Dichloroacetic acid

Endpoint summary

Description of key information

A key 90-day toxicity study in dogs was selected based on lowest LOAEL (12.5 mg/kg-day) for various target organs (metabolism, hepatic, neurologic, reproductive). Adverse effects noted at the low-dose group included mild to severe testicular degeneration, along with mild to moderate hepatic vacuolization and mild vacuolization of the myelinated white tracts of the cerebrum and cerebellum in males and females. Data from humans administered DCA as a pharmaceutical indicate that doses of 25-50 mg/kg-day produce neurological effects, including sedation and peripheral neuropathy. Data from supporting studies confirmed the target organs for hazard in rats and dogs, and in addition data from mechanistic studies indicated peroxisome proliferation and induction of carnitine acetyl-transferase, palmityl-CoA oxidase and the PPA-protein in the liver of rats and mice.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

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.

Reason / purpose for cross-reference:

reference to same study

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

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

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

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.

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.

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.

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.

Critical effects observed:

not specified

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEL

12.5 mg/kg bw/day

Study duration:

subchronic

Species:

dog

Quality of whole database:

Reliable

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The study of Cicmanec et al. 1991 (endpoint 07.05.01_04) was selected as the key study as it identifies the lowest LOAEL (12.5 mg/kg-day) for various target organs (metabolism, hepatic, neurologic, reproductive). In this study, groups of five male and five female beagle dogs were administered oral doses (0, 12.5, 39.5, and 72 mg/kg/day) of DCA in capsules for 90 days. Adverse effects noted from the low-dose group (12.5 mg/kg-day) onwards including mild to severe testicular degeneration, along with mild to moderate hepatic vacuolization and mild vacuolization of the myelinated white tracts of the cerebrum and cerebellum in males and females. Other findings included haematological and serum changes in mid- and high-dose dogs beginning at Day 30, and hind limb partial paralysis in many high-dose dogs. In addition, suppurative bronchopneumonia and chronic pancreatitis were noted in many high-dose and some mid-dose subjects. A NOAEL was not determined in this study. Data from humans administered DCA as a pharmaceutical indicate that doses of 25-50 mg/kg-day produce neurological effects, including sedation and peripheral neuropathy. Based on these considerations, the study of Cicmanec et al. (1991) is judged to identify a LOAEL that is likely to be appropriate for humans, and is selected as the principal study. The effects of concern are testicular degeneration accompanied by mild histopathological alterations in the liver and brain (EPA, 2003).

The following 90-day (13-week) studies in rats were considered to be important supporting studies:

•       The study of Katz et al. 1981 (endpoint 07.05.01_08), in which DCA was daily administered to 10-15 male and female rats/groups by oral gavage at dose levels of 125, 500 and 2000 mg/kg bw/day. Findings included decreased body weight gains, significant serum changes (e.g. decreases in blood glucose and lactate and increased creatinine levels), increased organ weights for liver, kidneys, and adrenal glands, brain lesions in the cerebrum and cerebellum (characterized by vacuolization of the myelinated white tracts), hind limb paralysis and frequent urination. All effects were reversible after cessation of treatment, except for the brain lesions which persisted during a 4-week recovery period. Based on these effects on organ weights and brain lesions, this study identified a LOAEL of 125 mg/kg-day, the lowest dose tested.

•       The study of Katz et al. 1981 (endpoint 07.05.01_09), who noted marked testicular degeneration and myelin vacuolization in dogs administered oral doses of 50 mg/kg-day or higher for 13 weeks.

•       The study of Mather et al. 1990 (endpoint 07.05.01_10), in which DCA was daily administered to 10 male rats/dosage group via the drinking water at 0, 0.05, 0.5, or 5 g/L (equivalent to dosage levels of approximately 0, 3.9, 35.5, or 345 mg/kg/day, respectively). Findings at the mid and/or high doses included decreased water consumption and decreased body weights, significant serum changes (e.g. decreases in proteins), and increased organ weights for liver, kidneys and spleen. Histopathological changes were only seen at the highest dose, i.e. for liver (focal hepatocellular enlargement, intracellular swelling, glycogen accumulation, with increased hepatic peroxisomal beta-oxidation activity) and kidney effects (diffuse degeneration of the tubular epithelium and glomeruli). No consistent effects were observed for immunological parameters, such as antibody production, delayed hypersensitivity, natural killer cell cytotoxicity, or production of PGE2 or IL-2. Based on hepatic and renal effects in male rats, this study defined a NOAEL and a LOAEL of 3.9 and 35.5 mg/kg-day.

•       The study of Yount et al. 1982 (endpoint 07.05.01_13), in which the metabolic and toxic effects of 2-chloropropionate and Dichloroacetate (both activators of the pyruvate dehydrogenase complex) were compared in male rats dosed orally in the feed at 0, 0.04 mol/kg of feed (4–2.5 mmol/kg/d, 515–322 mg/kg/day). 

Both compounds decreased growth rate and food consumption and caused neurotoxic effects. Both compounds brought about hind limb weakness, slower nerve conduction velocities and decreased diameter of tibial nerves. 2-Chloro-propionate treatment caused testicular abnormalities manifested by testicular maturation arrest and degeneration of germ cells. 2-Chloropropionate-treated rats had significantly lower plasma triacylglycerol-levels than control or dichloreacetate-treated rats. In mature rats, total serum ketone bodies were increased by dichloreacetate but not significantly elevated by 2-chloropropionate. Although 2-chloropropionate may lack sufficient safety to warrant chronic use in humans, it is a useful research tool for studying the metabolic effects of activation of the pyruvate dehydrogenase complex. Since 2-chloropropionate is not converted to oxalate and is not as ketogenic as dichloroacetate, 2-chloropropionate may be useful clinically in situations requiring only short-term therapy.

Other studies were available, however they were considered as disregarded studies for hazard assessment as either the groups were small, dosage was very high (>1000 mg/kg bw) and/or duration was only short (14 days). The additional information can be considered to provide additional mechanistic information, whereas hazard was already identified by key and support studies.

•       The study of Bath et al. 1990 (endpoint 07.05.01_1), in which DCA was daily administered for 90 days in 5 male rats at one dose level 80.5 mM (ca. 1100 mg/kg bw/day). The DCA group showed hepatomegaly with increased liver weights and decreased testes weights compared to the controls. Light microscopic examination of the major organs revealed variable degrees of alterations in the lung, liver, testes and brain.

•       The study of Bruckner et al. 1989 (endpoints 07.05.01_2 and 07.05.01_3), in which rats and mice were dosed via the drinking water at 1000, 3000 and 5000 mg/L for 14 days. The study showed induction of carnitine acetyl-transferase, palmityl-CoA oxidase and the PPA-protein in the liver of rats and mice (parameters for the induction of peroxisome proliferation).

•       The study of Davis 1986 (endpoint 07.05.01_5), in which male and female rats were dosed via the drinking water at 0, 0.03, 0.125, 0.5, and 1.875 g/L (0,10, 40 150, and 600 mg/kg/day). Renal phosphate-dependent glutaminase activity was increased at the highest concentration, and urinary ammonia was also increased. These changes indicated renal adaptation to an acid load. DCA, in pharmacological doses, impairs glucogenogenesis from lactate in part by decreasing lactate availability. Similar tendencies were observed in the present studies; however, female rats showed a biphasic response. At lower DCA concentrations, tissue lactate and plasma glucose concentrations were increased, whereas at higher concentrations of DCA, the expected decreases were observed.

•       The study of DeAngelo et al. 1986 (endpoints 07.05.01_6 and 07.05.01_7), in which rats and mice were daily dosed via the drinking water at 1000, 3000 and 5000 mg/L for 14 days. DCA induced peroxisome proliferation in rats and mice at all dose levels tested.

•       The study of Stacpoole et al.1979 (endpoints 07.05.01_11 and 07.05.01_12), in which rats and dogs were daily dosed with Dichloroacetate for 90 days at 0, 125, 500, 2000 mg/kg bw/day (rats) and 0, 50, 75, 100 mg/kg bw /day (dogs). DCA induced peroxisome proliferation in rats and mice at all dose levels tested. In rats, dose-related hind-limb paralysis, germinal epithelial degeneration of the testis, and vacualation of the myelinated white tracts of the cerebrum were observed. The paralytic effects resolved in all animals on discontinuation of the drug, but persistence of histologic changes in both testes and central nervous system was noted in some animals during a four-week recovery period. In dogs, hind-limb muscular weakness, testicular epithelial degeneration and vacuolation of Leydig cells and of both cerebral and cerebellar myelinated white tracts were among the changes observed. Of particular note was the development in some animals of ocular lesions, consisting of bilateral lenticular opacities, bulbar conjunctivitis, superficial corneal vascularization and a tendency to keratoconjunctivitis sicca. Of these ocular changes, the lenticular opacities proved irreversible.

 

Finally, some studies were available where DCA was administered by other routes. In contrast to the toxicity associated with its chronic, oral administration, DCA given intravenously was without demonstrable adverse effects, therefore the studies were also considered to be disregarded for hazard assessment.

•       The study of Stacpoole al. 1979 (endpoint 07.05.04_1), in which Dichloroacetate was daily administered in rats by intravenous route for 38 days at 0-250 mg/kg bw.

•       The study of Stacpoole al. 1979 (endpoint 07.05.04_2), in which Dichloroacetate was daily administered in dogs by intravenous route for 30 days at 0-100 mg/kg bw.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:

The study of Cicmanec et al. 1991 (endpoint 07.05.01_04) is selected as the key study as it identifies the lowest LOAEL (12.5 mg/kg-day) for various target organs (metabolism, hepatic, neurologic, reproductive).

Repeated dose toxicity: via oral route - systemic effects (target organ) digestive: liver; neurologic: brain (multiple sections); urogenital: other

Justification for classification or non-classification

DCA is classified according the EU labelling regulations Commision Directive 93/21/EEC as HARMFUL with symbol 'Xn' and risk phrase R 25 'HARMFUL IF SWALLOWED'. According to CLP regulation (No. 1272/2008 of 16 December 2008), STOT Category 2 classification is proposed with Signal word 'Warning' and hazard statement H373 'MAY CAUSE DAMAGE TO ORGANS (BRAIN, LIVER, TESTES) THROUGH PROLONGED OR REPEATED EXPOSURE (ORAL, 90 DAYS)'.

Based on these results and according to the EC Directive (No.93/21/EEC), R33 'Danger for cummulative effects' is proposed.