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Guidelines for Canadian Drinking Water Quality: Guideline Technical Document - Haloacetic Acids
10.0 Health effects in laboratory animals and in vitro test systems
10.4 Mutagenicity and genotoxicity
10.4.1 Monochloroacetic acid
There was no evidence of genotoxic potential in mutagenicity studies in bacteria using Salmonella typhimurium (Rannug et al., 1976; NTP, 1992; BG Chemie, 1993; Giller et al., 1997; ECETOC, 1999). Mostly positive results were seen in assays using mammalian cells (mouse lymphoma assay) (Amacher and Turner, 1982; McGregor et al., 1987) , but these may be due to changes in pH or cytotoxicity (ECETOC, 1999). In vitro DNA repair/damage assays involving Escherichia coli, S. typhimurium and cultured mammalian cells were largely negative (Gross et al., 1982; Ono et al., 1991; Chang et al., 1992; NTP, 1992; BG Chemie, 1993; Giller et al., 1997; ECETOC, 1999), except for one study looking at DNA strand breaks in Chinese hamster ovary cells, which was positive (Plewa et al., 2002). Clastogenic studies were mostly negative with MCA (Galloway et al., 1987; Sawada et al., 1987; Giller et al., 1997), except for one study in which MCA (acid) induced sister chromatid exchange in Chinese hamster ovary cells without S9 only (Galloway et al., 1987). In an in vivo bone marrow assay, positive results were seen via the intraperitoneal route, but not by the oral or subcutaneous route (Bhunya and Das, 1987). Details were lacking in this study. Sperm shape abnormalities were seen in an intraperitoneal injection study in mice only at the top two doses, but this study was poorly reported (Bhunya and Das, 1987).
10.4.2 Dichloroacetic acid
There was mostly negative evidence of genotoxic potential in mutagenicity studies in bacteria using S. typhimurium (Herbert et al., 1980; Matsuda et al., 1991; DeMarini et al., 1994; Fox et al., 1996; Giller et al., 1997; Meier et al., 1997), and equivocal results were seen with mammalian cells (Fox et al., 1996; Harrington-Brock et al., 1998). Clastogenic studies demonstrated largely negative results (Fox et al., 1996; Giller et al., 1997; Meier et al., 1997). DNA repair assays with bacteria were generally positive, but involved DCA as a free acid. DNA damage assays (strand breaks) involving mammalian cells (in vitro and in vivo) were largely negative (Chang et al., 1992; Plewa et al., 2002), except for one oral study involving mice and rats where the acid was used (Nelson and Bull, 1988; Nelson et al., 1989). Dose-related sperm head abnormalities were seen in mice following oral gavage with DCA (sodium salt) at doses of 1125-4500 mg/kg bw per day (Meier et al., 1997).
10.4.3 Trichloroacetic acid
There was no evidence of genotoxic potential in mutagenicity studies in bacteria (IARC, 1995; Kargalioglu et al., 2002; NTP, 2003a), except in one modified Ames study, which was weakly positive (Giller et al., 1997). Weakly positive results were also seen in the one gene mutation study with mammalian cells (Harrington-Brock et al., 1998). In vitro DNA damage assays with mammalian cells were negative (Chang et al., 1992; Plewa et al., 2002), but mixed results were seen when TCA was administered in vivo (Nelson and Bull, 1988; Nelson et al., 1989; Chang et al., 1992). TCA gave mostly negative results for DNA damage/repair with bacterial systems (Ono et al., 1991; Giller et al., 1997).
Clastogenic studies (in vitro and in vivo) also demonstrated equivocal results (Bhunya and Behera, 1987; MacKay et al., 1995; Giller et al., 1997; Meier et al., 1997). Harrington-Brock et al. (1998) reported that positive results for clastogenicity and mutagenicity may be seen in in vitro mammalian assays as a result of low pH, especially in the presence of metabolic activation.
Sperm shape abnormalities in mice were equivocal with intraperitoneal administration but negative with oral dosing (Bhunya and Behera, 1987). Gap-junctional intercellular communication was seen in liver cells of mice (in vitro) (IARC, 1995).
10.4.4 Monobromoacetic acid
There is some evidence to suggest that MBA is weakly mutagenic. Mixed results were seen in the Ames assay with MBA (Saito et al., 1995; Kohan et al., 1998; Kargalioglu et al., 2002; NTP, 2003b). Kargalioglu et al., (2002) reported in their Ames assay that MBA was more mutagenic than DBA or MCA. There was also slight evidence of genotoxic potential in in vitro DNA damage assays. MBA (acid) failed to induce primary DNA damage in an SOS chromotest (E. coli) with and without S9 and failed to increase the frequency of micronuclei in a newt micronucleus test (Giller et al., 1997). In contrast, MBA induced DNA strand breaks in the absence of S9, following a 1-hour treatment, using L-1220 mouse leukaemia cells (Stratton et al., 1981). The number of strand breaks increased further when the chemical was removed (Stratton et al., 1981). The authors suggested that MBA may act as an alkylating agent due to the presence of strand breaks (indicator of direct DNA damage).
10.4.5 Dibromoacetic acid
There is some evidence to suggest that DBA is weakly mutagenic. Mixed results were seen with the Ames test with S. typhimurium (Saito et al., 1995; Giller et al., 1997; Morita et al., 1997; Kohan et al., 1998; Kargalioglu et al., 2002; NTP, 2007). Positive results were seen for genotoxic potential in in vitro DNA damage assays. DBA induced primary DNA damage in an SOS chromotest using E. coli with and without metabolic activation (Giller et al., 1997), and induced DNA strand breaks in Chinese hamster ovary cells as measured in a comet assay (Plewa et al., 2002). There was also some evidence of clastogenicity (increased frequencies of micronucleated normochromatic erythrocytes using peripheral blood) in male mice, but not in female mice, when DBA was administered orally via drinking water as part of a 13-week study (NTP, 2007). No clastogenic activity was seen in the newt micronucleus test (Giller et al., 1997).
10.5 Reproductive and developmental toxicity
10.5.1 Monochloroacetic acid
No reproductive studies were identified. No adequate developmental studies were conducted with MCA. Two studies are reported below. However, they lack important information; one study originates from an abstract, and the other one uses only one dose.
One developmental assay (oral gavage with MCA acid in distilled water at 0, 17, 35, 70 or 140 mg/kg bw per day from gestation days 6 to 15) using Long-Evans rats was published in an abstract by Smith et al. (1990). This study suggested developmental effects (laevocardia in the high-dose group) in the presence of maternal toxicity. No statistical data were provided in the abstract, and no final study was published to confirm these results.
In a second developmental study (using only one dose), a group of 10 pregnant Sprague-Dawley rats was given MCA (neutralized) in drinking water at a concentration of 1570 mg/L (193 mg/kg bw per day) on gestation days 1-22 (Johnson et al., 1998). The control group consisted of 55 females. A significant decrease in body weight gain was observed in exposed dams relative to controls. The average amount of drinking water consumed on a daily basis per maternal rat was lower in treated dams than in the control group. No adverse reproductive, developmental or teratogenic effects were reported; however, a complete fetal examination for internal or skeletal abnormalities was not conducted.
10.5.2 Dichloroacetic acid
10.5.2.1 Developmental studies
Several studies on the potential developmental toxicity of DCA in female rats have been conducted and are detailed below and summarized in Table 11.
| Doses (mg/kg bw per day) | Species | Developmental effects | LOAEL/ NOAEL | Reference |
|---|---|---|---|---|
| 0, 14, 140, 400,900, 1400, 1900 or 2400 (GDa 6-15) | Long-Evanshooded rats (19-21/dose) |
|
NOAEL for developmental toxicity: 14 mg/kg bw per day | Smith et al., 1992 |
Four studies:
|
Long-Evans rats (7-11/dose) |
|
Epstein et al., 1992 | |
| 0 or 300 (GD 6-15) | Sprague-Dawley rats (19-20/dose) |
|
Fisher et al., 2001 | |
In a developmental study (Smith et al., 1992), pregnant Long-Evans hooded rats (19-21 per dose) were administered DCA (neutralized) by gavage in two separate studies at dose levels of 0, 14, 140 or 400 or of 0, 900, 1400, 1900 or 2400 mg/kg bw per day on days 6-15 of gestation, inclusively. There were dose-related reductions in adjusted maternal weight gain (140 mg/kg bw per day and above), dose-related increases in relative liver weights (all doses) and dose-related increases in relative kidney and spleen weights (400 mg/kg bw per day and above). Treatment-related maternal lethality was seen at 1400 mg/kg bw per day and above (1/19 [5.3%], 2/19 [10.5%] and 5/21 [24%], respectively). The pregnancy rates, total number of implants per litter and preimplantation losses were not affected by treatment. There was a dose-related increase in post-implantation loss rate (900 mg/kg bw per day and above), and the number of live fetuses per litter was reduced at the highest dose (at which significant maternal toxicity [lethality] was observed). A dose-related decrease in fetal body weight and crown-rump length was observed at 400 mg/kg bw per day and above. Dose-related increases in external (1400 mg/kg bw per day and above), total soft tissue (140 mg/kg bw per day and above), cardiovascular (400 mg/kg bw per day and above), urogenital (1400 mg/kg bw per day and above) and orbital (900 mg/kg bw per day and above) malformations were observed. Lower frequencies of urogenital (bilateral hydronephrosis, renal papilla, stage one) and orbital anomalies were seen compared with other malformations. The main fetal target for DCA was the heart and major vessels. The most common heart defect in fetuses occurred between the ascending aorta and the right ventricle and was identified as high interventricular septal defect. The second most common heart malformation was overt interventricular septal defect. The authors set the NOAEL for developmental toxicity at 14 mg/kg bw per day.
Four studies were performed with pregnant Long-Evans rats (7-11 per dose) exposed to DCA (neutralized) to determine the most sensitive developmental period and also to characterize heart defects (Epstein et al., 1992). In the first study, rats were treated by oral intubation with 1900 mg DCA/kg bw per day on gestation days 6-8, 9-11 or 12-15; in the second study, with 2400 mg/kg bw per day on gestation day 10, 11, 12 or 13; and in the third study, with 3500 mg/kg bw per day on gestation day 9, 10, 11, 12 or 13. No significant maternal toxicity was observed in the first three studies. The mean percentages of heart malformations were significantly higher (statistically) in the group treated with 1900 mg DCA/kg bw per day on gestation days 9-11 (7.2% per litter) and 12-15 (15.1% per litter) compared with the combined controls. The cardiac defects seen were either high interventricular septal defect or a conventional interventricular septal defect. Lower incidences of cardiac malformations were observed with the 2400 mg DCA/kg bw per day dose (gestation day 10: 2.5% per litter; gestation day 12: 3.3% per litter; gestation days 11 and 13: 0%). The incidences on gestation days 10 and 12 were significantly different from the combined controls. At the higher dose, 3500 mg/kg bw per day, the incidences of heart defects were not increased, but this study showed that dosing on gestation days 9, 10 and 12 (3.6%, 2.9% and 2.9% per litter, respectively) would produce these defects, and the incidences were significantly different from combined control (0.5% per litter). No heart defects were seen on gestation day 11 or 13 (Epstein et al., 1992).
In a fourth study (Epstein et al., 1992) to characterize heart defects, six dams were orally intubated with 1900 mg DCA/kg bw per day on days 6-15 of pregnancy, and 56 fetuses from the six litters as well as eight control fetuses from four litters were harvested for light microscopic examination of the heart. Examination revealed that 25 of 56 fetuses (45%) had cardiovascular defects; 24 had hearts characterized by high interventricular septal defects (five of these hearts also presented a membranous-type interventricular septal defect), and one had an isolated occurrence of an interventricular septal defect, membranous type.
In a more recent study, Fisher et al. (2001) gavaged a group of 20 pregnant female Sprague-Dawley rats with DCA (neutralized) at 300 mg/kg bw per day during gestation days 6-15. A control group (n = 19) was dosed with water. No malformations of the heart were seen, contrary to those reported in previous studies by Smith et al. (1992) and Epstein et al. (1992). It is possible that this study was not sensitive enough, given the high background of heart malformations (on a per litter basis) seen in the water controls (31.6%; higher than in the treated animals: 30%), which might have masked the effects in the DCA treatment group. The Smith et al. (1992) study showed only one or two cardiovascular defects at doses below 300 mg/kg bw per day. It is also possible that the strain differences in the rats and differences in the purity of the test agents used may account for the incongruent findings between the two set of studies.
10.5.2.2 Reproductive studies
Adult Sprague-Dawley male rats (n = 24 per dose) were given single oral doses of DCA (neutralized) at 0, 1500 or 3000 mg/kg bw to study testicular toxicity and then sacrificed (n = 8 per time period) on day 2, 14 or 28 (Linder et al., 1997a). No clinical signs of toxicity were seen in treated rats. Body weights were not statistically different from controls at any of the three time points. Mild effects on spermiation (i.e., delays) and changes in the degree of resorption of residual bodies were seen at both doses; these effects persisted to varying degrees throughout the observation period (Linder et al., 1997a).
In a parallel study by the same author, another group of male rats (n = 8-26) was given multiple oral doses of DCA (neutralized) at 0, 18, 54, 160, 480 or 1440 mg/kg bw per day for up to 14 days (Linder et al., 1997a). No clinical signs of toxicity were observed in the treated animals, except for reduced weight gain, which was seen at the three top doses by day 14. Delayed spermiation and formation of atypical residual bodies were observed in all treated males, except at the lowest dose. An increased number of fused epididymal sperm occurred after day 5 at doses of 160 mg/kg bw per day and above. A decrease in percentage of motile sperm was seen on day 9 at 480 mg/kg bw per day and above and on day 14 at 160 mg/kg bw per day and above. On day 14, epididymal sperm count was decreased at 160 mg/kg bw per day and above, while a decrease in epididymal sperm weight was seen at 480 mg/kg bw per day and above. Distorted sperm heads and acrosomes were seen in step 15 spermatids after 14 days at 480 mg/kg bw per day and above. Testicular lesions developed with greater severity as the duration of the dosing and dose levels increased.
Long-Evans male rats (n = 8-19) were gavaged daily with DCA (sodium salt) at 0, 31.3, 62.5 or 125 mg/kg bw for 10 weeks (Toth et al., 1992). On day 70, each male was mated overnight with one untreated, pro-estrous female to assess fertility. Males were then sacrificed on day 75 and females on day 14 of gestation. Pregnancy and implantation rates were not significantly different from the female controls. However, in high-dose females, a reduction in the number of live implants was observed. In males, decreased weights were seen in the epididymis and preputial glands at 31.3 mg/kg bw per day and above and in the accessory organs (prostate and seminal vesicles) at the highest dose (125 mg/kg bw per day). At the two highest doses, sperm morphology was affected and epididymal sperm counts were decreased. Effects on the testis and spermiation (a reduction in late-step spermatid head counts) were seen only in the highest dose group. The authors set a LOAEL of 31.3 mg/kg bw per day for DCA (sodium salt) (equivalent to 26.7 mg DCA/kg bw per day), based on adverse male reproductive effects on the accessory organs and sperm (Toth et al., 1992).
In contrast, in a shorter 7-week subchronic drinking water study, Sprague-Dawley rats had normal testicular histopathology and sperm production when exposed to DCA (sodium salt) at 1100 mg/kg bw per day (Stacpoole et al., 1990). Normal histopathology and testis weights were also seen in male Sprague-Dawley rats (10 per dose) when treated with DCA (neutralized) in their drinking water at lower doses of 0, 50, 500 or 5000 mg/L (0, 4, 35 or 350 mg/kg bw per day) as part of a 90-day drinking water study (Mather et al., 1990).
In a 3-month gavage study (Katz et al., 1981), DCA (sodium salt) was administered to groups of male and female Sprague-Dawley rats (10 per sex per dose) at dose levels of 0, 125, 500 or 2000 mg/kg bw per day. An additional five rats per sex dosed with 0 or 2000 mg/kg bw per day were allowed a recovery period of 4 weeks. No adverse reproductive effects were observed in female rats, whereas adverse effects were observed in males only at the two highest doses. Testicular germinal epithelial degeneration was observed in 40% of mid-dose males and 100% of high-dose males. High-dose males showed aspermatogenesis and formation of syncytial giant cells in the germinal epithelium, whereas no spermatozoa were seen in the epididymis ducts. Syncytial giant cells were also seen in 20% of males from the mid-dose group. In the high-dose recovery group (n = 5), half of the males showed germinal epithelium regeneration, 75% were aspermatogenic and all showed loss of germinal epithelium.
As part of another 90-day study (Cicmanec et al., 1991), 4-month-old male and female beagle dogs (five per sex per dose) received DCA (neutralized) in gelatin capsules at 0, 12.5, 39.5 or 72 mg/kg bw daily. No significant weight changes were seen in the ovaries or the testes compared with controls. No effects of DCA on uterine histopathology in treated females were seen. Testicular changes (syncytial giant cell formation and degeneration of testicular germinal epithelium) were observed in all treated males, and prostatic glandular atrophy was observed in the mid- and high-dose males. A dose-related increase in severity of testicular lesions were seen at the mid- and high-dose groups.
In a 13-week subchronic study (Katz et al., 1981), similar testicular effects were observed in 10- to 12-month-old male beagle dogs (3-4 per dose) administered DCA (sodium salt) in capsules at doses of 0, 50, 75 or 100 mg/kg bw per day. Testicular changes in the germinal epithelium (degeneration of germinal epithelium, vacuolation of Leydig cells and formation of syncytial giant cells) as well as prostate gland atrophy were seen in all treated males. One male from the high-dose group was allowed to recover for 4 weeks post-treatment; the prostate appeared normal, and there was evidence of germinal epithelium regeneration with spermatogenesis.
DeAngelo et al. (1996) reported, as part of a modified carcinogenesis bioassay with male F344 rats, that testicular and relative testicular weights were slightly increased in the mid-dose group (500 mg/L), but absolute testes weights were decreased in the high-dose group (1600 mg/L).
10.5.3 Trichloroacetic acid
No reproductive studies were identified
In a developmental study, pregnant Long-Evans rats (n = 20-26) were treated with TCA (neutralized) at 0, 330, 800, 1200 or 1800 mg/kg bw per day (as sodium salt) by gavage on gestation days 6-15. No treatment-related maternal deaths were observed during the study. A dose-dependent increase in the frequency of resorptions per litter (34%, 62% and 90% of implants resorbed at dose levels of 800, 1200 and 1800 mg/kg bw per day, respectively) was observed at maternally toxic doses (decreased weight gain and dose-related increases in spleen and kidney weights). At all dose levels, a dose-related reduction in fetal weight and length as well as a dose-related increase in the frequency of soft tissue anomalies (from 9% at 330 mg/kg bw per day to 97% at 1800 mg/kg bw per day) were seen. Soft tissue anomalies were found mostly in the cardiovascular system and consisted of interventricular septal defects and laevocardia; however, the authors reported that this strain of rats is somewhat susceptible to laevocardia. At the two highest doses, an increase in skeletal malformations (principally in the orbit) was also observed. The maternal LOAEL is considered to be 330 mg/kg bw per day based on weight changes in the spleen and kidney. The developmental LOAEL is considered to be 330 mg/kg bw per day based on an increase in the frequency of soft tissue anomalies (Smith et al., 1989). No NOAEL was determined; however, a benchmark dose of 218 mg/kg bw per day was calculated for developmental toxicity (Health Canada, 2004b) for purposes of comparison with other end-points seen with TCA.
Previous developmental studies with TCE have shown an increase in congenital cardiac lesions in rats; however, it was suggested that metabolites of TCE were possibly responsible for these effects (Johnson et al., 1998). As a result, researchers conducted developmental studies with several metabolites of TCE, including TCA, to identify the responsible metabolites. The two studies that included TCA are described below but are inconclusive for the observed heart defects, since they used different methodologies and obtained conflicting results.
In a study by Johnson et al. (1998), pregnant Sprague-Dawley rats were given TCA (neutralized) in drinking water at concentrations of 0 mg/L (n = 55) or 2730 mg/L (n = 11) (0 or 290 mg/kg bw per day) on gestation days 1-22. A significant decrease in body weight gain was observed in treated dams, relative to controls. Developmental effects included a statistically significant increase in the number of resorptions, in the number of implantation sites and in cardiac soft tissue malformations (10.53% versus 2.15% for controls).
Fisher et al. (2001) gavaged a group of 19 pregnant female Sprague-Dawley rats with TCA (neutralized) at 300 mg/kg bw per day during gestation days 6-15. A control group (n = 19) was dosed with water. Mean maternal body weight gain was significantly less than controls on gestation days 7-15 and 18-21. A statistically significant reduction in fetal body weight (on a per fetus basis and on a per litter basis) was seen in the treated group. No malformations of the heart were seen at 300 mg/kg bw per day, contrary to those reported in the previous study by Smith et al. (1989).
10.5.4 Monobromoacetic acid
No well-conducted developmental studies were done with MBA. Randall et al. (1991) published an abstract on a study with MBA (acid) in Long-Evans rats suggesting possible soft tissue malformations in pups in the presence of maternal toxicity; however, the final study was never published.
No adverse reproductive effects were observed in male Sprague-Dawley rats when administered MBA (neutralized) at 0 or 100 mg/kg bw in a single dose or at 0 or 25 mg/kg bw per day for 14 days (Linder et al., 1994a).
10.5.5 Dibromoacetic acid
No effect on early pregnancy was seen in groups of mature female Holtzman rats (eight per dose per experiment) when gavaged with DBA (neutralized) at 0, 62.5, 125 or 250 mg/kg bw per day during gestation days 1-8. A group of dams was sacrificed on gestation day 9 or 20. The only effect, an increase in serum 17ß -estradiol at the highest dose, was seen in dams sacrificed on gestation day 9 (Cummings and Hedge, 1998).
No adequate developmental studies were conducted. However, two developmental screening assays (oral gavage with neutralized DBA) using CD-1 mice were published in abstracts by Narotsky et al. (1996, 1997). Although these studies suggested developmental effects in the presence/absence of maternal toxicity, no statistical data were provided in either abstract, and no final study was published to confirm these results.
Dose-related alterations of estrous cyclicity in female Sprague-Dawley rats were observed at doses of 90 and 270 mg/kg bw per day when rats were dosed for 14 consecutive days in drinking water with DBA (neutralized) (Balchak et al., 2000), but no alterations were seen at lower doses (10 or 30 mg/kg bw per day) in the same study or during a 20-week exposure to a dose of 5, 16 or 33 mg/kg bw per day (Murr and Goldman, 2005).
In a two-generation drinking water study, groups of Sprague-Dawley rats (30 per sex per dose) were treated with DBA (97% purity in deionized water) continuously via the drinking water at concentrations of 0, 50, 250 or 650 mg/L (equivalent to 0, 4.4-11.6, 22.4-55.6 and 52.4-132.0 mg/kg bw per day, respectively) (Christian et al., 2002). Doses were determined based on a range-finding reproductive/developmental study by Christian et al. (2001). Reduced water consumption was seen at all dose levels of the parental (P) and F1 generations. In the top dose group of the P and F1 generations, clinical signs associated with reduced water consumption, reduced body weights and weight gains and reduced food consumption were observed. Food consumption was also decreased in the F1 mid-dose group. Body weight decreases were also seen with all doses of the F1 generation during lactation; as a result, weaning was delayed until day 29. Small delays in sexual maturation (preputial separation, vaginal patency) at the top dose in F1 rats and a significant decrease in anogenital distance on lactation day 22 in F2 male pups at the middle and high doses were also attributed to a general retardation of growth associated with a significant reduction in body weight. Reproductive performance and development of female rats were unaffected at all doses in both P and F1 generations. In males, sperm motility, count and density and abnormal sperm were also unaffected by treatment. However, histopathology of the reproductive organs of P and F1 males in the mid- and high-dose groups revealed altered sperm production (a dose-related increase in retained step 19 spermatids in Stage IX and X tubules, and the presence of abnormal residual bodies in affected seminiferous tubules) and some epididymal tubule changes. Reproductive effects were seen in high-dose F1 males: a significant increase in the unilateral malformation of the reproductive tract (small or absent epididymides, and small testis) was seen. The authors identified a parental NOAEL for general toxicity of 50 mg/L (4.4-11.6 mg/kg bw per day) based on an increase in absolute and relative kidney and liver weights in the absence of histopathology. A U.S. EPA draft report (U.S. EPA, 2005a) has proposed a reproductive/developmental LOAEL of 250 mg/L (or 22.4-55.6 mg/kg bw per day) based on abnormal spermatogenesis in P and F1 males and a reproductive/developmental NOAEL at 50 mg/L (4.4-11.6 mg/kg bw per day).
Testicular effects were observed in a 13-week drinking water study (Melnick et al., 2007; NTP, 2007) in which male B6C3F1 mice and F344 rats were exposed to DBA (neutralized to pH 5) at doses of 0, 125, 250, 500, 1000 or 2000 mg/L (equivalent to 0, 16, 30, 56, 115 and 230 mg/kg bw per day for mice, and 0, 10, 20, 40, 90 and 166 mg/kg bw per day for rats). Testicular atrophy of the germinal epithelium as well as significant reductions in testicular weight, sperm motility and sperm concentrations were observed in high-dose male rats, along with a significant increase in hypospermia. Delayed spermiation with atypical residual bodies was observed at the two highest doses in male mice and only in the middle doses (500 and 1000 mg/L) in rats. The lowest dose showing testicular effects in rats is 500 mg/L. NTP, (2007) reported a NOEL for testicular lesions of 250 mg/L in rats. Similar effects (such as decreased testis weights, delayed spermiation or atypical residual bodies) were observed when rats (1000 mg/L and above) and mice (500 mg/L and above) were dosed for two weeks (Melnick et al., 2007; NTP, 2007).
Spermatotoxic/testicular effects consisting of testicular atrophy and sperm alteration (motility, morphology, count, fused or abnormal sperm, increased retention of step 19 spermatids, atypical residual bodies) were also observed in Sprague-Dawley rats given single or repeated oral doses of TCA (neutralized or acid) (Linder et al., 1994a, b, 1995; Vetter et al., 1998; Tsuchiya et al., 2000; Holmes et al., 2001).
Reproductive ability was compromised in male rats when gavaged with DBA (neutralized) (Linder et al., 1995). Abnormal sperm morphology, decreased sperm counts and motility and behaviour changes (during mating) in the males were noted.
Preliminary results of recently published abstracts (Klinefelter et al., 2000; Veeramachaneni et al., 2000; Bodensteiner et al., 2001; Veeramacheneni, 2002) suggest disruption in pubertal development, reproductive function, spermatogenesis and fertility in male rats and/or rabbits and a reduction of the population of primordial follicles in female rabbits when DBA (neutralized) is administered with the first exposure in utero from gestation day 15 throughout life. No final study was published to confirm these results.
