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Oral administration of phenol resulted in the death of 4 pregnant rats at 60 mg/kg, 5 pregnant rats at 120 mg/kg, and 14 pregnant rats at 240 mg/kg. These dead pregnant rats exhibited back hair loss and convulsions.
The rats that survived gestational exposure to phenol in each dosing group gained body weight as expected (Table 1). Maternal body weight, body weight gain, and gravid uterus weight showed no significant dose-dependent changes. However, the net body weight significantly increased at 30 mg/kg compared with that in the vehicle control group.
Table 1. Maternal body weight, gravid uterus weight, and body weight change in female rats receiving phenol per os from gestation day (GD) 5 to 20
Variable Vehicle
control
(n = 19)Positive
control
(n = 18)Groups exposed to phenol [mg/(kg∙d)] 15 (n = 20) 30 (n = 19) 60 (n = 20) 120 (n = 17) 240 (n = 6) Subjects (dams) Total treated 20 20 20 20 25 25 20 Deaths 0 0 0 0 4 5 14 Nonpregnant at sacrifice 1 2 0 1 1 3 0 Pregnant at sacrifice 19 18 20 19 20 17 6 Maternal body weight (g) GD 0 301.3 ± 19.9 320.3 ± 20.2 288.1 ± 27.5 308.3 ± 18.6 291.7 ± 24.4 296.1 ± 22.1 308.3 ± 14.3 GD 6 311.2 ± 20.6 329.7 ± 19.8 322.0 ± 26.1 329.6 ± 20.3 318.2 ± 27.7 320.4 ± 26.6 327.7 ± 16.4 GD 9 324.9 ± 24.0 343.6 ± 24.1 340.7 ± 27.3 338.7 ± 21.4 334.4 ± 29.3 330.9 ± 30.5 323.0 ± 27.5 GD 12 329.6 ± 25.1 347.1 ± 22.6 343.4 ± 23.2 351.9 ± 24.1 344.8 ± 28.0 337.6 ± 28.5 328.7 ± 26.9 GD 15 351.4 ± 26.3 368.9 ± 25.0 378.8 ± 28.3 363.3 ± 28.5 362.7 ± 27.5 354.2 ± 28.7 338.7 ± 33.6 GD 20 387.1 ± 38.4 393.1 ± 28.9 402.6 ± 27.9 428.4 ± 40.1 406.0 ± 26.8 413.8 ± 40.1 365.7 ± 60.4 Gravid uterus weight (g) 62.83 ± 17.65 40.69 ± 12.70** 65.34 ± 9.40 56.12 ± 19.70 65.26 ± 12.73 55.93 ± 17.85 40.72 ± 16.39 Body weight gain (g)a 81.2 ± 17.8 63.4 ± 17.6 80.6 ± 32.8 98.8 ± 28.4 87.9 ± 16.9 93.4 ± 29.2 38.0 ± 46.4 Net body weight gain (g)b 18.3 ± 17.1 22.8 ± 10.2 15.3 ± 33.7 42.7 ± 20.1** 22.6 ± 7.2 37.5 ± 34.7 −2.7 ± 38.1 Note. Values are expressed as mean ± SD. aBody weight on GD 20 - body weight on GD 6. bBody weight on GD 20 - body weight on GD 6 - gravid uterine weight. **Significant differences from the vehicle control group, P < 0.01. -
There was no significant difference between the phenol dose group and the vehicle control group in the number of corpora lutea and implantation. However, the preimplantation loss rate was 6.2% in the 240 mg/kg group, and no preimplantation loss was observed in the other test groups. The ratio of live fetuses in the 240 mg/kg group was significantly lower than that in the vehicle control group. However, the ratios of fetal resorption and postimplantation loss in the 240 mg/kg group were significantly higher than those in the vehicle control group. Fetal gender ratio in the 30 and 240 mg/kg groups deviated from 1:1 (Table 2).
Table 2. Developmental toxicity in rat fetuses prenatally exposed to phenol
Variable Vehicle
controlPositive
controlGroups exposed to phenol [mg/(kg∙d)] 15 30 60 120 240 All littersa 19 18 20 19 20 17 6 Number of implantation sitesb 15.1 ± 3.1 15.2 ± 3.0 15.5 ± 1.8 14.3 ± 3.7 14.4 ± 2.6 15.3 ± 22 15.2 ± 7.5 Number of corpora luteab 15.1 ± 3.1 15.2 ± 3.0 15.5 ± 1.8 14.3 ± 3.7 14.4 ± 2.6 15.3 ± 2.2 16.2 ± 6.6 Preimplantation loss (%)c 0.0 0.0 0.0 0.0 0.0 0.0 6.2* Number of fetuses 286 274 310 255 288 260 91 Number of live fetuses 269 227 308 249 279 258 76 Ratio of live fetuses (%) 94.1 82.9 99.4 91.5 96.9 99.2 83.5 Number of resorbed fetuses 16 19 2 2 9 2 15 Ratio of fetal resorption (%) 5.6 6.9 0.6 0.7 3.1 0.8 16.5 Number of dead fetuses 1 28 0 4 0 0 0 Ratio of dead fetuses (%) 0.4 10.2 0.0 1.6 0.0 0.0 0.0 Postimplantation loss (%)d 5.9 17.2 0.6 2.4 3.1 0.8 16.5 Sex ratio (M:F) 147:122 78:149 145:163 80:169 117:162 104:154 29:47 Note. aIncluded all pregnant females at euthanasia. bValues are expressed as mean ± SD. cPreimplantation loss (%) = [(number of corpora lutea - number of implantation sites) / number of corpora lutea] × 100%. dPostimplantation loss (%) = [(no. resorbed + dead fetuses) / no. implantations] × 100%. *Significant differences from the vehicle control group, P< 0.05. -
Results of fetal clinical observations are shown in Table 3 and Figure 1. Two rat fetuses showed ecchymoma in the vehicle control group (Figure 1A). Encephalocele and small brain were observed in all rat fetuses in the positive control group (Figure 1B). No overt signs were noted in the phenol treatment groups.
Figure 1. External examination of fetus treated with phenol during gestation. (A) Ecchymoma of the fetal lower limb. (B) The left side of the figure shows fetal encephalocele; the right side of the picture shows a normal rat fetus.
Table 3. External examination of rat fetuses prenatally exposed to phenol
Variable Vehicle
controlPositive
controlGroups exposed to phenol [mg/(kg∙d)] 15 30 60 120 240 Number of litters for external examination 19 18 20 19 20 17 6 Number of fetuses for external 269 227 308 249 279 258 76 Litters exhibiting abnormal findings (number/percentage) 2/10.5 18/100.0 0/0.0 0/0.0 0/0.0 0/0.0 0/0.0 Fetuses exhibiting abnormal findings (number/percentage) 2/0.7 227/100.0 0/0.0 0/0.0 0/0.0 0/0.0 0/0.0 Type of external abnormalities Ecchymoma 2/0.7 0/0.0 0/0.0 0/0.0 0/0.0 0/0.0 0/0.0 Encephalocele 0/0.0 227/100.0 0/0.0 0/0.0 0/0.0 0/0.0 0/0.0 As shown in Figure 2, the fetal body length, fetal body weight, and placental weight of the positive control group were significantly lower than those of the vehicle control group.
Figure 2. Effects of phenol on fetal body weight, placenta weight, fetal body length, and fetal tail length. (A) Fetal body weight. (B) Placenta weight. (C) Fetal body length. (D) Fetal tail length. Data are expressed as mean ± SEM. **Compared with the vehicle control group, P < 0.01.
In the phenol-treated groups, starting from the 30 mg/kg dose group, placental and fetal weights were significantly lower than those of the vehicle control group. Fetal body length was significantly higher in the 15 and 60 mg/kg dose group than in the vehicle control group (P < 0.05), but was not significantly different between the other dose groups and the vehicle control group. Fetal tail length was significantly longer in the 15, 60, and 120 mg/kg dose groups than in the vehicle control group, but was not significantly different between the other dose groups and the vehicle control group.
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No treatment-related fetal malformations were observed. Results of visceral examinations are shown in Table 4. Different degrees of cardiac (auricle blood clot) and liver abnormalities were observed in each experimental group.
Table 4. Visceral examination of rat fetuses prenatally exposed to phenol
Variable Vehicle
control
Positive control
Groups exposed to phenol [mg/(kg∙d)] 15 30 60 120 240 Number of litters for visceral examination 19 18 20 19 20 17 6 Number of fetuses for visceral examination 133 110 151 121 134 128 37 Litters affected (number/percentage)a 7/36.8 17/94.4 10/50.0 4/22.2 13/65.0 8/47.1 4/66.7 Fetuses with malformationsb (number/percentage) 11/8.3 85/77.3 17/11.3 5/4.1 19/14.2 24/18.8 9/24.3 Type of visceral variations Ventricular dilatation and hemorrhage, small brain 0/0.0 66/60.0 0/0.0 0/0.0 0/0.0 1/0.8 0/0.0 Auricle blood clot 10/7.5 19/17.3 17/11.3 5/4.1 19/14.2 23/18.0 8/21.6 Redundant liver, abdominal hernia, reduced liver lobe, abnormalities 1/0.8 3/2.7 0/0.0 0/0.0 1/0.7 1/0.8 1/2.7 Small right kidney 0/0.0 3/2.7 0/0.0 0/0.0 0/0.0 0/0.0 0/0.0 Cutaneous dropsy 0/0.0 49/44.5 0/0.0 0/0.0 0/0.0 0/0.0 0/0.0 Note.aIncluded litters with one or more affected fetuses. bA single fetus might be presented more than once when listing individual defects. The following visceral variations were observed: vehicle control group: auricle blood clot in 10 fetuses, redundant liver in 1 fetus; 15 mg/kg group: auricle blood clot in 17 fetuses; 30 mg/kg group: auricle blood clot in 5 fetuses; 60 mg/kg group: auricle blood clot in 19 fetuses, reduced liver lobe in 1 fetus; 120 mg/kg group: auricle blood clot in 23 fetuses, abnormality in 1 live fetus; 240 mg/kg group: auricle blood clot in 8 fetuses, redundant liver in 1 fetus. The malformation ratios of fetuses in the phenol-treated groups (from low to high doses) were 11.3%, 4.1%, 14.2%, 18.8%, and 24.3%, respectively.
In addition to the above-mentioned changes in visceral abnormalities, other abnormalities were also observed in the positive control group, including small brain, intraventricular hemorrhage, ventriculomegaly, and peritoneal interstitial hemorrhage, as well as small right kidney and cutaneous dropsy. Some typical visceral variations are shown in Figure 3.
Figure 3. Visceral malformations of fetus treated with phenol during gestation. (A) Normal viscera of the fetus. (B) Small brain and cutaneous dropsy. (C) Ventriculomegaly. (D) Abdominal hernia. (E) Ventricular hemorrhage. (F) Small brain and paranasal sinus expansion. (G) Auricle blood clot. (H) Redundant liver.
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Results of skeletal examination are shown in Table 5. Compared with the vehicle control group, the positive control group showed cranial bone loss, abnormal cervical vertebra, abnormal thoracic vertebra, abnormal lumbar vertebra, appendix vertebral fracture and widening, limb bone loss, rib loss, short bones, and broken bones. The above-mentioned abnormalities were not observed or only occasionally observed in the phenol-treated groups and the vehicle control group.
Table 5. Skeletal examination of rat fetuses prenatally exposed to phenol
Variable Vehicle control
Positive control
Groups exposed to phenol [mg/(kg∙d)] 15 30 60 120 240 Number of litters for skeletal examination 19 18 20 19 20 17 6 Number of fetuses for skeletal examination 136 117 157 128 145 130 39 Litters affecteda (number/percentage) 3/15.8 18/100.0 3/15.0 3/15.8 2/10.0 2/11.8 1/16.7 Fetuses with malformationsb(number/percentage) 3/2.2 116/100.0 8/5.1 4/3.1 2/1.4 4/3.1 3/7.7 Type of skeletal variations Abnormal sagittal suture large 2/1.5 53/45.7 0/0.0 2/1.6 0/0.0 0/0.0 0/0.0 Abnormal occipital bone aplasia 1/0.8 107/92.2 9/5.2 0/0.0 2/1.4 2/1.5 3/7.9 Other abnormal cranium 3/2.3 102/87.9 0/0.0 2/1.6 0/0.0 0/0.0 2/5.3 Abnormal cervical vertebra 0/0.0 7/6.0 0/0.0 0/0.0 0/0.0 0/0.0 0/0.0 Abnormal thoracic vertebra 0/0.0 11/9.5 0/0.0 0/0.0 0/0.0 0/0.0 0/0.0 Lumbar vertebral centra, ossification, incomplete 0/0.0 22/19.0 1/0.6 0/0.0 0/0.0 0/0.0 0/0.0 Abnormal sacral coccygeal vertebra 0/0.0 27/0.2 0/0.0 0/0.0 0/0.0 0/0.0 0/0.0 Abnormal rib 0/0.0 97/83.6 0/0.0 1/0.8 0/0.0 0/0.0 0/0.0 Abnormal sternum 0/0.0 21/0.2 0/0.0 0/0.0 0/0.0 0/0.0 0/0.0 Abnormal limb bone 0/0.0 85/73.3 0/0.0 0/0.0 0/0.0 2/1.5 0/0.0 Note.aIncluded litters with one or more affected fetuses. bA single fetus might be presented more than once when listing individual defects. The following skeletal variations were observed: vehicle control group: large sagittal suture in 2 fetuses, occipital bone aplasia in 1 fetus, interparietal bone aplasia in 3 fetuses; 15 mg/kg group: occipital bone aplasia in 9 fetuses, lumbar vertebral centra incomplete in 1 fetus; 30 mg/kg group: large sagittal suture in 2 fetuses, interparietal bone aplasia in 2 fetuses, short 13th rib in 1 fetus; 60 mg/kg group: occipital bone aplasia in 2 fetuses; 120 mg/kg group: occipital bone aplasia in 2 fetuses, left or right metacarpal bone loss in 2 fetuses; 240 mg/kg group: occipital bone aplasia in 3 fetuses, interparietal bone aplasia in 2 fetuses. Some typical visceral variations are shown in Figure 4.
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The method of this verification experiment was the same as that in the US EPA, but we added 15 and 240 mg/kg as the lowest and highest dose groups, respectively. However, the toxic effects of phenol observed in this study were different from those stated in the US EPA.
In a study by Jones-Price et al.[11], a group of 20 to 22 female CD-1 rats were administered 30, 60, or 120 mg phenol/(kg∙d) by gavage on GD 6 to 15. No maternal effects were observed after treatment at any dose, but dose-dependent and significant (P < 0.001) reductions in fetal body weights compared with the control values were observed in the 120 mg/(kg∙d) dose group. No structural abnormalities were noted in any dose group. The results of this previous study suggested a NOAEL and LOAEL for developmental toxicity of 60 and 120 mg/(kg∙d), respectively [10, 11]. Based on the above result, the RfD was derived as follows: RfD = NOAEL/UF. NOAEL = 60 mg/(kg∙d); UF = 100.
NOAEL was based on the absence of developmental effects in rat fetuses exposed to phenol. Uncertainty factor (UF) was chosen in accordance with the developmental toxicity guidelines proposed by the EPA.
To verify this NOAEL, we designed the experiment based on a study by Jones-Price et al.[11], but added two more dose groups bringing the total, number of phenol dose groups to six: 0, 15, 30, 60, 120, and 240 mg/(kg∙d). In this study, the bone and visceral malformation rates of fetuses in five dose groups did not significantly change compared with those in the vehicle control group. However, in the 60 mg/(kg∙d) dose group, the dead pregnant rats exhibited back hair loss and convulsions, with mortality rate exceeding 10%, which suggested that phenol exerted potent maternal toxicity at this dosage. Moreover, fetal and placental weight significantly decreased after phenol treatment starting at a dose of 30 mg/(kg∙d) compared with those in the vehicle control group (P < 0.01). Therefore, it can be concluded the developmental NOAEL of phenol was 15 mg/(kg∙d), considering the significantly decreased fetal body weight and placental weight after phenol treatment at 30 mg/(kg∙d).
Results of other development toxicity studies on phenol are shown in Table 6[31]. In another study by Jones-Price et al.[32], CD-1 mice were administered 0, 70, 140, and 280 mg/(kg∙d) phenol at GD 6–15; the results showed decreased average fetal bw/litter and fetuses with malformations, and the developmental NOAEL was determined to be 140 mg/(kg∙d). In a study of Kavlock[33], Sprague-Dawley rats were administered phenol by gavage at doses of 0, 100, 333, 667, and 1,000 mg/(kg∙d) at GD 11; malformation was reported and the developmental NOAEL was determined to be 333 mg/(kg∙d). Narotsky and Kavlock et al. [34] administered 0, 40, and 53.3 phenol mg/(kg∙d) to Fischer rats at GD 6–19; reduced number of live pups/litter and fully resorbed litters were observed, and the developmental NOAEL was 40 mg/(kg∙d). In a study by Argus[35], Sprague-Dawley rats were administered 0, 60, 120, and 360 mg/(kg∙d) phenol at GD 6–15, and the results showed decreased average fetal bw/litter and developmental NOAEL of 120 mg/(kg∙d). Ryan et al.[36] performed a two-generation reproduction study in rats treated with phenol via drinking water; the results showed that litter survival and offspring body weight (preweaning) decreased in the 5,000 mg/L group in both generations. Taken together, all these previous studies showed different NOAELs. The current study was conducted according to the latest OECD Guideline for The Testing of Chemicals, Prenatal Developmental Toxicity Study[25], and accumulated data on the developmental toxicity of phenol.
Table 6. Data on the in vivo developmental toxicity of phenol obtained from the literature[31]
Species Exposure day (s) Dose [mg/(kg∙d)] Developmental endpointa Developmental NOAEL [mg/(kg∙d)] Reference Sprague-Dawley rats GD 11 0, 100, 333, 667, 1,000 Malformationsb reported at the two highest dosesc 333 [33] Sprague-Dawley rats GD 6–15 0, 30, 60, 120 Decreased average fetal b.w./litter 60 [11] Sprague-Dawley rats GD 6–15 0, 60, 120, 360 Decreased average fetal b.w./litter 120 [35] Fischer rats GD 6–19 0, 40, 53.3 Reduced live pups/litter and fraction litters fully resorbed 40 [34] CD-1 mice GD 6–15 0, 70, 140, 280 Decreased average fetal b.w./litter Fraction fetuses malformed 40 [32] Sprague-Dawley rats 10–11 weeks prior to mating through weaning 0, 200, 1,000, 5,000 ppm
[0, 20, 93, 350 mg/(kg∙d)]Fraction of nonliving offspring postnatal day 4 and postnatal days 7–21d 70 (males),
93 (females)[36] Note.aIn each study, phenol was administered by oral gavage. bHindlimb paralysis and/or short or kinky tails. cNot analyzed for statistical significance. dEffects possibly related to decreased maternal water intake due to flavor aversion. Phenol toxicity is related with two main processes: the unspecified toxicity related to the hydrophobicity of the individual compound and the formation of free radicals[37]. Thus, the developmental toxicity mechanism of phenol remains unclear. One possible cause may be oxidative stress. Phenol induces lipid peroxidation, which is responsible for damage and finally degradation of cell membranes[37]; it is also readily oxidized to quinone radicals, which tends to be more reactive. Catechols have the tendency to cause DNA damage or arylation, destroy proteins, and disrupt electron transport in energy-transducing membranes[38].
Owing to the negative effects of phenolic pollutants on public health and the ecological system, phenol has been designated as a priority pollutant by the US EPA and the National Pollutant Release Inventory of Canada[39-41]. As phenol possesses hazardous health effects, which can be both acute and chronic, and commonly exists as its derivatives, such as Bisphenol A, chlorophenols, and phenolic endocrine-disrupting compounds[39, 41], subsequent studies are needed to analyze and identify more phenolic pollutants and their environmental fate in natural environments.
In summary, to confirm the HA for phenol, we verified the NOAEL of phenol accepted by the US EPA. This study accumulated animal experimental data to provide a new basis for revision of the Standards for Drinking-water Quality and to further establish our own health advisories for short-term health risks of phenol in China.
doi: 10.3967/bes2020.055
Verification on the Developmental Toxicity of Short-term Exposure to Phenol in Rats
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Abstract:
Objective To verify the health advisory for short-term exposure to phenol. Methods The method of this validation experiment was the same as the US Environmental Protection Agency (EPA) methodology for toxicology experiments used to determine phenol drinking water equivalent level (DWEL). Pregnant female Sprague-Dawley rats were administered phenol in distilled water by gavage at daily doses of 15, 30, 60, 120, and 240 mg/kg body weight (b.w.) from implantation (the 6th day post-mating) to the day prior to the scheduled caesarean section (the 20th day of pregnancy). The following information was recorded: general behavior; body weight; number of corpus luteum, live birth, fetus, stillbirth, and implantation; fetal gender; body weight; body length; tail length; and abnormalities and pathomorphological changes in the dams. Results In the 60 mg/kg b.w. dose group, the mortality of pregnant rats increased with increasing doses, suggesting maternal toxicity. Fetal and placental weights decreased as phenol dose increased from 30 mg/kg b.w., and were significantly different compared those in the vehicle control group, which suggested developmental toxicity in the fetuses. However, the phenol-exposed groups showed no significant change in other parameters compared with the vehicle control group (P > 0.05). Conclusion Despite using the same method as the US EPA, a different NOEAL of 15 mg/(kg·d) was obtained in this study. -
Key words:
- Phenol /
- Short-term exposure health advisory /
- Verification
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Figure 3. Visceral malformations of fetus treated with phenol during gestation. (A) Normal viscera of the fetus. (B) Small brain and cutaneous dropsy. (C) Ventriculomegaly. (D) Abdominal hernia. (E) Ventricular hemorrhage. (F) Small brain and paranasal sinus expansion. (G) Auricle blood clot. (H) Redundant liver.
Table 1. Maternal body weight, gravid uterus weight, and body weight change in female rats receiving phenol per os from gestation day (GD) 5 to 20
Variable Vehicle
control
(n = 19)Positive
control
(n = 18)Groups exposed to phenol [mg/(kg∙d)] 15 (n = 20) 30 (n = 19) 60 (n = 20) 120 (n = 17) 240 (n = 6) Subjects (dams) Total treated 20 20 20 20 25 25 20 Deaths 0 0 0 0 4 5 14 Nonpregnant at sacrifice 1 2 0 1 1 3 0 Pregnant at sacrifice 19 18 20 19 20 17 6 Maternal body weight (g) GD 0 301.3 ± 19.9 320.3 ± 20.2 288.1 ± 27.5 308.3 ± 18.6 291.7 ± 24.4 296.1 ± 22.1 308.3 ± 14.3 GD 6 311.2 ± 20.6 329.7 ± 19.8 322.0 ± 26.1 329.6 ± 20.3 318.2 ± 27.7 320.4 ± 26.6 327.7 ± 16.4 GD 9 324.9 ± 24.0 343.6 ± 24.1 340.7 ± 27.3 338.7 ± 21.4 334.4 ± 29.3 330.9 ± 30.5 323.0 ± 27.5 GD 12 329.6 ± 25.1 347.1 ± 22.6 343.4 ± 23.2 351.9 ± 24.1 344.8 ± 28.0 337.6 ± 28.5 328.7 ± 26.9 GD 15 351.4 ± 26.3 368.9 ± 25.0 378.8 ± 28.3 363.3 ± 28.5 362.7 ± 27.5 354.2 ± 28.7 338.7 ± 33.6 GD 20 387.1 ± 38.4 393.1 ± 28.9 402.6 ± 27.9 428.4 ± 40.1 406.0 ± 26.8 413.8 ± 40.1 365.7 ± 60.4 Gravid uterus weight (g) 62.83 ± 17.65 40.69 ± 12.70** 65.34 ± 9.40 56.12 ± 19.70 65.26 ± 12.73 55.93 ± 17.85 40.72 ± 16.39 Body weight gain (g)a 81.2 ± 17.8 63.4 ± 17.6 80.6 ± 32.8 98.8 ± 28.4 87.9 ± 16.9 93.4 ± 29.2 38.0 ± 46.4 Net body weight gain (g)b 18.3 ± 17.1 22.8 ± 10.2 15.3 ± 33.7 42.7 ± 20.1** 22.6 ± 7.2 37.5 ± 34.7 −2.7 ± 38.1 Note. Values are expressed as mean ± SD. aBody weight on GD 20 - body weight on GD 6. bBody weight on GD 20 - body weight on GD 6 - gravid uterine weight. **Significant differences from the vehicle control group, P < 0.01. Table 2. Developmental toxicity in rat fetuses prenatally exposed to phenol
Variable Vehicle
controlPositive
controlGroups exposed to phenol [mg/(kg∙d)] 15 30 60 120 240 All littersa 19 18 20 19 20 17 6 Number of implantation sitesb 15.1 ± 3.1 15.2 ± 3.0 15.5 ± 1.8 14.3 ± 3.7 14.4 ± 2.6 15.3 ± 22 15.2 ± 7.5 Number of corpora luteab 15.1 ± 3.1 15.2 ± 3.0 15.5 ± 1.8 14.3 ± 3.7 14.4 ± 2.6 15.3 ± 2.2 16.2 ± 6.6 Preimplantation loss (%)c 0.0 0.0 0.0 0.0 0.0 0.0 6.2* Number of fetuses 286 274 310 255 288 260 91 Number of live fetuses 269 227 308 249 279 258 76 Ratio of live fetuses (%) 94.1 82.9 99.4 91.5 96.9 99.2 83.5 Number of resorbed fetuses 16 19 2 2 9 2 15 Ratio of fetal resorption (%) 5.6 6.9 0.6 0.7 3.1 0.8 16.5 Number of dead fetuses 1 28 0 4 0 0 0 Ratio of dead fetuses (%) 0.4 10.2 0.0 1.6 0.0 0.0 0.0 Postimplantation loss (%)d 5.9 17.2 0.6 2.4 3.1 0.8 16.5 Sex ratio (M:F) 147:122 78:149 145:163 80:169 117:162 104:154 29:47 Note. aIncluded all pregnant females at euthanasia. bValues are expressed as mean ± SD. cPreimplantation loss (%) = [(number of corpora lutea - number of implantation sites) / number of corpora lutea] × 100%. dPostimplantation loss (%) = [(no. resorbed + dead fetuses) / no. implantations] × 100%. *Significant differences from the vehicle control group, P< 0.05. Table 3. External examination of rat fetuses prenatally exposed to phenol
Variable Vehicle
controlPositive
controlGroups exposed to phenol [mg/(kg∙d)] 15 30 60 120 240 Number of litters for external examination 19 18 20 19 20 17 6 Number of fetuses for external 269 227 308 249 279 258 76 Litters exhibiting abnormal findings (number/percentage) 2/10.5 18/100.0 0/0.0 0/0.0 0/0.0 0/0.0 0/0.0 Fetuses exhibiting abnormal findings (number/percentage) 2/0.7 227/100.0 0/0.0 0/0.0 0/0.0 0/0.0 0/0.0 Type of external abnormalities Ecchymoma 2/0.7 0/0.0 0/0.0 0/0.0 0/0.0 0/0.0 0/0.0 Encephalocele 0/0.0 227/100.0 0/0.0 0/0.0 0/0.0 0/0.0 0/0.0 Table 4. Visceral examination of rat fetuses prenatally exposed to phenol
Variable Vehicle
control
Positive control
Groups exposed to phenol [mg/(kg∙d)] 15 30 60 120 240 Number of litters for visceral examination 19 18 20 19 20 17 6 Number of fetuses for visceral examination 133 110 151 121 134 128 37 Litters affected (number/percentage)a 7/36.8 17/94.4 10/50.0 4/22.2 13/65.0 8/47.1 4/66.7 Fetuses with malformationsb (number/percentage) 11/8.3 85/77.3 17/11.3 5/4.1 19/14.2 24/18.8 9/24.3 Type of visceral variations Ventricular dilatation and hemorrhage, small brain 0/0.0 66/60.0 0/0.0 0/0.0 0/0.0 1/0.8 0/0.0 Auricle blood clot 10/7.5 19/17.3 17/11.3 5/4.1 19/14.2 23/18.0 8/21.6 Redundant liver, abdominal hernia, reduced liver lobe, abnormalities 1/0.8 3/2.7 0/0.0 0/0.0 1/0.7 1/0.8 1/2.7 Small right kidney 0/0.0 3/2.7 0/0.0 0/0.0 0/0.0 0/0.0 0/0.0 Cutaneous dropsy 0/0.0 49/44.5 0/0.0 0/0.0 0/0.0 0/0.0 0/0.0 Note.aIncluded litters with one or more affected fetuses. bA single fetus might be presented more than once when listing individual defects. Table 5. Skeletal examination of rat fetuses prenatally exposed to phenol
Variable Vehicle control
Positive control
Groups exposed to phenol [mg/(kg∙d)] 15 30 60 120 240 Number of litters for skeletal examination 19 18 20 19 20 17 6 Number of fetuses for skeletal examination 136 117 157 128 145 130 39 Litters affecteda (number/percentage) 3/15.8 18/100.0 3/15.0 3/15.8 2/10.0 2/11.8 1/16.7 Fetuses with malformationsb(number/percentage) 3/2.2 116/100.0 8/5.1 4/3.1 2/1.4 4/3.1 3/7.7 Type of skeletal variations Abnormal sagittal suture large 2/1.5 53/45.7 0/0.0 2/1.6 0/0.0 0/0.0 0/0.0 Abnormal occipital bone aplasia 1/0.8 107/92.2 9/5.2 0/0.0 2/1.4 2/1.5 3/7.9 Other abnormal cranium 3/2.3 102/87.9 0/0.0 2/1.6 0/0.0 0/0.0 2/5.3 Abnormal cervical vertebra 0/0.0 7/6.0 0/0.0 0/0.0 0/0.0 0/0.0 0/0.0 Abnormal thoracic vertebra 0/0.0 11/9.5 0/0.0 0/0.0 0/0.0 0/0.0 0/0.0 Lumbar vertebral centra, ossification, incomplete 0/0.0 22/19.0 1/0.6 0/0.0 0/0.0 0/0.0 0/0.0 Abnormal sacral coccygeal vertebra 0/0.0 27/0.2 0/0.0 0/0.0 0/0.0 0/0.0 0/0.0 Abnormal rib 0/0.0 97/83.6 0/0.0 1/0.8 0/0.0 0/0.0 0/0.0 Abnormal sternum 0/0.0 21/0.2 0/0.0 0/0.0 0/0.0 0/0.0 0/0.0 Abnormal limb bone 0/0.0 85/73.3 0/0.0 0/0.0 0/0.0 2/1.5 0/0.0 Note.aIncluded litters with one or more affected fetuses. bA single fetus might be presented more than once when listing individual defects. Table 6. Data on the in vivo developmental toxicity of phenol obtained from the literature[31]
Species Exposure day (s) Dose [mg/(kg∙d)] Developmental endpointa Developmental NOAEL [mg/(kg∙d)] Reference Sprague-Dawley rats GD 11 0, 100, 333, 667, 1,000 Malformationsb reported at the two highest dosesc 333 [33] Sprague-Dawley rats GD 6–15 0, 30, 60, 120 Decreased average fetal b.w./litter 60 [11] Sprague-Dawley rats GD 6–15 0, 60, 120, 360 Decreased average fetal b.w./litter 120 [35] Fischer rats GD 6–19 0, 40, 53.3 Reduced live pups/litter and fraction litters fully resorbed 40 [34] CD-1 mice GD 6–15 0, 70, 140, 280 Decreased average fetal b.w./litter Fraction fetuses malformed 40 [32] Sprague-Dawley rats 10–11 weeks prior to mating through weaning 0, 200, 1,000, 5,000 ppm
[0, 20, 93, 350 mg/(kg∙d)]Fraction of nonliving offspring postnatal day 4 and postnatal days 7–21d 70 (males),
93 (females)[36] Note.aIn each study, phenol was administered by oral gavage. bHindlimb paralysis and/or short or kinky tails. cNot analyzed for statistical significance. dEffects possibly related to decreased maternal water intake due to flavor aversion. -
[1] Wu F, Meng W, Zhao X, et al. China embarking on development of its own national water quality criteria system. Environ Sci Technol, 2010; 44, 7992−3. doi: 10.1021/es1029365 [2] Vernet C, Philippat C, Calafat AM, et al. Within-Day, Between-Day, and Between-Week Variability of Urinary Concentrations of Phenol Biomarkers in Pregnant Women. Environ Health Perspec, 2018; 126, 037005. doi: 10.1289/EHP1994 [3] Braun JM. Early-life exposure to EDCs: role in childhood obesity and neurodevelopment. Nat Rev Endocrinol, 2016; 13, 161. [4] World Health Organization. Guidelines for drinking-water quality: third edition. WHO, 2004. [5] World Health Organization. Guidelines for drinking-water quality: fourth edition. WHO, 2014. [6] U.S. Environmental Protection Agency. National Primary Drinking Water Regulations. Safe Drinking Water Act, 2002. [7] Jin Y. GB 5749-2006 Definition of Standards for drinking water quality[M]. Beijing: China Standards Press, 2007. [8] Ministry of Health of the People's Republic of China. Standards for drinking water quality. 2006. [9] Zavaleta JO, Cantilli R, Ohanian EV. Drinking water health advisory program. Ann.ist.super.sanita, 1993; 29, 355−8. [10] Office of Water U.S. Environmental Protection Agency Washington, DC. Health advisories for IOCs and SOCs. 1992. [11] Jones-Price CN, TA Ledourx, JR Reel, et al. Teratological evaluation of phenol (CAS no. 108-95-2) in CD rats. Research Triangle Park, NC: National Institute of Environmental Health Siences, 1983. [12] U.S. Environmental Protection Agency. 2002 Edition of the drinking water standards and health advisories. Environmental Protection Agency. 2002. [13] U.S. Environmental Protection Agency. Drinking water regulations and health advirories. 1994. [14] U.S. Environmental Protection Agency. Drinking Water Regulations and Health Advisories. 1995. [15] U.S. Environmental Protection Agency. Drinking Water Regulations and Health Advisories. 1996. [16] U.S. Environmental Protection Agency. Drinking Water Standards and Health Advisories. 2000. [17] U.S. Environmental Protection Agency. 2006 Edition of the Drinking Water Standards and Health Advisories. 2006. [18] U.S. Environmental Protection Agency. 2009 Edition of the Drinking Water Standards and Health Advisories. 2009. [19] U.S. Environmental Protection Agency. 2004 Edition of the Drinking Water Standards and Health Advisories. 2004. [20] U.S. Environmental Protection Agency. 2011 Edition of the Drinking Water Standards and Health Advisories. 2011. [21] U.S. Environmental Protection Agency. 2012 Edition of the Drinking Water Standards and Health Advisories. 2012. [22] U.S. Environmental Protection Agency. 2018 Edition of the Drinking Water Standards and Health Advisories Tables. 2018. [23] U.S. Environmental Protection Agency. Phenol; CASRN 108-95-2. Integrated Risk Information System, 2002. [24] Sc aw. Consensus Recommendations on Effective Institutional Animal Care and Use Committees. Laboratory Animal Science, 1987. [25] Organization for Economic Co-operation and Development. OECD guideline for the testing of chemicals prenatal developmental toxicity study. OECD. [2001-1-22]. [26] Staples RE, Schnell VL. Refinements in rapid clearing technic in the Koh-alizarin red s method for fetal bone. Stain Technol, 1964; 39, 61. [27] Wilson JG. Methods for administering agents and detecting malformations in experimental animals. Teratology Principles & Techniques, 1965; 262−77. [28] Nishimura K. A Microdissection method for detecting thoracic visceral malformations in mouse and rat fetuses. Congenit Anom Kyoto, 1974; 14, 23−40. [29] Elsayed EA. A Methodology for the Health Sciences. IIE Trans, 1997; 29, 806−7. [30] Biostatistical analysis. Prentice-Hall, 1974. [31] Spenkelink, Be rt, Pu nt, et al. Combining in vitro embryotoxicity data with physiologically based;kinetic (PBK) modelling to define in vivo dose-response curves for; developmental toxicity of phenol in rat and human. Arch Toxicol, 2013; 87, 1709−23. doi: 10.1007/s00204-013-1107-4 [32] Jones-Price C, Ledoux TA, Reel JR, et al. Teratologic evaluation of phenol (CAS No. 108-95-2) in CD-1 mice. NTP Study TER80129. Research Triangle Institute, Research Triangle Park, NC.1983(b). [33] Kavlock RJ. Structure-activity relationships in the developmental toxicity of substituted phenols: in vivo effects. Teratology, 1990; 41, 43−59. doi: 10.1002/tera.1420410106 [34] Narotsky MG, Kavlock RJ. A multidisciplinary approach to toxicological screening: II. Developmental toxicity. J Toxicol Environ Health, 1995; 45, 145−71. doi: 10.1080/15287399509531987 [35] Argus. Oral (gavage) developmental toxicity study of phenol in rats. Protocol number: 916-011. Horsham, Pennsylvania. 1997. [36] Ryan BM, Selby R, Gingell R, et al. Two-generation reproduction study and immunotoxicity screen in rats dosed with phenol via the drinking water. Int J Toxicol, 2001; 20, 121. doi: 10.1080/109158101317097700 [37] Michałowicz J, Duda W. Phenols--Sources and Toxicity. Polish J. of Environ. Stud, 2007; 16. [38] Anku WW, Mamo MA, Govender PP. Phenolic compounds in water: sources, reactivity, toxicity and treatment methods. Phenolic compounds-natural sources, importance and applications, 2017; 420−43. [39] Alshabib M, Onaizi SA. Effects of surface active additives on the enzymatic treatment of phenol and its derivatives: a mini review. Curr Pollut Rep, 2019; 5, 52−65. doi: 10.1007/s40726-019-00105-8 [40] Baken KA, MA SR, Merijn S, et al. Toxicological risk assessment and prioritization of drinking water relevant contaminants of emerging concern. Environ Int, 2018; 118, 293−303. doi: 10.1016/j.envint.2018.05.006 [41] Villegas LGC, Mashhadi N, Chen M, et al. A short review of techniques for phenol removal from wastewater. Curr Pollut Rep, 2019; 2, 157−67.