Dichloroacetic acid

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1991

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Although some details are missing, the study is consdired to be reliable, relevant and adequate.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference 1

Reference Type:

publication

Title:

Unnamed

Year:

1991

Reference 2

Reference Type:

secondary source

Title:

Unnamed

Year:

2003

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 409 (Repeated Dose 90-Day Oral Toxicity Study in Non-Rodents)

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Dichloroacetic acid

EC Number:

201-207-0

EC Name:

Dichloroacetic acid

Cas Number:

79-43-6

Molecular formula:

C2H2Cl2O2

IUPAC Name:

2,2-dichloroacetic acid

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): Dichloroacetic acid
- Other: purchased from Sigma Chemical Company (St. Louis, MO) as reagent grade.

Test animals

Species:

dog

Strain:

Beagle

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Laboratory Research Enterprises of Kalamazoo, Michigan
- Age at study initiation: 4-month old
- Weight at study initiation: male beagle dogs (weight range 8.6- 13.6 kg); female beagle dogs (weight range 6.1-9.4 kg)
- Housing: individually in stainless-steel cages
- Diet (e.g. ad libitum): Purina Hi-pro dog chow ad libitum
- Water (e.g. ad libitum): distilled drinking water ad libitum
- Acclimation period: 1 month

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 25 ± 2°C
- Humidity (%): 40-60%
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: capsule

Vehicle:

other: NaOH

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Due to its low pH and corrosive properties it was neutralized with Na OH to a final pH of 7.4. The stock solution was pipetted into gelatin capsules for dosing.
Oral dosing was performed daily, 7 days per week, between 9:00 and 10:00AM, for a total of 90 days. The DCA was prepared each day just prior to dosing and was administered at least 1 hr before the dogs were fed. Following dosing the dogs were observed for 10 min to ensure that the capsule had been swallowed. Doses of DCA throughout each week were determined on the basis of individual body weights which were measured the preceding Friday.

Duration of treatment / exposure:

90 d

Frequency of treatment:

daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 12.5, 39.5, 72 mg/kg bw /d
Basis:
actual ingested

No. of animals per sex per dose:

5

Control animals:

yes

Details on study design:

The study design incorporated three treatment groups: 12.5, 39.5, and 72 mg/kg. DCA was administered orally in gelatin capsules daily 7 days a week. The control group received gelatin capsules containing distilled water only.
Five male and five female dogs were assigned to each experimental group.

Examinations

Statistics:

Statistical analysis of final body and organ weights and organ weights per l00 g body wt was performed as a one-factor (dose) analysis of variance (ANOVA) with Tukey's multiple comparison procedure; this analysis tested for an overall dose-related effect. The assumption of the homogeneity of variance necessary for the validity of ANOVA procedures was upheld by Levene's test. ANOVA procedures with contrast comparisons were used in the pairwise analyses. A linear trend analysis was also done for each response measure using ANOVA.
The pathology lesion data were analyzed by the exact test for trend. This generalization of the Fisher-Irwin test was used to test for a positive linear dose-related trend in the number of animals with particular lesions. A one-tailed Fisher exact test was used in the pairwise comparisons of each dose group with its appropriate control. Given the sample sizes per treatment group, it is noted that the power of the tests to detect a dose-related effect is low relative to the trend test.
The distribution of hematology and serum enzyme data contained many extreme data points. For this reason no analysis of raw data values was performed. Instead, the numbers of animals outside the normal range were examined.
The e:xact test for trend and the one-tailed Fisher exact test were also used in the analysis.
Within the data tables significant differences are presented on a gradient scale: *p = 0.03-0.05, **p = 0.01-0.03, ***p < 0.0 l.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

One female of the high-dose group died at Day 50 and two high-dose males died at Days 51 and 74. Dyspnea was the most significant clinical sign observed. Bilateral conjunctivitis accompanied by a slight clear ocular discharge was a commonly observed sign. Slight bilateral posterior paresis was observed beginning about Day 50 in one female and two males in the high-dose group. Diarrhea was observed sporadically in dogs in the mid- and high-dose groups.

Mortality:

mortality observed, treatment-related

Description (incidence):

One female of the high-dose group died at Day 50 and two high-dose males died at Days 51 and 74. Dyspnea was the most significant clinical sign observed. Bilateral conjunctivitis accompanied by a slight clear ocular discharge was a commonly observed sign. Slight bilateral posterior paresis was observed beginning about Day 50 in one female and two males in the high-dose group. Diarrhea was observed sporadically in dogs in the mid- and high-dose groups.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

High-dose males exhibited a weight loss of 16%. Middose males and high-dose females showed a 9% loss and middose females showed an 11% loss in body weight during the 90-day study.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

effects observed, treatment-related

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Reduced total erythrocyte counts and hemoglobin levels. One of the male dogs with a markedly elevated total leukocyte count of 33,000/mm3 on Day 45 died at Day 51.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

ALT, AST, and LDH showed significant changes

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

increased relative liver weights, relative kidney weights, relative lung weights significantly increased; relative brain weights increased; no significant weight changes were found in testes or ovaries.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

The lungs were mottled and showed moderate red discoloration. The kidneys were pale and were discolored ye1low-brown. White frothy material was present in the trachea and the liver showed mild yellow discoloration.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Within the brain, vacuolization of white myelinated tracts was observed in all DCA-treated dose groups; Hepatic, 1ung, pancreatic, and testicular pathologic changes; Prostatic glandular atrophy; Thymic atrophy

Details on results:

CLINICAL SIGNS AND MORTALITY
Dyspnea was the most significant clinical sign observed. It was noted around Day 45 in 2 males and 2 females in the middose groups and in 8 of 10 dogs in the high-dose groups. Forced expiratory effort was the primary characteristic of dyspnea noted in the affected dogs.
In general, once dyspnea appeared it became noticeably worse within 5-7 days and general depression and reduced activity were noted. When the affected dogs did move there was an obvious increase in respiratory rate, and efforts at forced expiration were so pronounced that abdominal heaving became apparent immediately. In some cases coughing would accompany difficult breathing. All dogs in the high-dose groups exhibited severe dyspnea by the end of the study.
Bilateral conjunctivitis accompanied by a slight clear ocular discharge was a commonly observed sign. This change was observed in 24 of 30 treated dogs during the initial month of the study and was occasionally noted in a few control dogs. The conjunctivitis progressed to a pronounced degree of swelling, with the ocular discharge becoming more purulent in 8 of 10 high-dose animals. The discharge remained clear throughout the study in affected low- and middose dogs.
Slight bilateral posterior paresis was observed beginning about Day 50 in one female and two males in the high-dose group. Among the affected dogs the paralysis was only partial and was not evident each time that the dog attempted to move. This sign was difficult to clearly verify because the dogs were reluctant to move and when they did they would drag the hind limbs intermittently rather than take coordinated steps. Once observed, the paralysis persisted but did not progress until near the termination of the study.
Diarrhea was observed sporadically in dogs in the mid- and high-dose groups. Feces of affected dogs were very watery and had a very fetid odor. Once diarrhea was noted it became progressively worse. The quantity of fluid lost each day in severely affected dogs was estimated to be 750 mL. In some animals f1uid therapy was needed to avoid severe dehydration.
One high-dose female died at Day 50 and two high-dose males died on Days 51 and 74. Based upon clinical observation of animals and microscopic examination of postmortem tissues, it was concluded that these deaths resulted from pneumonia and dehydration.

BODY WEIGHT AND WEIGHT GAIN
High-dose males exhibited a weight loss of 16%. Middose males and high-dose females showed a 9% loss and middose females showed an 11% loss in body weight during the 90-day study. Changes in body weights for the various dose groups are depicted in Figs. 1 and 2.

FOOD CONSUMPTION AND WATER CONSUMPTION
Reduction in food and water consumption was noted in dogs of all treatment groups but not the control groups. However, large individual variation precludes a conclusive statement regarding these changes. Anorexia did not appear to be dose related in that great variation was also observed in some low-dose subjects.

HAEMATOLOGY
All hematologic results are summarized in Tables 1 and 2. Reduced total erythrocyte counts and hemoglobin levels were noted in both high-dose males and females on Day 30 of the study. At Days 45, 60, 75, and 90, hemoglobin values were also below normal for middose males. By trend analyses, decreases in total erythrocyte counts and hemoglobin levels were found to be statistically significant at Days 30, 45, 60, and 90 for both sexes and
at Day 75 for males. One of the male dogs with a markedly elevated total leukocyte count of 33,000/mm3 on Day 45 died at Day 51.

CLINICAL CHEMISTRY
The clinical chemistry results are presented in Tables 3 and 4. Among the parameters tested only ALT, AST, and LDH showed significant changes and therefore only these values are presented. Trend analysis revealed significant changes in the females tor LDH at Days 30 and 45. By trend analysis, there was a significant change for AST and ALT at Day 60 for male dogs. At days 75 and 90 there was significant change for LDH in the males.

ORGAN WEIGHTS
At necropsy, the weights of liver, kidney, testes/ovaries, heart, lung, and brain were determined and summary data for each study group are presented in Tables 5 and 6. The liver weights (expressed as percentages of body weight) of both males and females were significantly higher than those of the controls at all doses of DCA. Percentage kidney weights in middose and high-dose females and males were significantly increased. The lung weights of high-dose animals of both sexes (expressed as a percentage of body weight) were significantly higher than those of controls. No significant weight changes were found in testes or ovaries. High-dose males and females showed increased relative brain weights.

GROSS PATHOLOGY
The following tissues were examined grossly and microscopically at necropsy: cerebrum, cerebellum, medulla, salivary gland, pancreas, axillary lymph node, pituitary, adrenals, thymus, mesenteric lymph node, thyroid, parathyroid, trachea, esophagus, heart, colon, jejunum, aorta, stomach, duodenum ileum, spleen, urinary bladder, lungs, sciatic nerve, spinal cord, kidneys, li er, ovaries/testes, uterus/prostate gland, skin, mammary gland,
eyes, sternum/bone marrow, femur, and gall bladder.
Many organs in the high-dose group showed distinct gross changes at necropsy. The lungs were mottled and showed moderate red discoloration. The kidneys were pale and were discolored ye1low-brown. White frothy material was present in the trachea and the liver showed mild yellow discoloration. The hepatic lesions were considered primary lesions and the changes in the kidneys and lungs were considered secondary. Although changes in the cerebrum and cerebellum were only observed microscopically, they were considered primary lesions. Changes in the pancreas and gall bladder were also primary.

HISTOPATHOLOGY: NON-NEOPLASTIC
Within the brain, vacuolization of white myelinated tracts was observed in all DCA-treated dose groups; in all cases the severity was mild (see Fig. 3). In some dogs this was present in both cerebrum and cerebellum, while in others it was noted only in cerebrum or cerebellum (Table 7). In addition, vacuolar change was observed in the medulla and spinal cord of some males and meningoencephalitis was present in one-high dose female; again,
the severity was mild.
Hepatic, lung, pancreatic, and testicular pathologic changes are summarized in Table 8. Chronic hepatitis was present in 2 high-dose males and one middose and 2 high-dose females.
Hemosiderosis was prevalent in mid and high-dose animals of both sexes. Although hepatic vacuolization was observed in most DCA-treated dogs, it was also present in some control animals. Mucosal epithelial vacuolization and hyperplasia of the gallbladder were prominent in all DCA-treated dogs (see Fig. 4). Pneumonia and bronchopneumonia were observed in nearly all DCA-treated dogs, being more severe in mid- and high-dose animals.
Marked suppuration was evident in the high-dose animals.
Pancreatic acinar degeneration associated with chronic inflammation was noted in many DCA-treated dogs in the mid- and high-dose groups (see Fig. 5). Testicular changes featuring syncytial giant cell formation and degeneration of germinal epithelium were present in nearly all DCA-treated males. The mid- and high-dose groups had an increased severity of lesions. The testicular lesions are thought to occur as a primary effect of DCA. The testes of affected males did not show lesions upon gross necropsy.
Prostatic glandular atrophy characterized by a significant reduction of glandular alveoli was noted in the mid- and high-dose groups.
Thymic atrophy was observed in most high-dose males and was characterized by a marked depletion of lymphoid tissue.

Effect levels

Dose descriptor:

LOAEL

Effect level:

12.5 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Based on visual organ effects (neurological changes, hepatic vacuolization, and testicular effects) and increased liver weights.

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

A LOAEL of 12.5 mg/kg-day can be identified, based on visual organ effects (neurological changes, hepatic vacuolization, and testicular effects) and increased liver weights.

Executive summary:

Male and female juvenile beagle dogs were dosed daily for 90 days with dichloroacetate (DCA). The compound was administered orally via gelatin capsules at doses of 0, 12.5, 39.5, and 72 mg/kg/day. Each dose group consisted of five males and five females. The dogs were observed clinically and blood samples were taken at 15-day intervals for hematologic and serum chemistry values. Decreased total erythrocyte count and hemoglobin levels were observed in mid- and high-dose dogs beginning at Day 30. Serum concentrations of LDH were elevated at Days 30 and 45 in females and at Day 75 in males treated with DCA at 72 mg/kg/day. One female of the high-dose group died at Day 50 and two high-dose males died at Days 51 and 74. Hind limb partial paralysis was observed in many high-dose dogs. Vacuolization of myclinated white tracts of cerebrum, cerebellum, and/or spinal cord was observed in many high-dose dogs as well as some mid- and low-dose subjects.

Degeneration of testicular germinal epithelium and syncytial giant cell formation was noted in males of all dose groups. Hepatic vacuolar change and chronic hepatitis appeared only in DCA-treated dogs. In addition, suppurative bronchopneurnonia and chronic pancreatitis were noted in many high-dose and some mid-dose subjects. A "no-adverse-effect level'' was not determined in this study.

A LOAEL of 12.5 mg/kg-day can be identified, based on visual organ effects (neurological changes, hepatic vacuolization, and testicular effects) and increased liver weights.