Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Monochloroacetic acid (MCA) is a strong acid and dissociates in biological media; many studies have used monochloroacetic acid neutralized with sodium hydroxide or its sodium salt, so that data of monochloroacetate can be used to also evaluate the genotoxicity for sodium monochloroacetic acid (and vice versa). In a dermal toxicity study (see section 7.2.3) with rabbit skin it was shown, in addition, that the toxicity of MCA is not caused by a surplus of H+ ions and that the influence of pH appeared to be secondary, because its direct reaction to biomolecules was the primary cause of cytotoxicity.

Link to relevant study records
Reference
Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
Sprague-Dawley rats were exposed (parental, F1, and F2 generations) from gestation day 0 of the parental generation to postnatal day (PND) 6 of the F2 generation to a realistically proportioned mixture of THMs and HAAs at 0, 500×, 1,000×, or 2,000× of the
U.S. Environmental Protection Agency’s maximum contaminant levels (MCLs).
GLP compliance:
not specified
Remarks:
Study was carried out by the National Health and Environmental Effects Research Laboratory, U.S. EPA, Research Triangle Park, North Carolina; however, in a publication it is seldomly noted whether a study has been carried out according to GLP or not.
Limit test:
no
Justification for study design:
Trihalomethanes (THMs) and haloacetic acids (HAAs) are regulated disinfection by-products (DBPs); their joint reproductive toxicity in drinking water is unknown.
Specific details on test material used for the study:
MCA was part of the concentrated drinking water (see Table 1 at illustrations). THM = Trihalomethanes; HAA = haloacetic acids (including MCA)
Chloroacetic acid (lot no. 05715PC, purity 99%)
Mixtures were prepared twice weekly. Prior to addition of THMs to the dosing solutions, pH was adjusted to values of 6–7 using sodium hydroxide. Dosing formulations not immediately placed on cages were stored at 4°C in light-protected polyethylene carboys.
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
Timed-pregnant Sprague-Dawley rats (Charles River Laboratories, Raleigh, NC) were obtained on gestation day (GD) 0. GD 0 was defined as the day that evidence of mating (copulatory plug or vaginal sperm) was detected. The dams, weighing 165–245 g and 10–14 weeks of age, were housed individually in polycarbonate cages. At weaning, male progeny were housed two per cage and females were housed three per cage. Dams and weanlings were uniquely identified with eartags and provided heat-treated pine shavings for bedding.
Animal rooms were maintained on a 12/12-hr light/dark cycle (lights on at 0600 hours). Room temperature and relative humidity were maintained at 22.2 ± 1.1°C and 50 ± 10%, respectively. Feed (Formulab Diet 5008; PMI® LabDiet®) and drinking water were provided ad libitum. Water was provided in amber glass bottles with Teflon®-lined caps and stainless steel sipper tubes equipped with stainless steel ball bearings. Animals used in this study were treated humanely and with regard for the alleviation of suffering. Procedures were approved by the Institutional Animal Care and Use Committee, and animals were maintained in a facility certified by the American Association for the Accreditation of Laboratory Animal Care.
Route of administration:
oral: drinking water
Vehicle:
water
Details on exposure:
Twenty-five parental (P0) animals were assigned to each treatment group using a nonbiased randomization procedure that assured a homogeneous distribution of body weight. The designated DBP mixture was the sole source of drinking water for the animals in each treatment group. Control animals received vehicle. The dams were exposed to their designated water through the weaning of their litters. The progeny (F1 generation) continued their exposure past puberty and breeding, through gestation of the F1 females, and up to postnatal day (PND) 6 of the F2 litters.
Details on mating procedure:
The study was started with pregnant females.
Following estrous cycle monitoring, each A-female (see further for explanation) was transferred to the cage of a randomly selected non sibling A-male of the same treatment group for up to 14 days. When evidence of mating (copulatory plug or vaginal sperm) was observed (GD0), the female was weighed and singly housed. Similarly, D-males were cohabited with two untreated females for up to 7 days. These females were necropsied on GDs 9–14 to evaluate pregnancy status. Uteri were
examined for the numbers of live and resorbed implantation sites, and ovaries were examined for the number of corpora lutea.
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
Rats were exposed (parental, F1, and F2 generations) from gestation day 0 of the parental generation to postnatal day (PND) 6 of the F2 generation.
Frequency of treatment:
Via drinking water
Dose / conc.:
7.04 mg/L drinking water
Dose / conc.:
14.07 mg/L drinking water
Dose / conc.:
28.15 mg/L drinking water
No. of animals per sex per dose:
24-25 females per dose at start
Control animals:
yes, concurrent vehicle
Details on study design:
F1 weanlings were randomly selected for different roles as the experiment continued. From each litter, one male and one female (designated as “A” animals) were selected for breeding to produce F2 litters. An additional male and female (“B” animals) were selected for examination of serum hormones at puberty; the males were killed on PND55, whereas the females were killed on the day of vaginal opening (VO). A third female (“C”) was killed on the day of estrus for examination of serum hormones. For 10 randomly selected litters per group, one male (“C”) was selected to provide epididymal sperm for in utero insemination, and another male (“D”) was bred to two untreated females.

In utero insemination. C-males, 10/group, were bred to untreated receptive females using artificial insemination. Briefly, within 15 min of sperm diffusion from the proximal cauda epididymis, each uterine horn of the anesthetized recipient female was injected with a volume containing 5 × 106 sperm, a value that results in approximately 75% fertility of control males. A single female was inseminated per male. Nine days later, inseminated females were killed, and corpora lutea (reflecting the number of ovulations) and uterine implantation sites were counted. The fertility of each male was expressed as implants divided by corpora lutea.
Positive control:
no
Parental animals: Observations and examinations:
Body weights were recorded at least twice per week throughout the experiment. Water consumption for each cage was also recorded at least twice per week.
Beginning on GD20, dams were observed periodically to determine the time of parturition. The stage of parturition (completed, in progress, first pup delivered) was also recorded.
Corpora lutea (reflecting the number of ovulations) and uterine implantation sites were counted.
Oestrous cyclicity (parental animals):
For 19 days beginning on PND46 or PND47, daily vaginal smears of A-females were examined microscopically for vaginal cytology. Estrous cycles were classified as regular (4 or 5 days), extended, or abnormal (Goldman et al. 2007). During the third week of this period, PNDs 57–65, C-females were killed on the day of estrus and evaluated for serum levels of estradiol and progesterone. Selected C-females, 10–11/group, were also evaluated for the number of released oocytes; oviducts were excised and oocytes were flushed and counted.
Sperm parameters (parental animals):
Cauda epididymal sperm motility and morphology were evaluated as described previously (Klinefelter et al. 2002) in adult (PNDs 89–93) males. In males assessed for fertility by artificial insemination (PNDs 96–100), SP22, a sperm membrane protein and biomarker of fertility (Klinefelter 2008), was quantified using an enzyme-linked immunosorbent assay (ELISA).
Litter observations:
F1 litters were examined on PNDs 0 (day of birth), 6, 13, 21, and 26. F2 litters were examined on PNDs 0 and 6. On PND0, pups were examined for evidence of nursing (i.e., abdominal milk bands) and were sexed, counted, and weighed. In addition, 15 F1 litters each from the control group and the high-dose group (2,000×) were selected randomly, and the anogenital distance (AGD) of each pup was measured. On PND6, pups were again sexed, counted, and weighed. F1 litter sizes were reduced on PND6 to a maximum of 10 pups (5 males and 5 females when possible). On PND13, each F1 pup was examined for eye opening and nipple retention. On PND21, F1 pups were sexed and weighed. Because of low pup weights in the high-dose group, weaning of all pups was delayed until PND26; at this time pups were sexed, weighed, and weaned.
F1 animals were examined daily for onset of puberty. Females were examined for VO starting on PND27 and were scored as closed, partially open, or fully open. On the day of VO (i.e., when fully open), each female was weighed; B-females were killed by decapitation and sera were collected for assay of progesterone, estradiol, and leptin. Males were examined for preputial separation (PPS) starting on PND34. Males were weighed on the day of PPS and daily on PNDs 41–47. PPS was scored as none, minimal, at least 50%, or complete. On PND55, B-males were weighed and killed, sera were collected for measurement of testosterone, and testes and epididymides were weighed and fixed.
Postmortem examinations (parental animals):
Full necropsies were conducted on P0 females at 26 days post-partum (upon weaning of their litters), on F1 A-males at PNDs 89–93, and on F1 A-females at PNDs 96–104 (after PND6 examinations of the F2 litters). Animals were weighed and killed by decapitation; trunk blood was collected and sera were prepared. Sera were frozen at –80°C for hormone analysis. Cranial, thoracic, abdominal, and pelvic viscera were examined grossly. Organ weights were recorded for the brain, kidneys, spleen, ovaries, testes, thymus, liver, lung, adrenal glands, pituitary gland, uterus with oviducts and cervix, epididymides, prostate, and seminal vesicles with coagulating glands (and fluids). Uterine implantation sites were counted. Uteri from nonparous females were stained with 2% ammonium sulfide to detect cases of full-litter resorption. Tissues from liver, lungs, kidneys, adrenals, thymus, spleen, stomach, duodenum, ileum, cecum, colon (proximal, middle, distal), mesenteric lymph nodes, trachea, esophagus, thyroid, pituitary gland, urinary bladder, prostate, seminal vesicle and coagulating gland, vagina, and ovaries, were fixed in buffered formalin.For males, the left testis and epididymis were fixed in Bouin’s fluid, and the right cauda epididymis was sampled for assessment of sperm motility and morphology.
Histology. For P0 females, F1 A-males, and A-females, fixed tissues from 10 randomly selected rats from the control and high-dose groups were embedded in paraffin blocks, sectioned, stained with hematoxylin and eosin, and examined microscopically. If results from this initial examination suggested a treatment effect, the specified tissue of the remaining rats were also processed and examined. For 10–11 F1 males and females per group, three colon segments were stained with new methylene blue for analysis of aberrant crypt foci.
For P0 and F1 A-females, the ovaries were examined quantitatively for primordial and primary follicles by examining 20 cross sections (5 μm thick) per ovary. Routine histopathological examination of the ovaries was conducted in conjunction with the enumeration of follicles.
Statistics:
Significance level: 0.05 Adjustments were not made for multiple end points. For all developmental and reproductive data, the litter was considered the experimental unit; for example, litter means and frequencies per litter were used as the experimental units for analyzing pup weights and pup examination data. Prenatal loss (the number of implants minus the number of viable pups at PND0), neonatal loss (the number of pups viable on PND0 but not on PND6), and peri
natal loss (the number of implants minus the number of live pups on PND6) were analyzed as percentages of the number of implants (prenatal and peri natal loss) or the number of live pups at PND0 (neonatal loss). Continuous data, counts per litter, and
proportions per litter were evaluated by analysis of variance (ANOVA) using the general linear models (GLM) procedure in SAS, Release 9.1 (SAS Institute Inc.). Proportions per litter (e.g., prenatal loss) underwent arcsine square root transformation
prior to GLM analysis. End points pertaining to survival were analyzed using one-tailed tests. Gestation lengths were analyzed using the Kruskall–Wallis test. Pup weight analysis used the number of live PND0 pups as a covariate. Similarly, analyses of the numbers of live pups used the number of implants as a covariate. Incidences per group (e.g., estrous cycles) were analyzed with Fisher’s exact test to compare each group with controls. AGD was analyzed by analysis of covariance with pup weight as a covariate and litter as a random effect using Proc Mixed (SAS).
Because of the potential bias inherent in the use of birth-based age for assessment of onset of puberty (Narotsky 2011), pubertal data were analyzed using both conception-based age and day-of-birth–based age. Birth-based age was defined as the number of days since birth that PPS or VO were observed, whereas conception-based age was defined as the number of days since GD22 that these landmarks were observed, regardless of the actual day of parturition.
Maternal body weights and water consumption of P0 females were significantly reduced compared with controls throughout gestation and lactation in the group receiving 2,000× water. At 1,000×, body weights were comparable with those of controls but water consumption was significantly reduced intermittently during gestation and consistently throughout lactation. At 500×, body weights and water consumption were comparable to those of controls.
Pregnancy rates were ≥ 96% in all groups, and all dams successfully maintained their pregnancies to term. Gestation lengths were comparable for all groups; all dams delivered on GD21 or GD22, and no abnormalities in parturition were noted.

Full necropsies were conducted on the P0 dams. No gross necropsy findings were attributed to treatment. Histological examinations of tissues from 10 randomly selected P0 rats from the control and 2,000× groups prompted follow-up examinations of adrenals and kidneys of all P0 females. Incidences of nephropathy and, in the adrenal cortex, hypertrophy of the zona glomerulosa and atrophy of the zona reticularis were significantly increased at 2,000× compared with controls, with severity increasing with dose for the adrenal observations. .
Histological examination of ovaries of 10 females each in the control and 2,000× groups of P0 dams revealed comparable numbers of primordial and primary follicles across groups.
Dose descriptor:
NOAEL
Effect level:
14.07 mg/L drinking water
Based on:
test mat.
Sex:
female
Basis for effect level:
other: decrased bw gain and water intake, and kidney and adrenal effects at 28.15 mg/L
During the 14-day breeding period, F1 breeding pairs showed no effects of treatment. One pair at 1,000× failed to mate, whereas all remaining pairs mated, most within the first 4 days of cohabitation. Pregnancy rates were comparable in all groups; all females were pregnant except for two controls and two females at 1,000×. All F1 dams delivered on GD21 or GD22, and gestation lengths were comparable across groups. No abnormalities in parturition were noted for the F1 dams.

Ten F1 males from each group were each cohabited with two untreated females for up to 7 days. All males mated with at least one female. The incidences of males mating, impregnating females, and siring live litters were comparable in all groups Midgestation examination of the females revealed comparable numbers of corpora lutea, implantation sites, live embryos, and resorption sites for all groups, as well as comparable attrition rates both pre- and postimplantation.

Ten F1 males from each group provided cauda epididymal sperm samples to inseminate untreated females in utero. There were nonsignificant increases in preimplantation loss and the incidence of infertile males increasing dose. Sperm from these males showed nonsignificant (p ≤ 0.0567) reductions in SP22, a sperm protein biomarker of fertility, at 500× and 1,000×, whereas values at 2,000× were comparable to controls.
Evaluations of sperm motion in adult F1 males indicated no effect on the percentage of motile sperm; however, compromised forward motion at 2,000× was observed, including a significant increase in beat cross frequency (rate of crossing the average path trajectory) and decreases in straightness (linearity of the spatial average path) and linearity (linearity of the curvilinear trajectory).
Full necropsies were conducted on the F1 males and females that were used for breeding. No gross necropsy findings were attributed to treatment. For F1 males, absolute—but not relative—organ weights for brain, pituitary, liver, kidneys, adrenals, thymus, and spleen were reduced at 2,000×, and for thymus at 500×. For F1 females, absolute adrenal and liver weights were reduced in the high-dose group and relative kidney weights were increased at 1,000× and 2,000×. For F1 animals, histological examinations of 10 randomly selected males and females of the control and 2,000× groups revealed no findings attributed to treatment. For 10–11 F1 animals per sex per group, colon samples (proximal, medial, and distal) were examined histologically for aberrant crypt foci; none were observed.
Histological examination of ovaries of 10 females each in the control and 2,000× groups of F1 dams revealed comparable numbers of primordial and primary follicles across groups.
Dose descriptor:
NOAEL
Effect level:
14.07 mg/L drinking water
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: effects on sperm forward motion at 28.15 mg/L
Body weights and water consumption of F1 males and females postweaning were significantly reduced at 2,000×. At lower concentrations, female body weights were comparable to controls, whereas male body weights at 1,000× were signifi-
cantly reduced only 1 week postweaning. Water consumption, however, was significantly reduced for both males and females at 1,000×. At 500×, female water consumption was reduced compared with controls at most intervals, but male consumption was significantly reduced only during the PND 55–58 interval.
No pup malformations were observed at any of the F1 litter examinations, and no treatment effects on viability were evident. The numbers of uterine implantation sites were comparable across all groups, as were the numbers of pups at each post-
natal examination. Attrition rates (i.e., prenatal loss, postnatal loss) were unaffected by treatment. Although pup weights were comparable between groups at PND0, at all subsequent litter examinations pup weights were significantly reduced in the 2,000× group. In the 1,000× group, pup weight reductions were significant in males at PND26 (weaning) and in females at PNDs 21 and 26. Pup weights at 500× were unaffected at all litter examinations.
All pups in 15 litters from the control and high-dose groups were each examined for AGD at the PND-0 examination. Values were comparable between the groups for both male and female progeny.
On PND13, F1 pups were examined for eye opening and nipple retention. The incidences of pups with both eyes open, or both eyes closed, were comparable for all groups.
Males that still had nipples were observed only in the treated groups. The mean ± SE percents affected per litter were 3.2 ± 1.9, 1.0 ± 1.0, and 6.0 ± 2.3 at 500×, 1,000×, and 2,000×, respectively. The incidence at 2,000× was significantly different from controls. For females, less-than-prominent nipples were observed only at 2,000×, but this incidence did not reach significance.
Onset of male puberty, indicated by the day of PPS, was significantly delayed at 1,000× and 2,000×, whereas a nonsignificant delay (p = 0.0588) was noted at 500×. Compared with controls, PPS was delayed 1.2, 2.8, and 5.7 days at 500×, 1,000×, and 2,000×, respectively. Body weights on the day of PPS were reduced only at 2,000×.
Hormone measurements from males necropsied on PND55 (when most males have reached puberty) revealed comparable serum testosterone levels across groups; however, significantly reduced concentrations of testosterone (< 50% of
control) were observed in the testicular interstitial fluid at 2,000×. For the females, onset of puberty, as indicated by the day of VO, was significantly delayed at 1,000× and 2,000×. Compared with controls, VO delays were 0.9, 1.4, and 5.8 days at 500×, 1,000×, and 2,000×, respectively. Body weights on the day of VO were comparable across groups Serum samples obtained on the day of VO revealed comparable levels of leptin and estradiol across groups, but progesterone levels were significantly reduced (~ 50% of control values) at 1,000× and 2,000× compared with controls. Leptin, an adipose hormone important in regulating food intake and metabolism and with a permissive role in the onset of puberty (Sanchez-Garrido and
Tena-Sempere 2013), was significantly correlated with body weight at 1,000× (R2 = 0.236, p < 0.05) and 2,000× (R2 = 0.457, p < 0.001).
Examination of vaginal cytology of two F1 females per litter for 19 days revealed regular 4- or 5-day cycles for all females except for females from one, two, four, and five litters at 0, 500×, 1,000×, and 2,000×, respectively. Among animals with irregular cycles, extended/abnormal diestrus was observed in two control littermates, two females (non-littermates) at 500×, five females (four litters) at 1,000×, and three females (three litters) at 2,000×. Extended estrus was observed in two
females (two litters) at 2,000×. All incidences were comparable across groups. For those females exhibiting regular estrous cycles, serum concentrations of progesterone and estradiol on the day of estrus were comparable across groups, as were the numbers of oocytes obtained from flushed oviducts.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
7.04 mg/L drinking water
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Reduced pup weight at 14 and 28 mg/L, reduced BW and water intake at 28 mg/L and effects on nipple retention, PPS, VO, testosterone and progesterone at 14 and/or 28 mg/L
Examination of F2 litters on PNDs 0 and 6 showed no treatment effects. The numbers of implantation sites and live pups at each examination were comparable between controls and treated litters. Except for one control litter, all litters survived to PND6. Attrition rates per litter (prenatal loss, neonatal loss) and pup weights were comparable between groups. AGD, examined in the control and 2,000× groups on PND0, showed no differences between groups for either males or females.
Except for filamentous tail observed in one control male, no malformations were observed in any of the F2 pups.
Dose descriptor:
NOAEL
Generation:
F2
Effect level:
>= 28.15 mg/L drinking water
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment-related effects at 28.15 mg/L (highest dose tested)
Reproductive effects observed:
yes
Lowest effective dose / conc.:
14.07 mg/L drinking water
Treatment related:
not specified
Relation to other toxic effects:
not specified
Dose response relationship:
yes
Relevant for humans:
no

The mixtures contained 7.04, 14.07 or 28.15 mg/L MCA. With an average drinking water intake of ca. 50 g (50 mL) per day, and an average BW of 250 g this would correspond to ca. 1.4, 2.8 or 5.6 mg MCA/kg bw/day.

Conclusions:
A mixture of the regulated THMs and HAAs (including MCA) at concentrations 500× greater than regulatory MCLs (Maximum Contaminant Level) had no adverse effects; furthermore, 2,000×, the highest concentration evaluated, did not affect the animals’ ability to reproduce. The lack of effects on prenatal survival and birth weight in this study contrast with associations reported in some epidemiological studies (e.g. epidemiological associations reported for low birth weight and spontaneous abortion in humans exposed to chlorinated water (Levallois et al. 2012; Niewenhuijsen et al. 2009)). Although reproduction per se was unaffected, retained nipples and sperm motility effects in males at 2,000× and pubertal delays in both sexes at ≥ 1,000× the regulatory MCLs indicate that a mixture of these regulated DBPs (Disinfection By-Products) can influence endocrine physiology; however, these findings may have been secondary to reduced water consumption and body weight (viz. delayed puberty at ≥ 1,000× may have been secondary to reduced water consumption. Male nipple retention and compromised sperm motility at 2,000× may have been secondary to reduced body weights). Moreover, it is not clear which chemical(s) of the mixture would be responsible for the effect(s) observed. According to the authors, in toxicity tests with individual chemicals, two regulated DBPs (dibromoacetic acid at 400 mg/L, bromodichloromethane at 150 mg/L) have been shown to delay puberty in rats (Christian et al. 2002a, 2002b; Klinefelter et al. 2004) and may have contributed to the effects seen here. Dibromoacetic acid has also been shown to reduce sperm quality in rats at 40 mg/L (Klinefelter et al. 2004) or 2 mg/kg by gavage (Kaydos et al. 2004; Klinefelter et al. 2004) and rabbits at 2 mg/kg in drinking water (Veeramachaneni et al. 2007). In vitro, dibromoacetic acid has been shown to decrease progesterone secretion in newly matured ovarian follicles (Goldman and Murr 2002) and may have contributed to the reduced pubertal progesterone levels observed here.
As the low dose of 500x MCL was a NOAEL, it can be concluded that drinking water containing 7.04 mg/L MCA (besides all other constituents) was without effects; this would correspond to ca. 1.4 mg/kg bw/day. It may even concluded that this level might be 4 times higher (viz. 5.6 mg/kg bw) because at 2000x there were no effects on reproduction and the effects observed (retained nipples and sperm motility effects in males at 2000× and pubertal delays in both sexes at >= 1000x) may have been secondary to reduced water consumption and body weight. Moreover, this level is higher than the NOAEL observed in a carcinogenicity study.
Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

There is a 2 -gen reproductive study of MCA available in rats as part of a study investigating reproduction toxicity of DPB (disinfection by-products). It was concluded that MCA did no induce effects on reproduction at the levels tested. Moreover, in the 90 -day studies in mice and rats no effects on the reproductive organs were observed.

The growth of mouse antral follicles and the concentration of estradiol in the medium by incubation with 0.25 to 1 mM MCA decreased, which was interpreted as toxicity to the ovaries of mice (Jeong et al. 2016). These concentrations also showed cytotoxicity in the in vitro genotoxicity studies.

Effects on developmental toxicity

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Principles of method if other than guideline:
Pregnant rats were dosed via drinking water from 1 day of pregnancy throughout pregnancy (21 days).
GLP compliance:
not specified
Limit test:
yes
Specific details on test material used for the study:
Not provided
Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
This study was conducted in Association for Assessment and Accreditation of Laboratory Animal Care accredited and Institutional Animal Care and Use Committee governed facilities at the University of Arizona Animal Care Center. Animals were quarantined for 7 days before study. Study groups consisted of virus free, young, sexually mature Hsd:Sprague Dawley SD rats (Harlan Sprague Dawley, Inc., Indianapolis, Indiana). Females, 225 +/- 30 g, were housed in pens of four, and the males, 300 +/- 50 g, were housed individually. All rats had access to water and Teklad 4% Mouse Rat diet (Teklad, Madison, Wisconsin) ad libitum. Each animal was identified by an ear notch code. The number of animals in each group was determined by a power calculation to detect a threefold increase in the malformations over controls.
Route of administration:
oral: drinking water
Vehicle:
water
Details on exposure:
Control animals for this study received distilled water throughout pregnancy. On day 1 of pregnancy, and continuing throughout pregnancy, their regular drinking water was replaced with treated water. Compounds tested included TCAA, MCAA, TCEth, CMC, TCAld, DCAld and DCVC. Solutions of the various compounds were prepared by dilution with distilled water and, if necessary, titrated with NaOH to a pH of approximately 7.0, a pH similar to that of the control water. Each water bottle was placed in a specially made metal casing to reduce light exposure and subsequent chemical breakdown. The amount of water consumed by each pen of animals (4 maximum) was monitored and recorded every 24 h. Bottles were cleaned and fresh solutions prepared daily. Levels of metabolites were based on the dosage equivalent to that expected if all of the high dose of TCE (1,100 ppm, the limit of solubility, and the maximal TCE dose tested) was to breakdown completely into that given metabolite. To achieve uniformity between the tri- and monochloroacetic acids, MCAA was also given at an equivalent dose.
Analytical verification of doses or concentrations:
not specified
Details on mating procedure:
Daily vaginal smears or impedance measurements (Estrous Impedance Monitor, Fine Scientific Instruments, Inc., Phoenix, Arizona) were obtained from all females to determine stages of the estrous cycle. When in proestrus, female rats were placed in a cage with one male overnight. The presence of a vaginal plug and/or spermatozoa in the vaginal smear the following morning was considered indicative of day 1 of pregnancy.
Duration of treatment / exposure:
Whole pregnancy period (21 days)
Frequency of treatment:
Continuously (via drinking water)
Duration of test:
Whole pregnancy period (21 days)
Dose / conc.:
1 570 mg/L drinking water
Remarks:
corresponds to 193 mg/kg bw/day
No. of animals per sex per dose:
10 rats in test group, 55 rats in control group
Control animals:
yes, concurrent vehicle
Details on study design:
For all groups, on day 22 of gestation, approximately 1 day before parturition, each pregnant rat was weighed before euthanasia in a carbon dioxide chamber. An examination was then conducted for any abnormalities (external and internal) and the gravid uterus and ovaries removed. The uterus was opened, exposing all fetuses, implantation sites (sites where the embryo implanted in the uterus, but did not mature beyond implantation, leaving only a metrial gland) and resorption sites (sites where fetal development began, but stopped at some point during gestation with only decaying fetal tissue remaining). The position of each site was recorded. Fetuses and placentas were examined in situ, then removed and individually examined externally for any morphologic abnormalities. All fetal placements, weights, placental weights, crown rump (C/R) lengths and any gross fetal abnormalities were evaluated by an experienced veterinarian.
Maternal examinations:
Each rat was carefully observed throughout pregnancy and weight gain was monitored and recorded daily.
Ovaries and uterine content:
The uterus was opened, exposing all fetuses, implantation sites (sites where the embryo implanted in the uterus, but did not mature beyond implantation, leaving only a metrial gland) and resorption sites (sites where fetal development began, but stopped at some point during gestation with only decaying fetal tissue remaining). The position of each site was recorded. Placentas were examined in situ. Pplacental weights were evaluated by an experienced veterinarian.
Fetal examinations:
Fetuses were examined in situ, then removed and individually examined externally for any morphologic abnormalities. All fetal placements, weights, crown rump (C/R) lengths and any gross fetal abnormalities were evaluated by an experienced veterinarian. Using an Optivisor (Donegan Optical Co., Inc., St. Lenexa, Kansas) for magnification, the thoracic and abdominal cavities were opened. All abdominal organs were inspected for any congenital abnormalities. Exposing the thoracic cavity allowed observation of the great arterial and venous connections to the heart in situ. Pulmonary and vena caval attachments were then incised, as distal to the heart as possible, and the heart removed. A 27-gauge needle was placed apically in the left
ventricle and the heart gently flushed with 2% glutaraldehyde solution. Each heart was then placed in an individual vial that was labeled with a seven digit code (for future “blind” assessment) and placed in the same solution for 24-h fixation. The
heart was then transferred to a 0.1 mol/L phosphate buffer solution for storage.
Individual hearts were dissected and evaluated using a Nikon SMZ-2T light microscope with an attached TV camera and monitor (Nikon, Chandler, Arizona). This allowed excellent visualization and manipulation. Initially, the heart was examined for any gross morphologic abnormalities from both dorsal and ventral aspects. The heart was then examined in a step by step protocol which is detailed by Dawson et al. (10). This method allows visualization of the atrial septum, aortic and pulmonary vessels, semi-lunar and atrioventricular valves and the ventricular septum. All confirmed abnormalities were agreed upon by the three investigators: a veterinarian, a pathologist and a pediatric cardiologist.
All abnormal specimens were then photographed using a Nikon N2020 camera mounted on the light microscope. Decoding of the hearts, with respect to treatment, occurred only after final examination of all hearts and fetuses.
Statistics:
For an effect size of a threefold increase over background heart defects, a power analysis of 90%, with an alpha error of 0.05 and a beta error of 0.1, determined that a sample size of 100 was needed for statistical significance.
Statistics for the individual fetal data were analyzed by using Fisher’s exact test. The Wilcoxon and exact permutation tests were used to determine the significance of the litter outcome.
Indices:
No info
Clinical signs:
no effects observed
Description (incidence and severity):
All maternal rats were healthy throughout the study and without evidence of toxicity.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Steady weight gain occurred throughout pregnancy in bot the control and MCA group.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Average amount of the control group was 46 mL/day; for the MCA group it was 21 mL/day.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
no effects observed
Description (incidence and severity):
Ovaries had normal morphologic features. All uterine morphological examinations were normal.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not specified
Details on results:
There were no pregnancy complications.
Number of abortions:
not specified
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
effects observed, non-treatment-related
Description (incidence and severity):
One dead fetus in the MCA group, 3 dead fetuses in the control group.
Changes in pregnancy duration:
not specified
Changes in number of pregnant:
no effects observed
Details on maternal toxic effects:
The uterus was examined for implantation sites, resorption sites, and live and dead fetuses. External inspection of live fetuses (n = 602 in control group and n = 131 in MCA group) and dead fetuses (n = 3 in control groups versus n = 1 in MCA group) demonstrated no gross morphologic congenital abnormalities in any group. No differences were found between the treated group and the control for the mean number of implantation sites and resorption sites using Fisher’s exact analysis.
Dose descriptor:
NOAEL
Effect level:
>= 193 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: absence of maternal toxicity at this level (only level tested)
Fetal body weight changes:
no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
not specified
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Skeletal malformations:
not examined
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
See below
Dose descriptor:
NOAEL
Effect level:
>= 193 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: absence of increased incidence of heart malformations at this level (only level tested)
Developmental effects observed:
no

Fetuses were analyzed by determining numbers of live or dead fetuses, fetal weight; placental weight, C/R length, and external morphology. No significant difference was found when comparing the treated and control fetal

group for these observations. There were no gross external or noncardiac internal congenital abnormalities found in the treated or control group.

Several different cardiac malformations were found and no one lesion or grouping predominated (see table attached). Variations of normal morphology similar to those found in humans were not classified as defects (for example, tricuspid valve leaflet contribution to complete coverage of a membranous ventricular defect). General types of cardiac defects were grouped in categories and listed by treatment in the table attached. Septation defects appeared to be more represented than a difference between lesions of the left and right sides of the heart.

When examining the fetuses on an individual basis the assumption was made that each fetus had an equal chance of developing a heart malformation independent of its litter mates. Because this may not be the correct assumption, fetal hearts were also evaluated on a per litter basis to determine the significance of any increase in number of cardiac abnormalities. The range of cardiac abnormalities in the MCA group was 4.55% with a control group value of 2.15% (not statistically significantly different).

Conclusions:
MCA at 193 mg/kg bw did not appear to be a specific cardiac teratogen in the fetus when imbibed by the maternal rat.
Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
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

An aqueous monochloroacetic acid solution was used in Long Evans rats from 6-15th day of pregnancy in doses of 0, 17, 35, 70 or 140 mg/kg bw and administered daily by bolus dosing (oral gavage). In the high dose group mothers, there was a reduction in body weight gain; the organ weights were not changed compared to the controls. Only in the fetuses of this dose group malformations of the cardiovascular system, especially the left ventricle, were statistically significantly increased. There were no skeletal malformations (Smith et al. 1990).

Ten Sprague-Dawley rats were given drinking water with 1570 mg/L neutralized MCA throughout their pregnancy, which corresponded to 193 mg/kg bw/day. Neither maternal toxicity nor developmental toxicity, and in particular, no significantly increased incidence of malformations of the heart was detected. The incidence of heart malformations in the control group was 2.15% and 4.55% in the MCA group (Johnson et al., 1998ab). The findings in the study by Smith et al. (1990) could not be reproduced. These may be due to the high bolus peak concentration.

Justification for classification or non-classification

According to MAK (2019): In a study of prenatal toxicity in rats, there was an increased incidence of malformations of the cardiovascular system at the highest gavage dose of 140 mg/kg bw/day MCA (Smith et al. 1990). The NOAEL was 70 mg/kg bw/day. These results could not be reproduced when neutralized MCA was administered with the drinking water at a dose of 193 mg/kg bw. The different toxicokinetics in the two types of application may contribute to the different NOAELs.

For the toxicokinetic transfer of the NOAEL of 70 mg/kg bw to a concentration in workplace air, the following are taken into account: the species-specific correction value (1:4) corresponding to the toxicokinetic difference between rat and humans, the experimentally determined almost complete absorption (98.5%), the body weight (70 kg) and the respiratory volume (10 m3) of humans as well as the assumed 100% inhalation absorption. This results in a corresponding concentration for the workplace of 121 mg/m3, which is approximately 60 times higher than the MAK value of 2 mg/m3 (0.5 ml/m3). As such MCA and its sodium salt are assigned to pregnancy group C. [Group C: There is no reason to fear damage to the embryo or foetus when MAK and BAT values are observed.]

Based on the available 2 -gen study and absence of effects on the reproductive organs in the 90 -day studies in mice and rats no classification is needed for reproductive toxicity.

Based on the absence of effects in the drinking water study using neutralized MCA at a high dose of 193 mg/kg bw throughout pregnancy no classification for developmental toxicity is needed. The cardiovascular effects seen at 140 mg/kg bw using oral gavage may be due to bolus application; the next lower level of 70 mg/kg bw was a NOAEL.

Additional information