Dichloroacetic acid

Toxicity to reproduction

Administrative data

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.

Data source

Reference

Reference 1

Reference Type:

publication

Title:

Unnamed

Year:

1992

Reference 2

Reference Type:

secondary source

Title:

Unnamed

Year:

2003

Materials and methods

Test guideline

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

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:

Dichloroacetic acid (Sigma ChemicaI Co., St. Louis, MO)

Test animals

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)

Administration / exposure

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

Doses / concentrations

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).

Examinations

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.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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

Details on results (P0)

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.

Effect levels (P0)

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

Results: P1 (second parental generation)

Effect levels (P1)

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

Results: F1 generation

Effect levels (F1)

Remarks on result:

other: no F1-generation produced

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

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.