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Toxicity to reproduction

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Administrative data

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

Data source

Reference
Reference Type:
publication
Title:
Reproductive toxicity of a mixture of regulated drinking-water disinfection by-products in a multigenerational rat bioassay
Author:
Narotsky MG, Klinefelter GR, Goldman JM, DeAngelo AB, Best DS, McDonald A, Strader LF, Murr AS, Suarez JD, George MH, Hunter III ES,
Simmons JE
Year:
2015
Bibliographic source:
Environ. Health Persp. 123(6), 564-570

Materials and methods

Test guideline
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.

Test material

Constituent 1
Chemical structure
Reference substance name:
Chloroacetic acid
EC Number:
201-178-4
EC Name:
Chloroacetic acid
Cas Number:
79-11-8
Molecular formula:
C2H3ClO2
IUPAC Name:
2-Chloro-ethanoic acid
Details on test material:
lot no. 05715PC
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.

Test animals

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.

Administration / exposure

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
Doses / concentrationsopen allclose all
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

Examinations

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.

Results and discussion

Results: P0 (first parental generation)

Details on results (P0)

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.

Effect levels (P0)

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

Results: P1 (second parental generation)

Details on results (P1)

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.

Effect levels (P1)

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

Results: F1 generation

Details on results (F1)

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.

Effect levels (F1)

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

Results: F2 generation

Details on results (F2)

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.

Effect levels (F2)

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)

Overall reproductive toxicity

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

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.

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