Dichloroacetic acid

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1992

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.

Data source

Reference

Reference 1

Reference Type:

publication

Title:

Developmental Toxicity of Dichloroacetate in the Rat

Author:

Smith et al.

Year:

1992

Bibliographic source:

TERATOLOGY 46: 217-223 (1992)

Reference 2

Reference Type:

secondary source

Title:

Unnamed

Year:

2003

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

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): Dichloroacetate; DCA
- Analytical purity: >99% purity
- Other: supplied by Sigma Chemical Co. (St. Louis, MO)

Test animals

Species:

rat

Strain:

Long-Evans

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories (Portage, MI)
- Age at study initiation: 65-80 days
- Housing: In groups of three in plastic cages with corn cob bedding (Bed O'Cobs, Anderson Cob Div., Maumee, OH). Sperm-positive females were considered to be in day 0 of pregnancy and were singly housed for the duration of the study.
- Diet (e.g. ad libitum): Purina Rodent Laboratory Chow No. 5001 (St. Louis, MO) ad libitum
- Water (e.g. ad libitum): distilled water ad libitum
- Acclimation period: Not provided

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.11 – 23.33°C
- Humidity (%):40-60%
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: distilled water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The compound was dissolved in water and adjusted to pH 7 with sodium hydroxide, such that the desired dosage, adjusted daily, could be administered at 10 mL/kg body weight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dosing solutions were prepared daily and their purity and stability confirmed using ion chromatography.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1
- Length of cohabitation: At 2:00 PM and checked for the presence of sperm at 9:00 AM on the following morning.
- Proof of pregnancy: sperm in vaginal smearreferred to as day 0 of pregnancy

Duration of treatment / exposure:

on gestational days 6 to 15

Frequency of treatment:

daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 14, 140, or 400 mg/kg-day DCA (calculated as the free acid)
Basis:
actual ingested
on gestational days 6 to 15

No. of animals per sex per dose:

19-20/group

Control animals:

yes, concurrent vehicle

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: The animals were observed daily for signs of toxicity.

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: Weights were collected at the beginning and end of gestation and daily during treatment.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): No data

POST-MORTEM EXAMINATIONS: Yes
Females dying prematurely were subjected to gross necropsy
- Sacrifice on gestation day 20: On day 20 of gestation, the dams were killed using CO, asphyxiation.
- Organs examined: Their livers, spleens, and kidneys were removed and weighed. Corpora lutea were counted using a dissecting microscope. The uterine horns were examined for the number and location of fetuses or resorption sites.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes. Corpora lutea were counted using a dissecting microscope.
- Number of implantations: Yes
- Number resorptions: Yes . The uterine horns were examined for the number and location of fetuses or resorption sites.

Fetal examinations:

- External examinations: Yes: all per litter: The fetuses were then removed, weighed, measured for crown-rump length, sexed, and evaluated for external abnormalities.
- Soft tissue examinations: Yes: Two-thirds of each litter were fixed in Bouin's solution for freehand razor blade sectioning (Wilson, '65).
- Skeletal examinations: Yes: 1/3 of each litter were fixed in alcohol, double stained with alizarin red S for ossified bone and Alcian blue for cartilage, and cleared in 2% KOH and glycerine (Inouye, '76; Kimmel and Trammell, '81).
- Head examinations: No data

Statistics:

Maternal body and organ weights (first study), mean fetal weights and crown-rump lengths, and litter sizes (first study) were analyzed for overall treatment differences using the one-way analysis-of-variance (ANOVA) procedure. Differences between the dose groups and the control group were examined using pairwise contrasts (Winer, '71). The presence of a significant dose response was tested with linear regression. The proportions of maternal deaths and pregnancies were compared using the Z-test for differences between proportions (Walpole and Myers, '78). Organ weights (second study), litter sizes (second study), sex ratios, and percentages of resorptions and fetuses with abnormalities were evaluated with the Kruskal-Wallis test for overall differences, with pairwise Mann-Whitney tests to compare each treatment with the control (Lehmann, ’75), and Jonckheere's test to analyze for a dose-related response (Lehmann, '75).

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
Secondary source: A significant decrease in maternal weight gain, adjusted for gravid uterine weight, was found in the mid- and high-dose dams (63 and 77% of control, respectively), as well as an increase in spleen and kidney weights at the highest dose. Absolute liver weight was significantly elevated for all dose groups compared to the control group, with 3, 8, and 14% increases observed, respectively. Dose-related hypertrophy in the liver, spleen and kidneys was reported in the two high-dose groups (no incidence data).

Effect levels (maternal animals)

1

Dose descriptor:

NOAEL

Effect level:

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

Basis for effect level:

other: other:

2

Dose descriptor:

LOAEL

Effect level:

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

Basis for effect level:

other: other:

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Secondary source: Reduced fetal crown-rump length (5% decrease) and fetal body weight (7% decrease) were significant in the high-dose group. A dose-related increase in soft 40 tissue anomalies, primarily cardiovascular, was reported in the 140 and 400 mg/kg-day groups.
The increase in soft tissue abnormalities was significant for the two highest dose groups and the cardiac abnormalities for the highest dose. An intraventricular septal defect between the ascending (bilateral hydronephrosis and renal papilla) and defects of the orbit also reported.

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

14 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

not specified

Basis for effect level:

other: soft tissue anomalies

Remarks on result:

other: see remarks

Remarks:

Reduced fetal crown-rump length (5% decrease) and fetal body weight (7% decrease) were significant in the high-dose group. A dose-related increase in soft 40 tissue anomalies, primarily cardiovascular, was reported in the 140 and 400 mg/kg-day groups. The increase in soft tissue abnormalities was significant for the two highest dose groups and the cardiac abnormalities for the highest dose. An intraventricular septal defect between the ascending aorta and the right ventricle was most commonly observed with less frequent urogenital defects (bilateral hydronephrosis and renal papilla) and defects of the orbit also reported. Collectively, these studies determined a NOAEL of 14 mg/kg-day and a LOAEL of 140 mg/kg-day DCA for developmental effects (soft tissue anomalies).

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

The principal fetal target for DCA was the heart and major vessels, with most affected fetuses displaying, after Wilson sectioning, a defect between the ascending aorta and the right ventricle. More detailed histopathological examination of this defect revealed it as a high interventricular septal defect (H-IVSD), with a range of tissue involvement (Epstein et al., '91). Overt interventricular septal defects were found to bethe second most common anomaly. Otherless frequently affected systems were the kidneys (bilateral hydronephrosis, renal papilla, stage one) and the orbit.

The developmental toxicity of DCA has not previously been reported, nor have there been developmental studies of the major metabolites, glyoxylate and oxalate. A related compound, trichloroacetic acid (TCA), induces a range of heart malformations in the developing rat, although not with the predominance of H-IVSD seen with this compound (Smith et al., '89).

The metabolic effects of DCA are widespread and very complex (Crabb et al., '81, Stacpoole, '89). Many of the actions of DCA in intermediary metabolism are thought to be the direct or indirect consequence of its activation of the mitochondrial pyruvate dehydrogenase complex (Whitehouse and Randle, '73). Its effects are not localized, and oral administration causes striking changes in plasma metabolite levels, including reductions in the levels of lactate. Of possible interest to this study may be the observation that DCA concentrates in rat myocardial mitochondria (Kerbey et al., '76) and also activates myocardial branched chain α- keto acid dehydrogenase, promoting leucine

oxidation (Sans et al., '80).The mammalian embryo during early organogenesis relies almost exclusively on glycolysis for energy, a process presumably disturbed by DCA and therefore a candidate for consideration as part of its mechanism of action. It is difficult to hypothesize, however, just how a compound with such widespread metabolic effects as DCA would, by this process, target

the embryonic heart almost exclusively. Clinical applications of DCA have been hindered by its known toxicity in the testes, the lens and in the nervous system, all tissues also depending heavily on glycolysis as an energy source.

Preliminary data from this laboratory suggest that DCA has free and rapid access to the fetus following oral administration. The half-life of the parent compound has been reported as 0.5 h in humans and 3 h in the (nonpregnant) rat (Lukas et al., '80), although the metabolic effects in humans evidently persist for considerable time following DCA withdrawal (Stacpoole et al., '78).

We have not yet measured the accumulation of DCA or its metabolites in the rat fetus, but we have found that repeated dosing appears to be necessary to produce the characteristic heart malformations (Epstein et al., '91). The effects of chronic DCA administration have been noted by others to differ from those following acute doses (Searle et al., '76). Thus, given the current source of inadvertent chronic human exposure, our present efforts center on estimating the extent and influence of cumulative toxicity in the embryo.

Applicant's summary and conclusion

Conclusions:

Collectively, these studies determined a NOAEL of 14 mg/kg-day and a LOAEL of 140 mg/kg-day DCA for developmental effects (soft tissue anomalies) and maternal effects (reduced body weight and organ hypertrophy).

Executive summary:

In an experiment by Smith et al. (1992), pregnant Long-Evans rats (19-20/group) were administered 0, 14, 140, or 400 mg/kg-day DCA by gavage on gestational days 6 to 15. A significant decrease in maternal weight gain, adjusted for gravid uterine weight, was found in the mid- and high-dose dams (63 and 77% of control, respectively), as well as an increase in spleen and kidney weights at the highest dose. Absolute liver weight was significantly elevated for all dose groups compared to the control group, with 3, 8, and 14% increases observed, respectively.

Dose-related hypertrophy in the liver, spleen and kidneys was reported in the two high-dose groups (no incidence data). Reduced fetal crown-rump length (5% decrease) and fetal body weight (7% decrease) were significant in the high-dose group. A dose-related increase in soft

40 tissue anomalies, primarily cardiovascular, was reported in the 140 and 400 mg/kg-day groups.

The increase in soft tissue abnormalities was significant for the two highest dose groups and the cardiac abnormalities for the highest dose. An intraventricular septal defect between the ascending aorta and the right ventricle was most commonly observed with less frequent urogenital defects (bilateral hydronephrosis and renal papilla) and defects of the orbit also reported. Collectively, these studies determined a NOAEL of 14 mg/kg-day and a LOAEL of 140 mg/kg-day DCA for developmental effects (soft tissue anomalies) and maternal effects (reduced body weight and organ hypertrophy).