Dichloroacetic acid

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

reproductive toxicity, other

Remarks:

based on test type (migrated information)

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.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Long-Evans

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories (Raleigh, NC)
- Age at study initiation: males, 100 days old: females, 70 days old
- Housing: Males were housed singly in plastic shoebox cages for at least 2 weeks before beginning treatment and females were housed two to a cage before and after the mating protocol of the study.
- Diet (e.g. ad libitum): Purina Lab Chow 5001 ad libitum
- Water (e.g. ad libitum): filter-purified tap water ad libitum
- Acclimation period: at least 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): approximately 25°C
- Humidity (%): 55%
- Photoperiod (hrs dark / hrs light): 12/12 (light commencing at 0600 hours)

Route of administration:

oral: gavage

Vehicle:

other: NaOH

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Dichloroacetic acid (Sigma ChemicaI Co., St. Louis, MO) was neutralized with NaOH to pH 7.0, diluted with deionized, distilled water, and administered by gavage for 10 consecutive weeks (up to experimental Day 70) at doses of 31.25, 62. 5, and 125 mg sodium dichloroacetate (NaDCA)/kg/day.
Control animals received 10 mL distilled water per kilogram body weight.

Details on mating procedure:

On experimental Day 70, fertility was assessed by allowing each treated male to mate overnight with one untreated, proestrus female. All males received three overnight mating experiences with ovariectomized, hormonally primed females prior to mating trials (Weeks 6, 7, and 9). Vaginal lavages were performed on the females the next morning to confirm the occurrence of mating. Males that failed to mate were allowed another opportunity with another proestrus female the following night. Sperm positive females were allowed to advance to Day [4 of gestation (vaginal lavage = Day 0]. Pregnant females were then examined for the number of live and dead implants, resorbed fetuses, and corpora lutea. Fertility was scored as "percentage implantation" [i.e., (implants/corpora lutea) X 100= % implantation].

Duration of treatment / exposure:

10 weeks

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
31.25, 62.5, 125 mg/kg bw /d
Basis:
actual ingested

No. of animals per sex per dose:

18 to 19/dose

Control animals:

yes

Details on study design:

- Dose selection rationale:
Pilot studies had been previously conducted for 10 weeks at dosages of 31.25, 62.5, 125, 500, and 1000 mg NaDCA/kg. Male rats gavaged with 500 or 1000 mg NaDCA/kg had become paralyzed and died after 4 to 6 weeks of dosing. Analysis of male reproductive endpoints from the remaining dose groups in this pilot study was confounded by the discovery of a high percentage of bilateral testicular degeneration in the control group and in the 31.25, 62.5, and 125 mg NaDCA/kg, dose groups. Since testicular weights in the highest viable dose group ( 125 mg NaDAC/kg) were so clearly dichotomized, with the higher weight group in the range of historical controls, inclusion of NaDCA-dosed animals for preliminary data analysis was based on the 80% confidence interval for testicular weights from "normal" control animals (1.36 to 1.71 g). Fifty-three percent of the controls and 54% of the treated animals were included in this interval (number of animals: 31.25 mg/kg, 10; 62.5 mg/kg, 12; 125 mg/kg, 12). Since technical problems precluded our evaluation of testicular spermatid head count in the present study, the following pilot study results are given: (in units of 106/g testis (±SD)) control, 100.4 (15.2); 31.25 mg/kg, 112.3 (14.4); 62.5 mg/kg, 113.7 ( 18.0);125 mg/kg, 100.1 (39.2).

Sperm parameters (parental animals):

Males were terminated on experimental Day 75. At least a 2-day Iapse following mating followed replenishment of epididymal reserves that may have been depleted in the mating trial. At termination the following organs were excised and weighed: liver, kidney, spleen, testes, accessory organs (prostate and seminal vesicles), preputial glands, and epididymis. One testis and cauda epididymis were each homogenized in 0.9% Na Cl, 0.01% TritonX-100 solution to obtain sperm head counts (Blazak et al., 1985). Sperm motion parameters were measured with the CellSoft (CryoResources, Ltd.,New York) computer-assisted sperm motion analysis system. For this analysis
the contralateral cauda epididymis was nicked and subsequently incubated for 3 min at 37°C in 10 mL Dulbecco's phosphate-buffered saline (+Ca2+ and Mg2+ ), pH 7.2, plus 10 mg/mL bovine serum albumin (Sigma, FractionV). An aliquot of the mixture was then diluted 10- to 20-fold and 10 µL was placed on a Petroff-Hausser chamber (Hausser Scientific, Philadelphia, PA; 20 µm depth). Sperm motion, as viewed on an Olympus BH-2 microscope ( 12.5 power, pseudo dark field optics) equipped with a "Fryer" (Fryer Co.Inc., Carpentersville, IL) stage warmer (3 7°C), was videotaped and analyzed using the CellSoft instrument. The CellSoft system settings were as follows: frames analyzed = 15; framing rate = 30 frames/sec; maximum velocity = 1100 µm/sec; threshold velocity = 20 µm/sec; minimum sampling for motility= 2 frames; minimum sampling for velocity = 3 frames; minimum sampling for straight-line velocity, linearity, amplitude of lateral head displacement (ALH), and beat/cross frequency = 11 frames (determined with an auxiliary computer program): minimum linearity for ALH = 3.5; pixel scale = 3.40 µm/pixel; at least 10 fields and 200 cells analyzed per sample; maximum average number of cells/field = 40: cell size range = 20-200 pixels (Toth el al., 1989).
One testis was prepared for histological examination by immersion in 10% neutral-buffered formalin for 24 hr. Following preparation of testis cross sections, fixation was done in 5% glutaraldehyde. Sections were stained with hematoxylin and periodic acid/Schiff's reagent and subsequently
embedded in glycol methacrylate (Chapin et al., 1985). Epididymides from additional study males (four control and four high dose animals) were also prepared for histological examination by perfusion fixation, staining with toluidine blue, and embedding in epoxy (Russell, 1983). For the evaluation of sperm morphology, air-dried slides of sperm from the vas deferens were prepared and stained with the triple stain of Bryan ( 1970).

Statistics:

Animal weights, organ weight, sperm motion endpoints, testicular spermatid head counts, and cauda epididymal sperm head counts were analyzed by analysis of variance with pairwise contrasts (two-sided) to compare the individual dose groups to the control group (Winer, 1971). Percentages of motile sperm and percentages of circularly swimming sperm were transformed with the arcsine transformation before analysis.
Percentages of individual sperm morphology groups were analyzed by the rank-based Wilcoxon test for pairwise differences (Lehmann, 1975). The Wilcoxon test was also used for analysis of pregnancy success (implantation rate) and the number of implants per pregnant female.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Reduced final animal weights relative to the controls were observed after treatment with 62.5 and 125 mg NaDCA/kg

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Reduced final animal weights relative to the controls were observed after treatment with 62.5 and 125 mg NaDCA/kg

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Animal and Organ Weights
Reduced final animal weights relative to the controls were observed after treatment with 62.5 and 125 mg NaDCA/kg and slower weight gain was seen for all NaDCA-treated males. The dose groups started the study at comparable weights (data not shown). At 31.25 mg NaDCA and higher,
relative liver weights were increased, while relative kidney andl spleen weights and absolute liver weight were increased at 62.5 and 125 mg NaDCA.
Of the male reproductive organs measured, only the testis did not show reductions in absolute weight. Reductions in absolute weights of the epididymis and preputial glands were observed at all NaDCA dose levels while the accessory organs (prostate and seminal vesicles) were reduced
at 125 mg NaDCA. Reductions in the relative weights o fthe epididymis and preputial glands resulted from NaDCA treatment at 62.5 mg NaDCA and higher, while relative cauda epididymis and accessory organ weights were unchanged. Relative testis weights were increased at 125 mg NaDCA.

Cauda Epididymal Sperm Head Counts; Sperm Morphology
Counts of cauda epididymal sperm heads were reduced at 62.5 and 125 mg NaDCA/kg. In the evaluation of sperm morphology, NaDCA treatment resulted in a reduction of the percentage of normal, intact sperm [borderline significantly different at 62.5 mg NaDCA (p = 0.06)]. However, this reduction reflected an increase in the percentage of normally shaped detached sperm heads rather than an increase in intact abnormal sperm forms.

Sperm Motion Analysis
The percentage of motile sperm as well as four descriptive sperm motion endpoints (curvilinear velocity, straight-line velocity, linearity, and amplitude of lateral head displacement) were reduced at 62.5 and 125 mg NaDCA.
The percentage of circularly swimming sperm and the beat/cross frequency were unchanged.

Histological Evaluation
No gross lesions (degenerated or atrophic seminiferous tubules, Leydig cell hyperplasia or hypoplasia) were evident in the cross sections of testes excised from males treated with 125 mg NaDCA/kg. However, there was evidence of retention of the late-step elongated spermatids beyond stage 8. Of the 9 high dose animals whose testes were examined histologically, all showed a predominance of retention of step 19 spermatids into stage 10 seminiferous tubules. At 62.5 mg NaDCA, 4 of 10 rats showed retention into stage 9 seminiferous tubules. Retention of late-step spermatids was not evident in the controls or in the 31.25-mg NaDCA dose group. Evidence of mature sperm heads drawn down toward the basal compartment in stage
10 tubules can be seen. Examination of the epididymal epithelium revealed normal cellular structures.

Fertility Assessment
NaDCA treatment did not result in a statistically significant reduction in the pregnancy rate. A reduction in the number of live implants per dam was observed at 125 mg NaDCA. However, none of the three NaDCA groups was found to be significantly different from the controls for implantation rate.

Dose descriptor:

LOAEL

Effect level:

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

Sex:

male

Basis for effect level:

other: Based on the organ weight changes reported for the preputial gland and epididymis, as well as impaired sperm formation

Dose descriptor:

LOAEL

Effect level:

31.25 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

reproductive function (sperm measures)

Remarks on result:

other: Based on the organ weight changes reported for the preputial gland and epididymis, as well as impaired sperm formation, a LOAEL of 31.25 mg/kg-day was identified

Remarks on result:

other: no F1-generation produced

Reproductive effects observed:

not specified

Conclusions:

Based on the organ weight changes reported for the preputial gland and epididymis, as well as impaired sperm formation, a LOAEL of 26.5 mg/kg-day was identified.

Executive summary:

Toth et al. (1992) studied the potential reproductive effects in male rats following subchronic oral exposure to DCA using lower doses than the earlier studies.

Dichloroacetic acid (Sigma ChemicaI Co., St. Louis, MO) was neutralized with NaOH to pH 7.0, diluted with deionized, distilled water, and administered by gavage for 10 consecutive weeks (up to experimental Day 70) at doses of 31.25, 62. 5, and 125 mg sodium dichloroacetate (NaDCA)/kg/day. Control animals received 10 mL distilled water per kilogram body weight.

Male Long-Evans rats (18 to 19/dose) were administered 0, 31.25, 62.5, or 125 mg/kg-day sodium dichloroacetate for 10 weeks by oral gavage. Reduced final animal weights relative to controls were observed at the mid- and high-dose groups. At 31.25 mg/kg-day NaDCA and higher, relative liver weights increased, while relative kidney and spleen weights and absolute liver weights were increased at 62.5 and 125 mg/kg-day NaDCA. Significant (p#0.05) reductions in the absolute weight of the preputial gland and epididymis were noted at all dose levels, but the absolute weight of the testis was not affected at any dose. At the two higher doses (62.5 and 125 mg/kg-day), there were significant (p#0.05) reductions in the percentage of motile sperm, effects on sperm motion (i.e., velocity, linearity, amplitude of lateral head displacement) and reduced epididymis sperm head counts. At 125 mg/kg-day, animals also had reduced accessory organ (prostate and seminal vesicle) weights and increased relative testis weights. Histological examination of testis cross sections did not reveal any gross lesions at any dose, and cellular structures in the epididymis epithelium appeared normal. Impaired spermiation was noted in 4 of the 10 mid-dose (62.5 mg/kg-day) animals and 9 of the 10 high-dose (125 mg/kg-day) animals, and was attributed to the retention of late-step spermatids in the seminiferous tubules, as observed histologically. This finding corroborated the observed reductions in epididymal, but not testicular late-step spermatid head counts. The fertility of treated males, although reduced in the high-dose group, did not differ significantly from controls at any dose level. Based on the organ weight changes reported for the preputial gland and epididymis, as well as impaired sperm formation, a LOAEL of 31.25 mg/kg-day was identified.

 

The LOAEL corresponds to 26.5 mg/kg-day dichloroacetic acid calculated as

result (sodium dichloroacetate) * MW(dichloroacetate)/MW(sodium dichloroacetate)

result (sodium dichloroacetate) * 127.928 /150.918

result (sodium dichloroacetate) * 0.8477.

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEL

26.5 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

reliable

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Key study for reproductive toxicity was performed by Toth et al. 1992 (endpoint 07.08.01_01) in which male Long-Evans rats were administered 0, 31.25, 62.5, or 125 mg/kg-day sodium DCA for 10 weeks by oral gavage. Reduced final animal weights relative to controls were observed at the mid- and high-dose groups. At 31.25 mg/kg-day NaDCA and higher, relative liver weights increased, while relative kidney and spleen weights and absolute liver weights were increased at 62.5 and 125 mg/kg-day NaDCA. Significant reductions in the absolute weight of the preputial gland and epididymis were noted at all dose levels, but the absolute weight of the testis was not affected at any dose. At the two higher doses (62.5 and 125 mg/kg-day), there were significant reductions in the percentage of motile sperm, effects on sperm motion (i.e., velocity, linearity, amplitude of lateral head displacement) and reduced epididymis sperm head counts. At 125 mg/kg-day, animals also had reduced accessory organ (prostate and seminal vesicle) weights and increased relative testis weights. Histological examination of testis cross sections did not reveal any gross lesions at any dose, and cellular structures in the epididymis epithelium appeared normal. Impaired spermiation was noted in 4 of the 10 mid-dose (62.5 mg/kg-day) animals and 9 of the 10 high-dose (125 mg/kg-day) animals, and was attributed to the retention of late-step spermatids in the seminiferous tubules, as observed histologically. This finding corroborated the observed reductions in epididymal, but not testicular late-step spermatid head counts. The fertility of treated males, although reduced in the high-dose group, did not differ significantly from controls at any dose level. Based on the organ weight changes reported for the preputial gland and epididymis, as well as impaired sperm formation, a LOAEL of 31.25 mg/kg-day was identified. The LOAEL corresponds to 26.5 mg/kg-day dichloroacetic acid.

Supporting studies for reproductive toxicity were also available:

·   Linder et al., 1997 (endpoint 07.08.01_02) studied testicular toxicity of DCA in adult male rats after single and multiple (up to 14 days) oral doses of 0, 18, 54, 160, 480, or 1440 mg/kg-day. Delayed spermiation and altered resorption of residual bodies were observed in rats given single doses of 1500 and 3000 mg/kg body weight; these effects persisted to varying degrees on posttreatment days 2, 14, and 28. Delayed spermiation and formation of atypical residual bodies also were observed on days 2, 5, 9, and 14 in rats dosed daily with 1440, 480, 160 or 54 mg/kg-day, respectively. Distorted sperm heads and acrosomes were observed in step 15 spermatids after doses of 480 and 1440 mg/kg-day for 14 days. Decreases in the percentage of motile sperm occurred after 9 days at doses of 480 and 1440 mg/kg-day, and after 14 days at 160 mg/kg-day. Increased numbers of fused epididymal sperm were observed on days 5, 9, and 14 in rats dosed with 1440, 480 and 160 mg/kg-day, respectively; other morphologic abnormalities occurred at 160 mg/kg-day and higher. On day 14, a significant decrease in epididymal weight was observed at 480 and 1440 mg/kg-day, and epididymal sperm count was decreased at 160 mg/kg-day and higher.

·  Bhat et al 1991 (endpoint 07.08.01_03) studied DCA in 5 male Sprague-Dawley rats given DCA at 1100 mg/kg (80.5 mM) for 90 days. Morphological changes were seen in the testes of dosed animals, with variable degrees of atrophy ( mild to severe). In the testes of two DCA-treated rats, the seminiferous tubules were severely atrophic and contained enlarged Sertoli cells, very few spermatocytes, and no mature spermatozoa. Interstitial hyperplasia, characterized by increased numbers of interstitial cells, was most pronounced in areas of severe atrophy. In three animals with mild to moderate testicular atrophy, individual seminiferous tubules showed disrupted spermatogenesis and the formation of multinucleated giant cells.

·  Katz et al. 1981 (endpoints 07.08.01_04 and 07.08.01_05) studied effects of sodium DCA after subchronic dosing in rats and dogs at 0, 125, 500, or 2000 mg/kg-day and , 50, 75, or 100 mg/kg-day, respectively. In rats, testicular germinal epithelial degeneration was seen in 40% of males at 500 mg/kg-day and all males at 2000 mg/kg-day; in all males at 2000 mg/kg-day, the testes appeared aspermatogenic and contained syncytial giant cells in the germinal epithelium, while the epididymis ducts were devoid of spermatozoa. Syncytial giant cells in the germinal epithelial were seen in 20% of the male rats dosed at 500 mg/kg-day. No effects were noted in the reproductive tissues of female rats. In dogs, prostate gland atrophy and testicular changes (degeneration of germinal epithelium, vacuolation of Leydig cells, formation of syncytial giant cells) were observed in all treated males. These effects were qualitatively judged by the authors to be dose-dependent. After a 5-week recovery period in one male, the prostate appeared normal and there was evidence of germinal epithelium regeneration with spermatogenesis.

·   Cicmanec et al., 1991 (endpoint 07.08.01_06) examined endpoints relevant to reproductive toxicity after subchronic dosing in male and female beagle dogs at 0, 12.5, 39.5, or 72 mg/kg-day of DCA in gelatin capsules. Testicular changes were reported in the males at all dose levels, including syncytial giant cell formation and degeneration of testicular germinal epithelium. Severity of the lesions increased in the mid- and high-dose animals. Prostate glandular atrophy characterized by a significant reduction of glandular alveoli was also noted in mid-and high-dose groups. The testes of affected males did not show lesions upon gross necropsy. Absolute and relative testicular weights were unaffected by DCA treatment. A reproductive LOAEL of 12.5 mg/kg-day, the lowest dose tested, was established in this study.

Short description of key information:

There is an extensive and consistent data base demonstrating the reproductive toxicity of DCA in rats and dogs. In male rats, DCA caused decreases in testicular weight at 1100 mg/kg bw and reduced viable sperm production from 62.5 mg/kg bw/day onwards. Reduced weights of the preputial gland and epididymis were also noted at lower dose levels of 31.25 mg/kg bw, whereas testis weight was not affected up to 125 mg/kg bw. The fertility of treated males up to 125 mg/kg bw/day, although reduced in the high-dose group, did not differ significantly from controls. A LOAEL of 31.25 mg/kg-day was identified from the key study. Testicular changes (degeneration of testicular germinal epithelium) and prostate glandular atrophy  were also seen in dogs, whereas testicular weights were unaffected; reproductive LOAEL of 12.5 mg/kg-day was retained in dogs.

Justification for selection of Effect on fertility via oral route:

The study in rats covered both morphological and functional parameters for reproductive toxicity after chronic dosing.

Effects on developmental toxicity

Description of key information

In female rats, DCA exposure to dose levels of 140 mg/kg-day during gestation can lead to impaired fetal maturation and result in soft tissue anomalies (primarily of cardiac origin) in the offspring.  The increase in soft tissue abnormalities was significant for the higher 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. 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). Chronic (life-time) postnatal exposure in in weanling rats at 137 mg/kg-day resulted in severe gait abnormalities, decreased hind limb grip strength, righting deficits, and tremors (>50% incidence) , and recovery was not evident. Treated rats also showed decreased forelimb grip strength, chest clasp, and an inhibited pupil response. Mechanistic in vitro studies in mouse whole-embryo cultures yielded neural-tube defects, heart and pharyngeal arch defects eye defects, rotational defects and somite dysmorphology. Apoptosis was demonstrated in the head, heart, midpiece, and hindpiece regions of embryos exposed to DCA for 24 hours, suggesting that DCA may inhibit protein kinase.  Some of the effects seen in vivo could be reproduced in the in vitro whole-embryo culture system.

Link to relevant study records

Reference

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.

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

not specified

Limit test:

no

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

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

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

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).

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).

Dose descriptor:

NOAEL

Effect level:

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

Basis for effect level:

other: other:

Dose descriptor:

LOAEL

Effect level:

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

Basis for effect level:

other: other:

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.

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).

Abnormalities:

not specified

Developmental effects observed:

not specified

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.

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).

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

14 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

reliable

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

A key study for developmental toxicity was performed by Smith et al. 1992 (endpoint 07.08.02_01). In a second part of their study 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. 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 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).

Supporting information was also further available from following sources:

· Smith et al. 1992 (endpoint 07.08.02_02) also studied developmental toxicity of DCA at higher dose levels of 900, 1400, 1900, or 2400 mg/kg-day from gestational days 6 to 15 in pregnant Long-Evans rats. Eight dams in the three high-dose groups died during treatment. Maternal weight gain, adjusted for gravid uterine weight, was significantly decreased to approximately 60% of the control value in all treatment groups. The absolute liver, spleen, and kidney weights significantly (p≤0.05) increased in all dose groups, with corresponding hypertrophy in these organs. The mean percentage of resorbed implants per litter was significantly elevated in all treated dose-groups. The number of live fetuses/litter was significantly reduced by 27% at 2400 mg/kg-day. All dose groups exhibited significant, but relatively small, dose-dependent reductions in fetal weight and fetal crown-rump. There was a significant increased incidence (dose-dependent) of soft tissue and cardiovascular anomalies in all treatment groups, and of external malformations beginning at the 1400 mg/kg-day group. No skeletal malformations were observed.

·  Epstein et al. 1992 (endpoint 07.08.02_05) investigated the time-sensitivity of DCA dosing on the development of the fetal rat. Pregnant Long-Evans rats were exposed, via oral intubation, to DCA as follows: 1900 mg/kg-day on consecutive gestation days 6 to 8, 9 to 11, or 12 to 15; single doses of 2400 mg/kg-day on gestation day 10, 11, 12, or 13; or single doses of 3500 mg/kg-day on gestation day 9, 10, 11, 12, or 13. No treatment effects on maternal body or organ weights were observed. Within the 1900 mg/kg-day exposure group, reduced mean fetal body weight was observed for days 6 to 8, and increased cardiac malformations for days 9 to 11 and 12 to 15. Single gestation day exposures increased the incidence of cardiac defects (2.5 to 3.3% and 2.9 to 3.6% at the 2400 and 3500 mg/kg-day doses, respectively).

· Moser et al. 1999 (endpoint 07.08.02_06) investigated the (postnal developmental) chronic-duration neurotoxic effects of DCA in weanling rats. Rats were exposed via drinking water to 2.5 or 3.5 g/L DCA for 24 months. However, exposures to the high dose were discontinued before the study ended because of excessive toxicity. In addition, the low dose was decreased at 6 weeks and at 10 weeks. Estimated intake levels over the exposure period were 235 mg/kg-day (for 6 months) and 137 mg/kg-day (for 24 months) for the high- and low-dose groups, respectively. Severe gait abnormalities, decreased hind limb grip strength, righting deficits, and tremors (>50% incidence) were evident in both dose groups throughout the 2-year period. Recovery was not evident even 18 months after exposure ended in the high-dose group. Treated rats also showed decreased forelimb grip strength, chest clasp, and an inhibited pupil response.

 

Following studies were rather performed for mechanistic purpose and were therefore disregarded from hazard assessment:

·  Hunter et al. 1996 (endpoint 07.08.02_03) exposed CD-1 mouse whole-embryo cultures to 0 to 14.7 mM DCA for 24 hours. The study authors found significant increases in neural-tube defects at treatment concentrations of 5.9 mM and above, heart and pharyngeal arch defects were seen at concentrations of 7.3 mM and above and eye defects, rotational defects and somite dysmorphology at concentrations of 11 mM and above.

·  Ward et al.2002 (endpoint 07.08.02_04) further investigated cell-cycle disruptions in mice neurulation-stage (gd8) embryos exposed to 11 mM DCA for 6, 12, 18, or 24 hours. DCA caused a slight increase in the number of heart cells in S phase and a slight decrease in those cells in G1 phase and a statistically-significant increase in sub-G1 events in embryos incubated ≥12 hours, which was interpreted to be an increase in the induction of apoptosis. This effect was consistent in the head, heart, midpiece, and hindpiece regions of embryos exposed to DCA for 24 hours.

The data suggest that DCA’s inhibition of protein kinase may be a mechanism for apoptosis. However, the inhibition of protein kinase C is unlikely to be the predominant mediator of DCA-induced embryotoxicity.

· Saillenfait et al., 1995 (endpoint 07.08.02_06) also studied the potential developmental toxicity of DCA in vitro using a rat whole embryo culture system at 0, 1.0, 2.5, 3.5, 5.0, 7.5, or 10 mM DCA. A significant, dose-dependent decrease in crown rump length was seen at 3.5 mM and above, while significant, dose-related decreases in yolk sac diameter, head length, somite (embryonic segment) number, protein content, and DNA content were seen at 2.5 mM and above. In addition, several defects which were nonexistent in the 0 and 1.0 mM groups were present to a substantial degree in the higher dose groups. At 2.5 mM, 30% of the embryos had brain defects, 45% had eye defects, and 10% had reduced embryonic axis. At 3.5 mM, 95% had brain defects, 75% had eye defects, 80% had reduced embryonic axis, 15% had reduced first branchial arch, 40% had otic system defects, and 15% had defective flexion. The results indicated a teratogenic effect from DCA in this system.

Justification for selection of Effect on developmental toxicity: via oral route:

The study was performed at lowest dose levels and could identify maternal and developmental NOAEL s and LOAELs.

Justification for classification or non-classification

The substance was demonstrated to be toxic for reproduction and development in experimental animals, however there is no evidence in humans for reproductive or developmental toxicity. In conclusion, Dichloroacetic acid is presumed to be a human reproductive toxicant. This classification corresponds to GHS/CLP Category 1B classification according to CLP regulation (No. 1272/2008 of 16 December 2008) with signal word 'Danger' and hazard statement: H360 – May damage fertility or the unborn child (soft tissue abnormalities; oral route). According to the Directive 67/548/EEC, Annex VI, DCA is classified Category 2 T R60-61.