Bis(2-chloroethoxy)methane

Repeated dose toxicity: dermal

Administrative data

Endpoint:

sub-chronic toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2002

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The study was performed by the NTP according to a method equivalent to OECD 411 and GLP. The publication and NTP report describe the findings in detail.

Data source

Reference

Reference 1

Reference Type:

publication

Title:

Cardiac damage in rodents after exposure to bis(2-chloroethoxy)methane

Author:

Dunnick, J. K., Lieuallen, W., Moyer, C., Orzech, D., Nyska, A.

Year:

2004

Bibliographic source:

Toxicol Pathol, Vol 32, Issue 3, pp. 309-17

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)

Principles of method if other than guideline:

- Ophthalmological examination was not performed.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Bis(2-chloroethoxy)methane

EC Number:

203-920-2

EC Name:

Bis(2-chloroethoxy)methane

Cas Number:

111-91-1

Molecular formula:

C5H10Cl2O2

IUPAC Name:

1-chloro-2-[(2-chloroethoxy)methoxy]ethane

Details on test material:

Chemical name: Bis(2-chloroethoxy)methane
Batch No.: lot B007269977
Supplier: Karl Industries, Aurora, Ohio,
Purity: 98.5% pure (National Toxicology Program, 2000).

Test animals

Species:

other: rats and mice

Strain:

other: F344 and B6C3F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Taconic Laboratories, Germantown, NY
- Age at study initiation: 5-6 weeks
- Weight at study initiation: Rats: males 93 +/- 3 g, females 89 +/- 3 g. Mice: males 22.8 +/- 0.2 g, females 18.6 +/- 0.2 g
- Fasting period before study: not applicable
- Housing: 1 per cage in polycarbonate cages
- Diet (e.g. ad libitum): Control and treated groups received irradiated NTP-2000 diet (Zeigler Brothers, Gardners, PA) ad libitum.
- Water (e.g. ad libitum): Tap water (City of Columbus municipal supply) via automatic watering system (Edstrom Industries, Waterford, WI), available ad libitum.
- Acclimation period: Rats: 11 (males) or 12 (females) days, Mice: 13 (females) or 14 (males) days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.6-23.9
- Humidity (%): 35-65
- Air changes (per hr): 10/hour
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
date of first dose: Rats: February 4 (males) or 5 (females), 2002 Mice: February 6 (females) or 7 (males), 2002
date of last dose: Rats: May 7 (males) or 8 (females), 2002 Mice: May 9 (females) or 10 (males), 2002

Administration / exposure

Type of coverage:

not specified

Vehicle:

ethanol

Details on exposure:

TEST SITE
- Area of exposure: back
- % coverage: no data
- Type of wrap if used: no data
- Time intervals for shavings or clipplings:weekly

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): The CEM solutions were applied to the shaved skin on the back of rats at 0.5 ml/kg body or to mice at 2.0 ml/kg to deliver doses of 0, 50, 100, 200, 400, or 600 mg/kg body weight.
- Concentration (if solution): Stock solutions, used within a month of preparation and stored in amber glass bottles at room temperature, were formulated at concentrations of 0, 100, 200, 400, 800, and 1,200 mg/ml in 95% ethanol for rats and at 0, 25, 50, 100, 200, or 300 mg/ml in 95% ethanol
for mice.
- Constant volume or concentration used: yes
- For solids, paste formed: not applicable

VEHICLE
- Justification for use and choice of vehicle (if other than water): solubility
- Amount(s) applied (volume or weight with unit): 95%
- Concentration (if solution): 95%
- Lot/batch no. (if required): no data
- Purity: no data

USE OF RESTRAINERS FOR PREVENTING INGESTION: no

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Periodic analyses of the dose formulations of bis(2-chloroethoxy)methane were conducted by the study laboratory using GC. During the 3-month studies, the dose formulations were analyzed three times; animal room samples of these dose formulations were also analyzed. Of the dose formulations analyzed, all 15 for rats and mice were within 10% of the target concentrations; all 30 animal room samples were within 10% of the target concentrations.

Duration of treatment / exposure:

Up to 14 weeks

Frequency of treatment:

Daily, excluding weekends and holidays.

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 50, 100, 200, 400, or 600 mg/kg body weight
Basis:

No. of animals per sex per dose:

10 mice and 10 rats. Ten additional rats/sex/dose level were included in the studies for clinical chemistry and hematology evaluations at weeks 1 and 3, and core animals were used for these evaluations at study termination.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: range finding study.
- Rationale for animal assignment (if not random): no data
- Rationale for selecting satellite groups: Ten additional rats/sex/dose level were included in the studies for clinical chemistry and hematology evaluations at weeks 1 and 3, and core animals were used for these evaluations at study termination.
- Post-exposure recovery period in satellite groups: not applicable
- Section schedule rationale (if not random): no data

Positive control:

Not applicable.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule for examinations: Observed twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule for examinations: clinical findings were recorded weekly and at the end of the studies.

DERMAL IRRITATION (if dermal study): No
- Time schedule for examinations:

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded initially, weekly, and at the end of the studies

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Ten additional rats/sex/dose level were included in the studies for clinical chemistry and hematology evaluations at weeks 1 and 3, and core animals were used for these evaluations at study termination.
- Anaesthetic used for blood collection: Yes (identity), Animals anesthetized with a carbon dioxide/oxygen mixture were bled from the retroorbital sinus.
- Animals fasted: No data
- How many animals: 20 sex/dose
- Parameters examined: hematocrit, erythrocyte count, mean corpuscular volume, hemoglobin concentration, packed cell volume, erythrocyte morphology, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, leukocyte count and differential reticulocyte count, and platelet count and morphology.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
- Animals fasted: No data
- How many animals: 20 rats/sex/dose
- Parameters examined: serum levels of sorbitol dehydrogenase, alanine aminotransferase, alkaline phosphatase, total bile acids, creatine kinase, urea nitrogen, creatinine, total protein and albumin.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

OTHER:
Reported in the publication: Immunohistochemistry for Cardiac Troponin T (cTnT)
Reported in the NTP report: At the end of the studies, sperm samples were collected from male animals in the 0, 100 (rats), 200, 400, and 600 (mice) mg/kg groups for sperm motility evaluations. The following parameters were evaluated: spermatid heads per testis and per gram testis, spermatid counts, and epididymal spermatozoal motility, count, and concentration. The left cauda, left epididymis, and left testis were weighed. Vaginal samples were collected for up to 12 consecutive days prior to the end of the studies from females administered 0, 50 (rats), 100, 200, or 400 (mice) mg/kg for vaginal cytology evaluations. The percentage of time spent in the various estrous cycle stages and estrous cycle length were evaluated.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
A complete gross necropsy and microscopic examination were performed on all animals. At necropsy, all organs and tissues were examined for grossly visible lesions. All major tissues were fixed in 10% neutral buffered formalin, processed, trimmed, embedded in paraffin, sectioned to a thickness of 4–6 µm, and stained with hematoxylin and eosin (H&E) for microscopic examination.

HISTOPATHOLOGY: Yes
All tissues from control, 400 and 600 mg/kg rats, 600 mg/kg mice and gross lesions were examined microscopically. This consisted of an examination of gross lesions, skin, mandibular and mesenteric lymph nodes, mammary glands, salivary glands, femur including diaphysis with marrow cavity, thymus, trachea, lungs and bronchi, heart and aorta, thyroid, parathyroids, esophagus, stomach, small intestine (duodenum, jejunum, ileum), large intestine (cecum, colon, rectum), liver, gall bladder (mice), pancreas, spleen, kidneys, adrenals, urinary bladder, seminal vesicles/prostate/testis/epididymis or ovaries/uterus, nasal cavity with nasal turbinantes, brain, pituitary, clitoral glands, preputial gland, Harderian glands, and eyes.
In addition, the hearts from all dosed groups of rats and mice were examined microscopically.
The liver from 400 mg/kg male and female mice, and the spleen and nose from 400 mg/kg female rats were examined microscopically.
The heart, liver, lungs, right kidney, thymus, and right testis were weighed at necropsy.

Other examinations:

Heart sections from 5 male F344/N rats from each of the 0, 200, 400, and 600 mg/kg dose groups were cut at 5 µm and stained with a primary antibody directed against cardiac troponin T (cTnT, mouse IgG, clone no. T1/61, Serotec, Ltd., Oxford, UK).

Statistics:

Organ and body weight data, which were approximately normally distributed, were analyzed using parametric multiple comparisons procedures of Dunnett (1955). Clinical chemistry and hematology data, which typically have skewed distributions, were analyzed using nonparametric multiple comparison methods of Shirley (1977) and Dunn (1964).

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Dermal irritation:

not examined

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
Treatment-related early deaths were seen in all male and female rats at 600 mg/kg, 2 of the female rats at 400 mg/kg, and 3 of the female mice at 600 mg/kg. All male mice survived the treatment periods. In general, clinical signs observed in these early death animals included prostration, ataxia, abnormal breathing, and lethargy, followed by rapid death within 18 hours. These early deaths in rats and mice were attributed to cardiac toxicity.

BODY WEIGHT AND WEIGHT GAIN
Final mean body weights of treated rats and mice were within 10% of control body weight.

FOOD CONSUMPTION
No data.

FOOD EFFICIENCY
No data.

WATER CONSUMPTION
No data.

OPHTHALMOSCOPIC EXAMINATION
No data.

HAEMATOLOGY
No changes in either rats or mice in hematology, or serum troponin T levels (data not shown) were considered attributable
to chemical administration.

CLINICAL CHEMISTRY
No changes in either rats or mice in clinical chemistry (data not shown) were considered attributable to chemical administration.

URINALYSIS
No data.

NEUROBEHAVIOUR
No data.

ORGAN WEIGHTS
In mice, increases occurred in the kidney weights at 400 and 600 mg/kg treated males and 600 mg/kg treated females and in the liver weights at 400 and 600 mg/kg-treated females. All other organ weights of treated and control rats and mice were similar.

GROSS PATHOLOGY
An enlarged heart was noted in a 100 mg/kg treated female rat. The stomach of a 600 mg/kg treated male rat developed an area of constriction. Otherwise, gross findings were interpreted to be spontaneous and unrelated to CEM administration.

HISTOPATHOLOGY: NON-NEOPLASTIC
Rats
Treatment-related toxic lesions were observed in the heart of male and female rats where dose-related, minimal to moderate increases in incidence and severity of myocytic cytoplasmic vacuolation and interstitial mononuclear cell infiltration were seen in both sexes treated with 600 and 400 mg/kg
and in females treated with 200 mg/kg/day. Increased incidence and severity of myocyte necrosis were seen in both sexes treated with 600 mg/kg and in females treated with 200 and 400 mg/kg.
Atrial thrombosis was seen only in 3 males treated with 600 mg/kg. Histopathological changes did not show any particular site specificity and were distributed in the left, middle and right sides of the heart. CEM-induced myocyte vacuolization consisted of a widespread accumulation of multiple vacuoles located within the myocyte sarcoplasm. The vacuoles were welldemarcated, clear, round, and variable sized. Vacuoles were primarily small, but occasionally coalesced into larger vacuoles. Interstitial vacuoles, representing fat, and possible edema, were interpreted as a background change, and not included as part of the treatment-related change. In some instances vacuoles may have been spontaneous, representative of minimal vacuolar change within a focus of spontaneous cardiomyopathy. Myocyte necrosis was characterized by small areas containing fragmented, angular, brightly eosinophilic myofibers with dark, shrunken nuclei. The same myocytes in some instances also contained variable numbers of clear vacuoles or vacuoles containing pale-staining fibrillar material. Mononuclear cell infiltration consisted of a widespread increase in nuclear density attributable to an increase in cells in the interstitum. These cells were not immediately identifiable, having indistinct borders, pale eosinophilic, sometimes fibrillar cytoplasm, and elongate nuclei. Occasionally the nuclei of these cells displayed a central condensed core of Anitschkow cells suggestive of a histiocytic origin. Typical background lesions of minimal spontaneous cardiomyopathy in the F344/N rat (Ruben et al., 2000) were also seen in all treatment and control groups. The incidence of this spontaneous cardiomyopathy was reduced in both sexes treated with 600 mg/kg/day and in females treated with 200 and 400 mg/kg. The “spontaneous” heart lesion was generally small (fewer than 30 mononuclear cells including macrophages), and focal to multifocal. The myocytes in these areas were often missing, or appeared to be hypereosinophilic, with hyalinized cytoplasm. The spontaneous heart lesion was distinguished from the CEM-induced myocytic changes because of the infrequent incidence of myocytic vacuolization. Fibrosis, often observed in spontaneous heart lesions in older F344/N rats, was not a major component of the CEM-induced heart lesion.
In the thymus, minimal to marked necrosis and atrophy were noted in both sexes treated with 600 mg/kg; sporadic cases of necrosis were noted in females treated with 400 mg/kg. In the spleen, minimal to marked lymphoid necrosis and atrophy were noted in both sexes treated with 600 mg/kg; lymphoid necrosis and atrophy were present in 2 females treated with 400 mg/kg. In the mesenteric lymph node, minimal to moderate lymphoid necrosis and atrophy were noted in both sexes treated with 600 mg/kg; sporadic cases of lymphoid necrosis were noted in female rats treated with 400 mg/kg. Minimal focal erosion of the mucosa in the glandular stomach was noted in a single female rat treated with 600 mg/kg.
The incidence of porphyrin pigmentation noted in the Harderian glands, was increased in both sexes treated with the 600 mg/kg/day. No damage in the glandular cells was associated with this pigment accumulation.
While the lesions in the heart of CEM treated rats were considered to be directly induced by the chemical, changes noted in treated rats in the thymus, lymphoid organs Harderian gland, and glandular stomach were considered to reflect an indirect stress effect.

Mice
Treatment-related heart lesions were seen in female, but not male mice where the heart lesions consisted of minimal to mild of myocytic cytoplasmic vacuolation in the 400 and 600 mg/kg groups. Myocyte vacuolization was identical to that seen in the rat described previously. Histopathological changes did not show any site specificity. Lymphoid necrosiswas seen in the mandibular and mesenteric lymph nodes, spleen and thymus, only in females treated with 600 mg/kg. This change was characterized by diffuse necrosis (pyknosis, karyrrhexis, and lysis of lymphocyte nuclei) of lymphocytes scattered throughout the tissue. This change was considered to be stress-related. A single case of lymphoid necrosis was noted in a female mouse treated with 200 mg/kg.

HISTOPATHOLOGY: NEOPLASTIC (if applicable)
No findings.

HISTORICAL CONTROL DATA (if applicable)
No data.

OTHER FINDINGS
Troponin Immunohistochemical Evaluation in Male Rats
The severity of cTnT loss between treated and control rats was indistinguishable between the 0 mg/kg and the 200 and 400 mg/kg dose levels. However, increased severity of cTnT loss was evident in the 600 mg/kg dose groups when compared to controls. The average percentage of cTnT loss was between 1–25% in the control and 200 mg/kg treatment groups, whereas the 400 mg/kg dose group exhibited a wider range of loss involving 1–50% of the myocardial tissue. In the 600 mg/kg group, troponin loss was observed in a higher percentage (50–99%) of the myocardial cells. No cTnT immunoreactivity was observed in areas of cytoplasmic vacuoles, although some of these appeared to be surrounded by irregular clumps of brown staining suggestive of dissociated troponin. This type of cTnT loss was typical in CEM-treated rat myocardium and served to distinguish cytoplasmic vacuoles. cTnT loss was also observed in areas of cardiomyopathy most commonly observed in the hearts of control and lower dose groups (200 and 400 mg/kg) of CEM-treated F344/N rats.
Individual or groups of myocardial cells with no vacuolization also demonstrated cTnT loss. Many, but not all, of these fibers demonstrated loss of myocardial striation. Myofibers exhibiting cTnT loss were separated from neighboring muscle cells by sharply defined demarcations that often coincided with intercalated discs. Some, but not all, of these myocardial cells contained marginated or pyknotic nuclei. No inflammatory cells were observed around or in these myofibers. These changes were commonly observed in the hearts of all control and treated groups. In this study, all male and female rats in the 600 mg/kg group either died early or were moribund sacrificed prior to the scheduled study termination. Significant autolysiswas not noted in any of the heart slides, and, therefore,was not thought to have confounded the immunohistochemical staining.

There were no significant differences in sperm parameters of male rats administered 100, 200, or 400 mg/kg and no significant differences in the estrous cyclicity of female rats administered 50, 100, or 200 mg/kg when compared to the vehicle controls. There were no significant differences in sperm parameters of male mice administered 200, 400, or 600 mg/kg and no significant differences in the estrous cyclicity of female mice administered 100, 200, or 400 mg/kg when compared to the vehicle controls.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day

Sex:

female

Basis for effect level:

other: Rats: heart damage

2

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day

Sex:

male

Basis for effect level:

other: Rats: heart damage

3

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day

Sex:

female

Basis for effect level:

other: Mice: heart damage

4

Dose descriptor:

NOAEL

Effect level:

600 mg/kg bw/day

Sex:

male

Basis for effect level:

other: Mice: heart damage

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Survival, body weight, histopathologic cardiac lesions, and treatment-related organ weight effects in Bis(2-chlorethoxy)methane mouse studies.

Dermal dose, mg/kg	0	50	100	200	400	600
Male mice
Survival	10/10	10/10	10/10	10/10	10/10	10/10
Final Body wts. % control	—	103	103	103	99	100
Kidney weights (g)	0.295 ±0.016b	0.301 ± 0.018	0.312 ±0.016	0.314 ±0.024	0.336 ±.023**	0.343 ±0.022**
Cardiomyopathy	1/10 (2.0)	0/10	0/10	0/10	0/10	0/10
Mineralisation	0/10	0/10	0/10	0/10	1/10 (1.0)	0/10
Female Mice
Survival	10/10	10/10	10/10	10/10	8/10	7/10 Week of death Week 6–1 deaths Week 8–1 deaths Week 11–1 deaths
Body wts. % control	—	107	104	106	105	—
Kidney weights (g)	0.185±0.007	0.188±0.008	0.183±0.012	0.187±0.009	0.194±0.013	0.209±0.009*
Liver weights (g)	1.307±0.082	1.440±0.111	0.1423±0.120	0.1421±0.136	1.476±0.104*	1.468±0.190**
Myocardial vacuolation
Cytoplasmic	0/10	0/10	0/10	0/10	4/10 (1.0)	5/10 (1.6)
Cardiomyopathy	0/10	0/10	0/10	0/10	0/10	1/10 (1.0)

 

Survival, body weight, and histopathologic cardiac lesions in Bis(2-chlorethoxy)methane rat studies.

Dermal dose, mg/kg	0	50	100	200	400	600
Male rats
Survival	10/10	10/10	10/10	10/10	10/10	0/10 Week of death Week 1–4 deaths Week 6–1 death Week 7–2 deaths Week 8–2 deaths Week 10–1 death
Final Body wts. % control	—	97	97	99	92	—
Heart lesions
Myocyte, vacuolation cytoplasmic	0/10	0/10	0/10	0/10	6/10 (1.3)a	10/10 (1.5)
Infiltration, mononuclear cell	0/10	0/10	0/10	0/10	7/10 (1.0)	10/10 (1.8)
Myocyte, necrosis	0/10	0/10	0/10	0/10	0/10	7/10 (3.7)
Fibrosis	0/10	0/10	0/10	0/10	0/10	1/10 (1.0)
Atrium, thrombosis	0/10	0/10	0/10	0/10	0/10	3/10 (1.4)
Cardiomyopathy (background F344 rat lesion)	10/10 (1.0)	8/10 (1.3)	10/10 (1.0)	10/10 (1.0)	2/10 (1.0)	1/10 (1.0)
Female rats
Survival	10/10	10/10	10/10	10/10	8/10	0/10 Week of death Week 11–2 deaths Week 4–2 deaths Week 5–5 deaths Week 6–3 deaths
Body wts. % control	—	100	98	99	101	—
Heart lesions
Myocyte, vacuolation cytoplasmic	0/10	0/10	0/10	2/10 (1.0)	5/10 (1.4)	8/10 (1.6)
Infiltration, mononuclear cell	0/10	0/10	0/10	7/10 (1.0)	6/10 (1.2)	10/10 (1.9)
Myocyte, necrosis	0/10	0/10	0/10	1/10 (1.0)	1/10 (1.0)	5/10 (1.4)
Cardiomyopathy	8/10 (1.0)	6/10 (1.0)	8/10 (1.0)	4/10 (1.0)	3/10 (1.0)	0/10

 

*p≤0.05; **p≤0.01.

aGrading of lesions: Minimal=1, mild=2, moderate=3, severe=4.

B±}SD.

 

Summary of the severity of TnT loss in Control and CEM-treated male F344/N rats.

	Dosage (mg/kg)
Heart	0	200	400	600
Myocardium, TnT loss	5/5 (1.0)	5/5 (1.0)	5/5 (1.6)	5/5 (3.6)

 

Applicant's summary and conclusion

Conclusions:

The target organ for toxicty for bis(2-chloroethoxy)methane (CEM) is the heart. CEM caused cardiac toxicity in male and female F344 rats and B6C3F1 mice. The lowest NOAEL is 100 mg/kg bw.

Executive summary:

The authors of this study report that an environmental agent, bis(2-chloroethoxy)methane (CEM), caused cardiac toxicity in male and female F344 rats and B6C3F1 mice exposed to the chemical by dermal administration at doses of 0, 50, 100, 200, 400 or 600 mg/kg 5 days a week for up to 14 weeks. Treatment-related deaths occurred in 10/10 male and 10/10 female rats at 600 mg/kg, in 2/10 female rats at 400 mg/kg, and in 3/10 female mice at 600 mg/kg. The heart lesions were more severe in rats than mice, and more severe in females than males. In rats, the no-observed-adverse-effect level (NOAEL) for the heart lesions was 200 mg/kg for males and 100 mg/kg for females; in mice, it was more than 600 mg/kg for males and 200 mg/kg for females. Multifocal, widespread vacuolization of the myocytes comprised the main morphological feature of the lesions, and only in rats was it accompanied by mononuclear cell infiltration, myocytic necrosis and atrial thrombosis. Hearts from male rats were immunohistochemically stained for troponin T (cTnT) protein. Loss of cytoplasmic cTnT correlated with histopathological damage only in the 600 mg/kg animals. CEM is metabolized to thiodiglycolic acid, a chemical that causes mitochondrial dysfunction. It is hypothesized that mitochondrial damage leads to the heart toxicity from bis(2-chloroethoxy)methane.