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Maternal Exposure to Triclosan Impairs ThyroidHomeostasis and Female Pubertal Development inWistar Rat OffspringPablo E. A. Rodríguez a & Mónica S. Sanchez a ba Subsecretaria CEPROCOR, Ministerio de Ciencia y Tecnología, Córdoba , Argentinab Laboratorio de Neurobiología Celular y Molecular, Instituto Investigación Médica Mercedesy Martín Ferreyra (INIMEC-CONICET) , Cordoba, ArgentinaPublished online: 06 Nov 2010.

To cite this article: Pablo E. A. Rodríguez & Mónica S. Sanchez (2010) Maternal Exposure to Triclosan Impairs ThyroidHomeostasis and Female Pubertal Development in Wistar Rat Offspring, Journal of Toxicology and Environmental Health, PartA: Current Issues, 73:24, 1678-1688, DOI: 10.1080/15287394.2010.516241

To link to this article: http://dx.doi.org/10.1080/15287394.2010.516241

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Journal of Toxicology and Environmental Health, Part A, 73:1678–1688, 2010Copyright © Taylor & Francis Group, LLCISSN: 1528-7394 print / 1087-2620 onlineDOI: 10.1080/15287394.2010.516241

MATERNAL EXPOSURE TO TRICLOSAN IMPAIRS THYROID HOMEOSTASISAND FEMALE PUBERTAL DEVELOPMENT IN WISTAR RAT OFFSPRING

Pablo E. A. Rodríguez1, Mónica S. Sanchez1,2

1Subsecretaria CEPROCOR, Ministerio de Ciencia y Tecnología, Córdoba, Argentina2Laboratorio de Neurobiología Celular y Molecular, Instituto Investigación Médica Mercedes yMartín Ferreyra (INIMEC-CONICET), Cordoba, Argentina

Although the effects of triclosan have been examined in male reproductive functions, itis unknown whether this potent antibacterial agent affects pregnancy and female pubertaldevelopment. Effects of maternal exposure to triclosan on thyroid homeostasis (TH) andreproductive-tract development in female Wistar rats were thus studied. Dams were exposeddaily to triclosan (0, 1, 10, or 50 mg/kg/d) from 8 d before mating to lactation day 21.Offspring were also exposed after weaning. In vivo triclosan estrogenic activity was screenedby uterotrophic assay and vaginal opening (VO), with first estrus and uterus and ovarianweight determined in offspring. Dam blood samples were taken during pregnancy and lac-tation to examine the effect of triclosan on TH. No apparent external signs of toxicity ordifferences in mean numbers of implantation sites were observed in treated rats. Triclosantreatment decreased total serum T4 and T3 in pregnant rats and also lowered sex ratio, low-ered pup body weights on postnatal day (PND) 20, and delayed VO in offspring. In addition,the highest dose of triclosan significantly reduced the live birth index (percentage) and 6-dsurvival index. Data indicate that triclosan impairs thyroid homeostasis and reproductive tox-icity in adult rats and produces fetal toxicity in offspring exposed in utero, during lactation,and after weaning.

Growing populations and the intensifi-cation of land and water use for indus-try and agriculture have increased the needto reuse wastewater, even for supplement-ing the drinking-water supply (Leatherland,2000; Sumpter, 2005). Over the last fourdecades, the impact of chemical pollu-tion focused almost exclusively on conven-tional “priority” pollutants, especially acutelytoxic/carcinogenic pesticides and industrialintermediates, because of their persistence inthe environment. However, other contami-nants including a variety of metals, carcino-genic organic compounds, synthetic chemicals,pharmaceutical, veterinary, and illicit drugs,

Received 24 June 2010; accepted 25 June 2010.We gratefully acknowledge Mr. Luis Valenzuela for his assistance with animal care and Dra. Nancy Salvatierra for statistical assistance.

This study was supported by MINCyT, CEPROCOR, Córdoba, Argentina. We thank Dr Paul Hobson, native speaker, for revision of thearticle.

Address correspondence to Mónica S. Sanchez, Ceprocor, Ministerio de Ciencia y Tecnología, Santa María de Punilla (5164),Argentina. E-mail: monicasilvina.sanchez@cba.gov.ar

cosmetic ingredients, and other personal careproducts and food supplements, together withtheir respective metabolites and transforma-tion products, are present in water supplies(Daughton & Ternes, 1999; Weyer & Riley,2001; Kolpin et al., 2002). All these prod-ucts are of concern because of the potentialrisk of affecting human health by exposureto contaminated drinking water (WHO/IPCS,2002).

Triclosan is an antibacterial agent found inmany detergents, dishwashing liquids, soaps,deodorants, cosmetics, lotions, antimicrobialcreams, and toothpastes, and is an addi-tive in many plastics and textiles. However,

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TRICLOSAN IMPAIRS FEMALE PUBERTAL DEVELOPMENT 1679

the safety of triclosan has come into ques-tion with respect to adverse environmentaland human health. While companies thatmanufacture products containing this chem-ical claim it is safe, the U.S. EnvironmentalProtection Agency (EPA) has registered it as apesticide with adverse consequences (Darbre,2006; Brain et al., 2008). In addition, it iswell established that as a consequence ofthe synthesis of triclosan, several toxic by-products such as polychlorinated dibenzo-p-dioxins (dioxins) and polychlorinated diben-zofurans (furans) are generated as contaminants(Beck et al., 1989; Nilsson et al., 1974). Forthis reason, each batch of triclosan producedneeds to be carefully analyzed for the pres-ence of these pollutants (Menoutis & Parisi,2002).

Some studies showed that triclosan pro-duced disruption of thyroid hormone home-ostasis in the bullfrog (Veldhoen et al., 2006)and rat (Crofton et al., 2007), while other inves-tigations demonstrated that triclosan and itsmetabolites exert a weak estrogenic (Ishibashiet al., 2004) or androgenic activity (Foran et al.,2000) in frogs.

Allmyr et al. (2008) noted that triclosanwas present in human plasma and milk atconcentrations that correlated to the amountfound in personal care products containing tri-closan. Although the toxicity of triclosan tomammals appears to be minimal, several invitro studies indicated that triclosan adverselyaffected metabolic processes and hormonalhomeostasis (Hanioka et al., 1996; Schuuret al., 1998; Wang et al., 2004; Jacobs et al.,2005; Veldhoen et al., 2006). The aims ofthis study were to determine the influenceof triclosan on the female reproductive tractfollowing exposure during critical periods ofdevelopment, and to examine drug poten-tial effects on thyroid hormone homeostasis.The protocol used was the one recommendedby EDSTAC (1998) for detecting reproduc-tive and developmental effects of endocrinealterations in rat offspring as a result of expo-sure of dams during gestation and lacta-tion, or by direct exposure of offspring afterweaning.

MATERIALS AND METHODS

Chemicals and DosesTriclosan (5-chloro-2-(2,4-dichloro-

phenoxy)phenol), of 99.6% purity, wasobtained from Aldrich Chemical Company(St Louis, MO). Concentrated triclosan stocksolutions were prepared weekly at high pH(1 M NaOH) (Greenman et al., 1997; Parikhet al., 2000), and were subsequently diluteddaily to an appropriate volume with deionizedwater and neutralized to pH 7 using 0.1 M HClto obtain the target concentrations of 1, 10, or50 mg/kg/d triclosan for each rat. To preventphotolysis of triclosan, stock solutions werestored in brown glass volumetric flasks andkept refrigerated at 4◦C before use (Songet al., 2007). Triclosan was administered viadrinking water to reduce any additional stresson treated animals. Drinking-water solutionswere prepared fresh daily during pre- andpostnatal studies. The control group receivedwater plus vehicle under the same conditions.Dose levels were selected based on previoustoxicology studies carried out by Crofton et al.(2007).

AnimalsNulliparous female Wistar rats bred at our

laboratory, aged 2–3 mo, weighing 250–270g, were housed under a 12-h light/darkcycle (lights on at 08:00) under temperature-controlled (22–24◦C) conditions. Rat feed(BATISTELLA, Cargill, Cordoba) and tap waterwere available ad libitum. Estrus cycles weremonitored through daily vaginal smears, for 2wk. Females (14 per group) were then rear-ranged and paired according to synchronizedestrus cycles (over 4 d), at which time triclosanexposure began. Eight days following the ini-tial triclosan exposure, females were bred byplacing two receptive females (i.e., late-stageproestrus) with a breeder male (300–350 g)late in the afternoon and removing the femalesthe next day at lights-on. Immediately, animalswere examined for the presence of sperm invaginal smears. When this was not observed, itwas judged that copulation did not occur and

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1680 P. E. A. RODRÍGUEZ AND M. S. SANCHEZ

rats were removed from the test. Subsequently,in order to maintain a constant number of ani-mals in each treatment, the size of each groupwas adjusted to 12 pregnant females randomlyselected for each dose. Following breeding,females were individually housed and main-tained on a high-calorie, high-protein diet for-mulation (BATISTELLA, Cargill, Cordoba), andwere continually exposed to triclosan through-out gestation and lactation.

Experimental GroupsThe experimental protocol is presented

schematically in Figure 1.

DamsTreatment began during estrus, 8 d before

mating. The presence of spermatozoa in vagi-nal smears the morning after being caged witha fertile male on the night of proestrus wasconsidered indicative of pregnancy and wascounted as d 0 of pregnancy. At 2 or 3 d beforedelivery, the rats were housed individually.Dams were weighed daily during gestation and

lactation and subsequently killed by decapita-tion on the day of weaning, and the number ofimplantation sites was determined visually.

Pregnancy OutcomeThe following data were collected: length

of gestation, litter size, sex ratio of each litter(number of male offspring/number of femaleoffspring), pup weight on selected postnatal days(PND 1, 5, 10, 15 and 20), live birth index (num-ber of live offspring × 100/number of offspringdelivered), offspring survival on PND 6 (numberof live offspring on d 6 × 100/number of liveoffspring), and weaning index (number of liveoffspring on d 21 × 100/number of offspringafter standardization). Clinical signs of intoxica-tion such as increased salivation, piloerection,tremors, and seizures were monitored daily.

Standardization of Litter SizesThe size of each litter was adjusted to 6

female pups on PND 6. Natural litters with sixor fewer pups were not standardized. Whenthere were fewer females than necessary for the

• Mother body weight• Tail blood extraction

F0 GD0 GD5 GD10 GD15 GD20 LD5 LD10 LD15 LD20Females Parturition Weaning Uterus Weight

Mating Gestation LactationNecropsy PND 25

Drinking water plus Vehicle o Drinking water plus TRICLOSAN

GD20 PD5 PD10 PD15 PD20

IP Injection Benzoate OestradiolPND (22-24)

IP Injection VehiclePND (22-24)

• Pup´sbodyWeight

Drinking water plus vehicle

Drinking water plus TRICLOSAN

• Dam body weight

• Offspring survival index

• Standarized litters of 6pups

• Pups from large litters were not fostered to smaller litters

F1 Female Uterotrophic assay

F1 Female -Puberal

Drinking water plus vehicle

Drinking water plus TRICLOSAN

• Pregnancy outcome• Dam body weight • Gestation length• Litter size• Sex ratio

• 8 days before Mating

• Vaginal opening• First estrous• Estrous ciclicity• Uterus and ovary

weight

FIGURE 1. Schematic diagram of the experimental protocol.

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TRICLOSAN IMPAIRS FEMALE PUBERTAL DEVELOPMENT 1681

uterotrophic and pubertal assays, a random dis-tribution was preferred for the pubertal assaydue to the longer time of treatment and greaterpossibility of animals dying.

Female OffspringFemale pups were separated for assessment

3 d after weaning (PND 21) (uterotrophic assay)or at puberty. Clinical signs of intoxication weremonitored daily.

Uterotrophic CohortOn PND 21, two females randomly

selected from litters exposed during gesta-tional and lactational periods were screenedfor estrogenic activity by uterotrophic assay.One of these was treated orally for 3 consec-utive days (PND 22–24) with the same treat-ment as its mother, while the other remaineduntreated. As a positive control for estrogenic-ity, 8 female pups from control dams weresubcutaneously (sc) injected with 17β-estradiol(500 μg/kg/d; Sigma Chemical Company,St. Louis, MO) dissolved in corn oil. Positivecontrol rats were injected for 3 consecutivedays and killed by cervical dislocation on themorning d 4, and body weights were deter-mined (Padilla-Banks et al., 2001). After mak-ing an incision in the skin and the abdomi-nal muscle, the uterine cervix was cut awayfrom the vagina fornix at the junction betweenthe uterine horns and ovaries (Odum et al.,1997). Because fluid imbibition is an estrogenresponse, care was taken to retain all uterineluminal fluid. The uteri were then removed bylifting tissue anteriorly and trimming away themesometrium. Wet uterine weight was deter-mined and expressed as relative weight (wetuterine weight × 100/body weight).

Pubertal CohortAt weaning (PND 21), the remaining

females of each litter (4 rats) were randomlydivided into nondosed and dosed groups.Nondosed females were only exposed tovehicle from PND 22 to PND 50. In additionto in utero and lactational exposure, dosed

females were administered triclosan or vehi-cle in drinking water from PND 22 to PND50. From PND 30, all females were exam-ined daily for vaginal opening (VO). Changes invaginal smears were observed from the day ofVO acquisition until necropsy to determine ageand body weight at first estrus. The evaluationof vaginal smears was based upon the follow-ing cytological criteria for staging the estruscycle: proestrus (presence of nucleated epithe-lial cells), estrus (presence of cornified epithelialcells), metestrus (presence of approximatelyequal numbers of leukocytes and epithelialcells), and diestrus (presence of leukocytes)(Everett, 1989). All females were killed bydecapitation at PND 50 and the uterus andovaries were removed and weighed. The uteriwere weighed with their fluids. Organ weightswere expressed as relative weights.

Blood SamplesBlood samples were taken from the tail vein

of triclosan-treated or control rats (n = 6 pergroup) under light ether anesthesia on d 5, 10,15, and 20 of pregnancy (from 11.30 to 13:00h) and on d 2, 5, 10, 15, and 20 of lacta-tion (from 11.30 to 13:00 h) to determine thepattern of T3 and T4 secretion during preg-nancy and lactation. Trunk blood was collectedand serum was separated by centrifugation at1800 × g for 15 min and stored at –70

◦C until

use (Hapon et al., 2003).

Determination of HormoneConcentrations in Rat SerumSerum concentrations of total T3 and T4

were determined using competitive enzymeimmunoassay (EIA) kits (Radim, Pomezia, Italy).All serum samples (without dilution) were mea-sured in duplicate for each assay. Detectionlimits were 0.16 ng/ml for T3 and 4.5 ng/mlfor T4.

Statistical AnalysisData from fetuses were collected as litter

means and expressed as mean ± SEM for thenumber of dams. Data from pups or dams

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1682 P. E. A. RODRÍGUEZ AND M. S. SANCHEZ

TABLE 1. Pregnancy Outcome of Dams Treated With Triclosan During Pregnancy and Lactation

Vehicle Triclosan (mg/kg/d)

Parameter 0 1.0 10.0 50.0

Litters (n) 12 12 12 12Gestation length (d) 21.5 ± 0.8 21.5 ± 0.7 21.8 ± 0.5 22.5 ± 0.4Litter size (n) 11.7 ± 1.2 10.9 ± 0.9 11.8 ± 1.4 11.3 ± 1.1Number of implants 12.5 ± 2.2 11.9 ± 3.7 13.7 ± 3.3 12.7 ± 2.5Sex ratio (male:female) 1.3 ± 0.3 0.6 ± 0.1 (∗) 0.4 ± 0.1 (∗) 0.7 ± 0.1 (∗)Live birth index (%) 100.0 ± 0.0 99.4 ± 0.9 95.2 ± 3.83 86.00 ± 6.9 (∗)6-d Survival index (%) 98.2 ± 2.0 92.4 ± 6.4 85.2 ± 4.5 72.2 ± 10.3 (∗)Weaning index (%) 95.4 ± 5.1 75.8 ± 10.8 83.2 ± 7.5 87.6 ± 6.4

Note. Values are expressed as means ± SD. Asterisk indicates significant difference compared to controls, p < .05.

were collected individually and expressed asmean ± SD for the number of animals. Datawere tested for homogeneity of variance andnormality by Bartlett’s test and the Wilk–Shapiro normality test, respectively. Data wereanalyzed by one- or two-way analysis of vari-ance (ANOVA). Whenever ANOVA indicatedsignificant effects (p ≤ .05), the parametric datawere analyzed using the Newman–Keuls testand nonparametric data by the Kruskal–Wallistest followed by Dunn’s test. The softwareused was InfoStat (2009: InfoStat versión 2009.Grupo InfoStat, FCA, Universidad Nacional deCórdoba, Argentina).

RESULTS

Maternal FindingsDams treated with triclosan showed no

apparent external signs of toxicity. A significantincrease in maternal body weight was notedover the duration of pregnancy, but was notsignificantly different from triclosan treatmentat any dose with respect to control (data notshown). There were no significant differencesin food consumption (g/d) among treated ratscompared to control. Fluid intake was higherduring lactation when the pups began to drinkwater.

Maternal and Reproductive OutcomeDataNo significant differences were observed

between control (14 rats) and treated groups

(14 per group) for pregnancy rate, gesta-tion length, litter size, number of implanta-tion sites, or weaning index (%) (Table 1).However, the highest dose of triclosan signifi-cantly decreased the live birth index (%) and6-d survival index (%). Intrauterine exposureof the fetus produced a marked decrease inthe sex ratio (male:female) at all triclosan doses(1, 10, and 50 mg/kg/d), with the number ofmale offspring significantly lower than num-ber of females (Table 1). Triclosan treatmentreduced pup weight significantly at PND 20in all treated animals with respect to control(Figure 2).

Postnatal days

0

Gra

ms (

X ±

SE

M)

0

5

10

15

20

25

30

35

CONTROL 1 mg/kg/day 10 mg/kg/day 50 mg/kg/day

*

5 10 15 20

FIGURE 2. Female pup body weight means before weaning.Number of animals ranged from 30 to 35 per group. Valuesare expressed as means ± SEM. Asterisk indicates significantdifferences with controls (p < .05).

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TRICLOSAN IMPAIRS FEMALE PUBERTAL DEVELOPMENT 1683

TABLE 2. Sexual Development of Female Rat Offspring Exposed to Triclosan During Pregnancy and Lactation

Vehicle Triclosan (mg/kg/d)

Parameter 0 1.0 10.0 50.0

Number of animals (n) 9 9 9 9Vaginal opening (d) 31.8 ± 0.8 36.6 ± 1.7 (∗) 37.6 ± 2.6 (∗) 35.6 ± 0.9 (∗)Body weight at day of vaginal opening (g) 65.4 ± 3.0 72.0 ± 3.7 (∗) 70.4 ± 5.0 (∗) 71.6 ± 3.1 (∗)First estrus (d) 40.6 ± 5.1 46.5 ± 3.2 45.5 ± 6.4 46.8 ± 2.7Body weight at day of first estrus (g) 88.9 ± 5.4 88.2 ± 5.8 87.5 ± 4.8 86.7 ± 5.8

Note. Values represent group mean ± SD. Asterisk indicates significant difference compared to controls, p < .05.

TABLE 3. Sexual Development of Female Rat Offspring Exposed to Triclosan During Pregnancy, Lactation, and up to Puberty

Triclosan (mg/kg/d)

Parameter Vehicle 1.0 10.0 50.0

Number of animals (n) 9 9 9 9Vaginal opening (d) 32.3 ± 1.2 39.4 ± 1.5 (∗) 38.0 ± 2.5 (∗) 37.5 ± 2.0 (∗)Body weight at day of vaginal opening (g) 64.5 ± 3.5 74.6 ± 2.7 (∗) 72.0 ± 1.5 (∗) 73.7 ± 3.4 (∗)First estrus (d) 42.9 ± 4.6 46.0 ± 3.5 45.6 ± 3.7 46.8 ± 2.9Body weight at day of first estrus (g) 88.4 ± 5.7 89.7 ± 6.5 87.5 ± 4.3 91.3 ± 5.7

Note. Values represent group mean ± SD. Asterisk indicates significant difference compared to controls, p < .05.

Age and Body Weight at Vaginal OpeningVaginal opening (VO) was significantly

delayed in female rats exposed to triclosan(1, 10, or 50 mg/kg/d) in utero and duringlactation, and in females additionally exposeduntil puberty, compared to the vehicle-treatedgroup (Tables 2 and 3). The three doses oftriclosan produced a significative increase inbody weight (taken the same day as VO wasproduced) of rats treated in utero and duringlactation, and in females additionally exposeduntil puberty. Administration in utero and lac-tation of triclosan or also until puberty didnot markedly affect the relative weight of theuteri or ovaries compared to control (data notshown).

Uterotrophic AssayAdministration of triclosan did not

markedly alter the relative uterine weightsof immature rats that received this antibacterialagent during gestation and lactation, nor thatof females also treated for 3 d after weaning(data not shown). Estradiol (positive control)significantly increased relative uterine weightwhen compared with the vehicle-treatedgroup.

Effect of Triclosan on SerumTriiodothyronine (T3) andTetraiodothyronine (T4) ConcentrationsDuring Pregnancy and LactationTriclosan significantly increased serum tri-

iodothyronine (T3) concentrations (Figure 3A).Our results are in agreement with thoseobtained by Hapon et al. (2003). In the con-trol condition, the serum T3 concentrationsremained roughly constant during pregnancyand lactation, with the exception of markedincreases from d 15 of pregnancy until thefirst day of lactation. Even though there was asignificant dose-response relation in triclosan-treated rats with reduced serum T3 concen-trations, the peaks before and after deliverywere still present although somewhat attenu-ated (Figure 3A).

Serum tetraiodothyronine (T4) concentra-tion values were significantly affected by tri-closan. In control rats, circulating T4 concen-trations decreased during pregnancy, reach-ing their lowest points before delivery. Therewas a sharp increase in T4 concentrationsafter parturition, in agreement with previ-ous results (Hapon et al., 2003; Fukudaet al., 1980). In the triclosan-treated rats (10and 50 μg/kg/d), there was a decreased

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1684 P. E. A. RODRÍGUEZ AND M. S. SANCHEZ

0

0,2

0,4

0,6

0,8

1

1,2

1,4

1,6

A

GD5

Days

T3 levels

(ng/m

l)

control

1mg/kg/day

10 mg/kg/day

50mg/kg/day

GD10 GD15 GD20 LD5 LD10 LD15 LD20

B

0

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30

35

40

45

Seru

m T

4 levels

(ng/m

l)

control

1mg/kg/day

10 mg/kg/day

50mg/kg/day

GD5

Days

GD10 GD15 GD20 LD5 LD10 LD15 LD20

FIGURE 3. Circulating T3 (A) and T4 (B) concentrations dur-ing pregnancy and lactation in control and triclosan-treated rats.Triclosan was administered in drinking water at concentrations of1, 10, or 50 mg/kg/d. The results represent the means ± SEM(n = 8 rats per group). Significant difference is indicated by p<.05 compared with the respective control, ∗ p <.05 comparedwith the respective control and group treated with Triclosan 1mg/kg/d, and p <.05 compared with the other groups.

dose-response of T4 levels with respect tocontrol and 1 mg/kg/d treatment. However,the increased in hormone concentrations pro-duced after parturition were still present,although they were markedly attenuated com-pared with control (Figure 3B).

DISCUSSION

Data demonstrate that triclosan exposuredecreased T3 and T4 serum concentrations in adose-dependent manner during pregnancy andgestation. The doses of triclosan used in thepresent study, which were capable of reducingthe levels of T4, are lower than those in other

experimental models (Zorrilla et al., 2009). Thisdifference may be associated with substantialphysiological changes occurring during preg-nancy. These physiological adaptations pro-duce an impact on toxicokinetics and may alsoalter toxicodynamics. The absorption, distribu-tion, metabolism, transfer between maternaland fetal compartments, and the eliminationof many xenobiotics occur during pregnancy(Mattison et al., 1991; Frederiksen, 2001).Thus, changes in body weight, total body water,plasma proteins, body fat, and cardiac outputmay alter the distribution of and sensitivity totriclosan during pregnancy.

The alterations in hormone patternsexerted significant effects on pregnancy andlactation, as reflected by pup growth. Part ofthe decline in growth of pups may be dueto a failure of thyroid hormone homeostasisin offspring produced by triclosan present inbreast milk. Further, triclosan might reducethe milk ejection reflex in rat mothers, thuscompromising the normal feeding of pups.Although triclosan is poorly excreted intomilk, at least in humans, milk concentrationsmight reach 10% of that in serum (Allmyret al., 2006; Dayan, 2007) and may induce adisruption in normal function of thyroid glandin pups, contributing to diminished growth anddevelopment. Therefore, growth impairmentmay be a consequence of undernourishmentby an inadequate milk supply, together withalterations in levels of T3 and T4 due to thepassage of triclosan to the milk, albeit in lowamounts. Once the pups open their eyes, atabout PND 17, rats are also able to drinkthe water or triclosan solution available forthe mothers and as a result may experienceeven more alterations to the thyroid function.Nevertheless, the growth retardation observedfrom PND 5 onward was clearly not due toingestion of triclosan solution by the pups.It is also not surprising that the growth of thelitters was significantly less with pup mortalityincreasing during lactation in the high-dosetriclosan-treated group.

Results demonstrated a dose-relateddecrease in the live birth index and in the 6-dsurvival index in the group given 50 mg/kg/d.

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TRICLOSAN IMPAIRS FEMALE PUBERTAL DEVELOPMENT 1685

This is a dose of one order of magnitude lowerthan that reported in another reproductivestudy, where no pup lethality was foundat a dose of 200 mg/kg/d (Denning et al.,1992). However, these discrepancies maybe explained on the basis of the substantialdifferences existing between the two protocols.First and foremost, in our opinion, the maindifference lies in the initiation of the treatment(administration of triclosan) with respect tomating. Denning et al. (1992) began treatmenton d 6 of gestation and ended on gestation day(GD) 15, whereas in our case it was started8 d before mating and continued during thebreeding of pups. Second and also important,the source and purity of the chemical must betaken into account (Menoutis & Parisi, 2002).The triclosan purity was not stated in Denninget al. (1992), while in the present study apurity of 99.6 % was used. Third, triclosanwas administered by gavage in Denning et al.(1992), whereas in our experiments were con-ducted with triclosan administered in drinkingwater. In addition, there are other variablesthat might justify differences found betweenboth studies, such as age of animals, light cycleand mating.

As observed in other studies showing thatenvironmental toxins alter male:female ratio(Jongbloet et al., 2001, 2002; Hood, 2005;Mackenzie et al., 2005), our results demon-strated that triclosan was capable of decreas-ing the sex ratio due to an increased birthof females to the detriment of males. Thisphenomenon may be associated with contam-inant exposure (Hertz-Picciotto et al., 2008;Grant, 1996; 2007; Grant et al., 2005; Lephart,1996).

The results of the present study sup-port the hypothesis that triclosan delays theonset of puberty by altering the hypothalamic–pituitary activity. In treated male rats, triclosandecreased the synthesis of luteinizing hor-mone (LH) and follicle-stimulating hormone(FSH), probably by involving the hypothalamo–pituitary–gonadal axis (Kumar et al., 2008,2009), with both of these hormones beingimportant for normal pubertal development(Grumbach & Kaplan, 1990; Layman, 2000).

Thus, because the onset of puberty is atransitional period with signaling changes inthe hypothalamic–pituitary–ovarian axis, thepostulation that triclosan mediates its effectsthrough the central nervous system (CNS)and/or hormonal control of gonadal functionmerits further studies.

In summary, this study shows that althoughtriclosan did not produce any apparent exter-nal signs of toxicity, triclosan disrupted thy-roid homeostasis and reproductive toxicity inF0 rats exposed orally during the gestationand lactational period. Results showed thatthis compound also produced fetal toxicity infemale offspring exposed in utero, during lac-tation, and after weanling, thereby producinga decrease in female-pup body weight andsignificant reductions in the sex ratio for alldoses used, with the highest dose applied in thepresent study also significantly decreasing fetalviability (live birth index, 6-d survival index).Furthermore, triclosan delayed VO in Wistarfemales in utero or lactational exposure and upto PND 50, regardless of the changes producedin body weight. While this study did not intendto identify a specific mechanism of action, ourdata are consistent with a possible effect onhormone function and/or control of gonadalfunction that warrants further investigation.

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