doses. However, clinical signs were mild in all cases and resolved with supportive care. These findings suggest that even at higher doses, mechlorethamine can be administered with manageable side effects, thereby preserving quality of life, a crucial consideration in veterinary oncology.

The MTD of mechlorethamine identified in this study is higher than the conventional dose; however, this outcome is not unexpected, given the results from older literature. In the earliest study, published in 1965, four dogs received mechlorethamine at 0.4 mg/kg IV. Mean total leukocyte counts declined from 6100/μL to a nadir of 3900/μL, and platelets decreased from 170,000/μL to a nadir of 55 000/μL [13]. No other AEs were reported, and no deaths occurred. Based on an estimated body weight of 30 kg, a dose of 0.4 mg/kg corresponds to 12.3 mg/m², which is identical to the highest dosage administered in our study. Four dogs received a dose of 1.0 mg/kg (equivalent to 30.8 mg/m² for a 30 kg dog); three died by day four post‐administration due to GI toxicity. Nadir leukocyte and platelet counts could not be determined. Dosages of mechlorethamine up to 0.75 mg/kg (equivalent to approximately 12.7 to 17.5 mg/m² based on the study population's reported weight range) were well‐tolerated in Beagles [14]. This elevated tolerance might be attributable to the use of young, healthy animals rather than the tumour‐bearing dogs evaluated in our study. In the same study, dosages of 1.0, 1.25, and 1.75 mg/kg were associated with the development of enteritis, leukopenia, and mortality. It is not reported whether any form of supportive care was administered to these subjects. An additional study published in 1989 evaluated nine research dogs, each weighing between 9 and 12 kg, that were administered 1.5 mg/kg of mechlorethamine [15]. All developed granulocytopenia within 4 days. No additional toxicity data were reported.

Combination chemotherapy protocols enhance cancer treatment by targeting multiple pathways, thereby improving effectiveness, reducing resistance, and inhibiting tumour growth. A key principle in designing these protocols is to include agents that are effective as single therapies [16, 17]. The MOPP protocol, developed in the 1960s by Frei, DeVita, and colleagues, was the first combination chemotherapy regimen using drugs with nonoverlapping toxicities. It marked a turning point in physician‐based cancer treatment, achieving durable remissions and cures in advanced‐stage Hodgkin lymphoma [18]. Although the MOPP protocol is well‐established for the treatment of human lymphomas and leukaemias, its initial use in dogs lacked supportive evidence demonstrating the efficacy of two of its components, mechlorethamine and procarbazine, as single agents [1]. In our study, 40% (16/40) of dogs exhibited a PR to single‐agent mechlorethamine, including 60% (6/10) of dogs that were naïve to both chemotherapy and corticosteroids. Overall, these findings demonstrate that mechlorethamine has meaningful single‐agent antitumor activity in dogs with lymphoma, thereby providing a scientific rationale for its continued inclusion in multi‐agent chemotherapy protocols, such as MOPP.

The conventional paradigm in oncologic pharmacology theorizes that cytotoxic agents exhibit steep dose–response relationships, whereby small variations in administered dose can lead to substantial differences in therapeutic efficacy [19, 20]. This has been well‐documented in human oncology; for instance, in women with early‐stage HER2‐negative breast cancer, receipt of less than 85% of the intended cumulative chemotherapy dose was associated with significantly worse survival outcomes compared to those receiving ≥ 85% [21]. In our study, PRs to mechlorethamine were observed across a wide dosage range, from 3.5 to 12.3 mg/m², suggesting that clinically meaningful antitumor activity can occur even at doses lower than the MTD. While a positive correlation between dose intensity and response has been demonstrated in veterinary oncology for specific agents, such as vinblastine in canine mast cell tumors, other studies have failed to identify consistent improvements in response rates or outcomes with dose escalation [7, 22]. These findings reinforce the concept that a range of biological factors might influence interindividual variability in drug response. Tumor‐intrinsic features, such as proliferation rate and resistance mechanisms, can alter sensitivity to treatment [23]. At the same time, host‐specific variables, including genetic polymorphisms that affect drug metabolism, age‐related changes in pharmacokinetics, and differences in drug clearance, might also play a role [23]. Understanding this variability is essential for optimizing treatment regimens and advancing personalized approaches to chemotherapy in veterinary medicine.

The numerically higher response rate observed in treatment‐naïve dogs compared to those previously treated with chemotherapy or steroids (60% vs. 33%) suggests that prior exposure to cytotoxic agents might reduce tumour cell sensitivity to mechlorethamine. Notably, none of the dogs classified as resistant to L‐CHOP chemotherapy responded to single‐agent mechlorethamine, in contrast to earlier reports demonstrating response rates of up to 65% in L‐CHOP–resistant populations, including complete responses in 31% of cases [1]. This discrepancy is particularly relevant given the common use of mechlorethamine‐containing combination protocols as rescue therapies. This might also suggest potential synergistic effects when mechlorethamine is incorporated into multi‐agent protocols, with its most significant benefit possibly occurring in combination therapy rather than as a single agent. However, these findings must be interpreted with caution due to the small sample size, heterogeneity in mechlorethamine dosing, and variability in prior treatment regimens in our patient population. The observed response rates might not be generalizable to a broader population and could be influenced by sampling bias or random variation.

With the now‐established higher maximum tolerated dose (MTD) of mechlorethamine as a single agent, future studies will focus on dose escalation in combination regimens, including MOPP. Like our work, these studies should systematically assess both treatment‐related toxicity and therapeutic efficacy. Although higher dosing might not necessarily produce additional clinical benefits, there is still potential for better treatment outcomes without a corresponding rise in AEs. However, the increased cost of administering higher doses of mechlorethamine might limit feasibility and accessibility for some owners. Additionally, while all cytotoxic drugs carry risk to those handling them, in pre‐clinical models, mechlorethamine is highly corrosive to the skin and eyes [24]. It causes significant irritation to the mucous membranes of the respiratory tract. Furthermore, mechlorethamine has been identified as carcinogenic, mutagenic, and teratogenic. Given its toxicological profile, strict safety precautions, including the use of appropriate personal protective equipment, are essential during the preparation of mechlorethamine for parenteral administration.

This study has several limitations. Dogs received only a single dose of mechlorethamine, which precludes assessment of response rates to repeated or sequential treatments. Additionally, the duration of response beyond 7 days could not be evaluated in dogs that achieved PR. Follow‐up hematologic evaluations were performed at a single time point post‐treatment; therefore, nadirs occurring outside this window might have been missed. In certain cases of post‐treatment thrombocytopenia, platelet clumping was observed on CBCs, which limited the accurate assessment of platelet numbers. The overall incidence and severity of thrombocytopenia remained low, and no dogs displayed clinical signs attributable to thrombocytopenia. The 3 + 3 dose escalation design employed in this study, while widely used for its simplicity and safety, has limited statistical power and might underestimate the true MTD [25]. Compared with model‐based or adaptive designs, it provides less precise estimation of dose‐toxicity relationships and might not efficiently identify the optimal biologic dose. Lastly, all dogs received prophylactic oral marbofloxacin due to concerns regarding the unknown risks of mechlorethamine dose escalation. While prophylactic antimicrobials in human oncology remain a topic of controversy, they are considered a cost‐effective strategy to reduce the risk of febrile neutropenia in high‐risk populations [26, 27]. In contrast, their role in veterinary oncology is less well defined. Previous work has shown that prophylactic administration of trimethoprim‐sulfadiazine can significantly reduce non‐hematologic toxicity, hospitalisation, and overall morbidity in dogs with osteosarcoma or lymphoma treated with doxorubicin, with or without L‐asparaginase [28]. Additionally, prophylactic antimicrobials have been used successfully in other veterinary phase I clinical trials when severe neutropenia was anticipated, supporting the use of this strategy to mitigate the risk of opportunistic infections during periods of myelosuppression [29, 30]. Prophylactic antimicrobials did not reduce the risk of febrile neutropenia in dogs receiving CCNU (1‐[2‐chloroethyl]‐3‐cyclohexyl‐1‐nitrosurea) [31]. The benefit of prophylactic antimicrobials in mitigating morbidity from mechlorethamine treatment remains uncertain. Given the absence of significant hematologic toxicity at the identified MTD and considering the importance of antimicrobial stewardship, the authors do not recommend routine prophylactic antimicrobial use in dogs treated with mechlorethamine at the MTD. The use of compounded mechlorethamine may be associated with limitations in both stability and potency. After reconstitution, the drug undergoes rapid degradation, retaining 96.7% of its activity at 45 min and 93.3% at 60 min post‐reconstitution [32]. To mitigate this change in stability, all treatments in this study were administered within 45 min of reconstitution by our pharmacy. Additionally, while compounded chemotherapy drugs are not always equivalent in potency with respect to the active pharmaceutical ingredient [33], data from the pharmacy used to compound mechlorethamine in our study showed that the purity of the parent compound was compliant with a range of 96.5%–102.5% (not shown).

In conclusion, mechlorethamine can be safely given to dogs at a dose of 10.7 mg/m² IV as a single‐agent treatment. GI toxicity was common but mild and resolved with minimal supportive care. No dogs were hospitalized, developed febrile neutropenia, or died as a result of treatment. Further studies are needed to determine whether increasing the mechlorethamine dose in combination treatments is safe and feasible, and if such approaches lead to better clinical outcomes than current standards of care.

Ethics Statement

The authors have nothing to report.

Conflicts of Interest

The authors declare no conflicts of interest.

Chadsey L. E., Hess P. R., and Intile J. L., “Phase 1 Dose Escalation of Single‐Agent Mechlorethamine in Dogs With Lymphoma,” Veterinary and Comparative Oncology 24, no. 1 (2026): 51–57, 10.1111/vco.70025.

Data Availability Statement

The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.