Female and male Wistar rats were used to determine the subchronic oral toxicities of two new ethyl-carbamates with ixodicidal activities (ethyl-4-bromphenyl-carbamate and ethyl-4-chlorphenyl-carbamate). The evaluated carbamates were administered in the drinking water (12.5, 25 and 50 mg/kg/day) for 90 days. Exposure to the evaluated carbamates did not cause mortality or clinical signs and did not affect food consumption or weight gain. However, exposure to these carbamates produced alterations in water consumption, hematocrit, percentages of reticulocytes, plasma proteins, some biochemical parameters (aspartate aminotransferase, gamma-glutamyl transpeptidase, cholinesterase, and creatinine activities), thiobarbituric acid reactive substances, and the relative weight of the spleen. Histologically, slight pathological alterations were found in the liver that were consistent with the observed biochemical alterations. The nonobserved adverse effect levels (NOAELs) of the evaluated carbamates were 12.5 mg/kg/day for both the female and male rats. The low severity and reversibility of the majority of the observed alterations suggest that the evaluated carbamates have low subchronic toxicity.
Carbamates are relatively simple molecules that are characterized by being carbamic acid esters. These molecules have been used as pesticides in agriculture, for human drug therapies (e.g., for the treatment of Alzheimer’s disease, myasthenia gravis, glaucoma, and as a prophylactic for organophosphate compound poisoning), and in veterinary medicine as antiparasitic drugs [
The toxicities of carbamates are highly variable; some, such as aldicarb (oral LD50 0.3 to 0.9 mg/kg), carbofuran (oral LD50 8 mg/kg), and carbaryl (oral LD50 12.5 mg/kg), are highly toxic [
Aspersion or immersion baths are the application pathways for the majority of the compounds that are used to control ticks among cattle. These methods have also been suggested for the application of these new carbamates [
Clinically healthy Wistar rats between 7 and 8 weeks old (41 females and 41 males) and weighing between 170 and 200 g were used. The rats were housed in polypropylene cages with same-gender companions. The environmental temperature was maintained at
The carbamates used in this study were designed and synthesized at the National Autonomous University of Mexico using a benzimidazole molecule as the structural base. The carbamates were synthesized by reacting aryl- and alkylamines with sodium hydride and benzene diethylcarbonate and were then purified using column chromatography. Next, the products were recrystallized. The compounds were structurally characterized based on their infrared spectra, hydrogen and carbon-13 nuclear magnetic resonance (purity 98%), and mass spectrometry [
Chemical structures and molecular weights of the evaluated carbamates.
Carbamate identification code |
Structure | Molecular weight (g/mol) |
---|---|---|
LQM 919 |
|
244 |
LQM 996 |
|
199.63 |
The rats were randomly assigned to 1 of 9 groups. The numbers of rats and treatments for each group are shown in Table
Distributions of the experimental groups.
Group | Treatment per day | Rats: number and sex |
---|---|---|
1 | Control group (0.1% DMSO) | 5 males and 5 females |
2 | LQM 919 (12.5 mg/kg/day) | 5 males and 5 females |
3 | LQM 919 (25 mg/kg/day) | 5 males and 5 females |
4 | LQM 919 (50 mg/kg/day) | 5 males and 5 females |
5 | LQM 919 (25 mg/kg/day), satellite group | 3 males and 3 females |
6 | LQM 996 (12.5 mg/kg/day) | 5 males and 5 females |
7 | LQM 996 (25 mg/kg/day) | 5 males and 5 females |
8 | LQM 996 (50 mg/kg/day) | 5 males and 5 females |
9 | LQM 996 (25 mg/kg/day), satellite group | 3 males and 3 females |
The carbamates were administered in the drinking water daily for 90 days. The concentrations of carbamates were adjusted every 7 days to account for changes in the weights of the rats and average daily water consumptions. At the end of the 90 days, the rats in groups 1, 2, 3, 4, 6, 7, and 8 were euthanized using humane procedures [
After euthanasia, all rats were necropsied, and the pathological findings were recorded. The liver, kidneys, brain, and spleen were weighed, and their relative weights were calculated (i.e., the % of body weight). Samples were taken from the heart, liver, stomach, intestine, kidneys, brain, spleen, testicles, ovaries, and uterus for histopathology. Furthermore, blood and liver samples were collected for biochemical analyses.
The rats were observed twice daily from 7 days before the commencement of exposure until the 90-day exposure to the carbamates was completed. The satellite groups were observed for additional 21 days. The skin, mucosa, eyes, respiratory frequencies, nasal secretions, salivation, presence of tremors, convulsions, changes in activity levels, posture, gait, sensory responses to stimuli, and strange behaviors were systematically evaluated according to the table proposed by Morton and Griffiths [
For the histopathological examinations, portions of organs were fixed in 4% paraformaldehyde and embedded in paraffin. The embedded organs were cut into 5
Plasma was obtained from the blood samples and frozen at −80°C until processing. The plasma levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), and gamma-glutamyl transferase (GGT) were measured using the methods described by Reitman and Frankel [
Samples of 0.5 cm3 in size were collected from the livers of all euthanized rats to estimate the oxidation-reduction states of the livers of the rats that were exposed to the carbamates. These samples were submerged in a buffer solution (PBS, pH 7.2, sodium azide 15 mM, PMSF 1 mM, Triton X-100 0.1% and EDTA 5 mM) and frozen in liquid nitrogen until they were processed. The tissues were thawed on ice, the original buffer solution was discarded, new buffer solution was added, and the samples were mechanically homogenized, sonicated (3 pulses, 10 seconds, 50% amplitude), and centrifuged (13000 g, 7° to 4°C). An equivalent volume of 2.5% perchloric acid was added to the supernatant (40
The data regarding weight gain, relative organ weights, food and water consumption, hematocrit, percentages of reticulocytes in the blood, plasma levels of AST, GGT, LDH, ALT, CHE, and creatinine, and TBARS concentrations in the liver were analyzed using a one-way ANOVA, and differences between the means were established with Fisher’s
The administration of the carbamates LQM 919 or LQM 996 did not cause mortality in any rats. No clinical signs or adverse effects associated with toxicity were observed in the rats that were exposed to the carbamates throughout the 90 days of exposure or during the additional observation time of the satellite groups.
The average daily food and water consumptions are shown in Figure
Food and water consumptions of the female and male Wistar rats that were subchronically (90 days) exposed to LQM 919 or LQM 996. Sg = satellite group during carbamate exposure (90 days), SgP = satellite group during the recovery time (21 days). Different letters indicate significant differences between the means (
The average weight gains of rats exposed to carbamates are shown in Figure
Weight gains of the female (f) and male (m) Wistar rats that were subchronically exposed to LQM 919 or LQM 996. Sg = satellite group.
The relative weights of the livers, kidneys, brains, and spleens of the various groups are shown in Table
Weights of the organs relative to the body weights (%) of the female and male Wistar rats that were subchronically exposed to LQM 919 or LQM 996.
Liver, kidney, brain, and spleen weights relative to body weight (%) | ||||||||
---|---|---|---|---|---|---|---|---|
Treatment | Liver | Kidney | Brain | Spleen | ||||
Females | Males | Females | Males | Females | Males | Females | Males | |
LQM 919 (50 mg/kg/day) | 3.43 (±0.18)a | 3.22 (±0.22)a | 0.36 (±0.02)a | 0.35 (±0.04)a | 0.75 (±0.07)a | 0.49 (±0.04)a | 0.38(±0.08)b | 0.28(±0.04)b |
LQM 919 (25 mg/kg/day) | 3.86 (±0.47)a | 3.08 (±0.03)a | 0.33 (±0.01)a | 0.33 (±0.01)a | 0.70 (±0.17)a | 0.41 (±0.14)a | 0.51(±0.17)b | 0.41(±0.14)b |
LQM 919 (12.5 mg/kg/day) | 3.35 (±0.33)a | 3.54 (±0.37)a | 0.35 (±0.04)a | 0.33 (±0.01)a | 0.65 (±0.04)a | 0.52 (±0.10)a | 0.35(±0.04)a | 0.22(±0.01)a |
LQM 919 (25 mg/kg/day) Sg | 3.83 (±0.01)a | 3.60 (±0.24)a | 0.34 (±0.04)a | 0.34 (±0.02)a | 0.67 (±0.05)a | 0.38 (±0.01)a | 0.26 (±0.00)a | 0.18 (±0.03)a |
|
||||||||
LQM 996 (50 mg/kg/day) | 3.96 (±0.34)a | 3.55 (±0.24)a | 0.33 (±0.02)a | 0.33 (±0.01)a | 0.71 (±0.08)a | 0.47 (±0.04)a | 0.47 (±0.06)b | 0.44 (±0.08)b |
LQM 996 (25 mg/kg/day) | 3.90 (±0.53)a | 3.60 (±0.03)a | 0.35 (±0.02)a | 0.35 (±0.05)a | 0.72 (±0.09)a | 0.48 (±0.05)a | 0.55 (±0.07)b | 0.39 (±0.08)b |
LQM 996 (12.5 mg/kg/day) | 3.69 (±0.39)a | 3.46 (±0.30)a | 0.33 (±0.03)a | 0.33 (±0.03)a | 0.69 (±0.10)a | 0.50 (±0.08)a | 0.37 (±0.04)a | 0.31 (±0.06)b |
LQM 996 (25 mg/kg/day) Sg | 3.53 (±0.40)a | 3.51 (±0.06)a | 0.34 (±0.00)a | 0.33 (±0.03)a | 0.72 (±0.15)a | 0.38 (±0.03)a | 0.25 (±0.01)a | 0.24 (±0.04)a |
|
||||||||
Control group | 3.59 (±0.21)a | 3.25 (±0.17)a | 0.33 (±0.01)a | 0.32 (±0.02)a | 0.69 (±0.08)a | 0.46 (±0.03)a | 0.26 (±0.02)a | 0.19 (±0.03)a |
Sg: satellite group. Different letters indicate statistical differences between means (
Tables
Effects of subchronic exposure to LQM 919 on selected parameters in male and female Wistar rats.
Parameter | Group | |||||||||
---|---|---|---|---|---|---|---|---|---|---|
Females | Males | |||||||||
Control (—) | 12.5 mg/kg/day | 25 mg/kg/day | 25 mg/kg/day (Sg) | 50 mg/kg/day | Control (—) | 12.5 mg/kg/day | 25 mg/kg/day | 25 mg/kg/day (Sg) | 50 mg/kg/day | |
AST (U/L) | 238.2 (±78.13) | 259.0 (±130.4) | 152.0 (±17.12) | 125.6 (±2.81) | 202.3 (±72.55) | 195.8 (±70.16) | 166.5 (±28.12) | 276.7 (±110.3) | 358.2 (±49.30)* | 237.1 (±62.47) |
ALT (U/L) | 61.52 (±19.55) | 51.34 (±12.73) | 68.68 (±29.46) | 76.13 (±5.54) | 59.20 (±9.31) | 60.64 (±32.21) | 76.05 (±1.96) | 63.27 (±12.37) | 45.97 (±10.45) | 73.18 (±8.11) |
LDH (U/L) | 924.2 (±285.6) | 851.3 (±192.9) | 410.5 (±95.73) | 370.7 (±62.00) | 664.6 (±435.2) | 739.5 (±334.1) | 558.0 (±225.9) | 553.7 (±246.8) | 660.7 (±263.0) | 872.0 (±464.4) |
GGT (U/L) | 1.71 (±2.28) | 1.08 (±1.14) | 0.77 (±0.90) | 0.38 (±0.02) | 0.18 (±0.26) | 0.34 (±0.25) | 0.51 (±0.42) | 0.43 (±0.75) | 0.25 (±0.42) | 1.33 (±0.99) |
CHE (U/L) | 953.9 (±526.7) | 1404 (±794.8) | 982.2 (±116.6) | 1132 (±444.2) | 1088 (±137.8) | 344.3 (±111.1) | 459.0 (±138.5) | 466.2 (±96.50) | 344.6 (±33.32) | 237.28 (±140.31) |
Creatinin (mg/dL) | 0.44 (±0.20) | 0.64 (±0.07) | 0.68 (±0.09) | 0.53 (±0.16) | 0.75 (±0.16)* | 0.54 (±0.12) | 0.65 (±0.14) | 0.65 (±0.15) | 0.46 (±0.04) | 0.74 (±0.01)* |
Total protein (g/dL) | 5.83 (±1.07) | 6.85 (±0.67) | 7.26 (±0.43) | 7.42 (±0.18) | 6.61 (±0.98) | 5.52 (±0.76) | 6.52 (±0.29) | 6.27 (±1.48) | 5.80 (±0.27) | 7.08 (±0.28)* |
Albumin (g/dL) | 2.36 (±1.05) | 1.98 (±0.87) | 3.71 (±0.32) | 2.32 (±1.44) | 3.09 (±0.77) | 1.72 (±0.86) | 2.72 (±0.95) | 1.87 (±0.10) | 2.36 (±0.28) | 1.99 (±0.91) |
Globulin (g/dL) | 3.47 (±0.89) | 4.87 (±1.45) | 3.55 (±0.13) | 5.09 (±1.51) | 3.52 (±1.35) | 3.80 (±0.27) | 3.81 (±1.67) | 4.40 (±1.56) | 3.45 (±0.55) | 5.09 (±0.82) |
Reticulocytes (%) | 4.21 (±0.35) | 6.13 (±0.74) | 5.78 (±0.72) | 3.87 (±0.79) | 6.66 (±1.49)* | 3.93 (±0.77) | 5.44 (±1.07)* | 6.15 (±0.53)* | 2.75 (±0.95) | 5.87 (±0.66)* |
Hematocrit (%) | 36.6 (±2.92) | 41.70 (±2.16) | 33.20 (±8.78) | 29.33 (±9.45) | 39.80 (±6.41) | 35.70 (±3.89) | 43.00 (±2.82) | 28.33 (±6.65) | 37.33 (±3.05) | 48.00 (±5.56)* |
A/G | 0.74 (±0.39) | 0.49 (±0.45) | 1.04 (±0.06) | 0.54 (±0.44) | 1.00 (±0.39) | 0.46 (±0.24) | 0.76 (±0.34) | 0.48 (±0.24) | 0.70 (±0.19) | 0.43 (±0.29) |
AST: aspartate aminotransferase, ALT: alanine aminotransferase, LDH: lactate dehydrogenase, GGT: gamma-glutamyltransferase, CHE: cholinesterase, A/G: albumin and globulin, Sg: satellite group. Values are presented as the means ± the SDs.
Effects of subchronic exposure to LQM 996 on selected parameters in male and female Wistar rats.
Parameter | Group | |||||||||
---|---|---|---|---|---|---|---|---|---|---|
Females | Males | |||||||||
Control (—) | 12.5 mg/kg/day | 25 mg/kg/day | 25 mg/kg/day (Sg) | 50 mg/kg/day | Control (—) | 12.5 mg/kg/day | 25 mg/kg/day | 25 mg/kg/day (Sg) | 50 mg/kg/day | |
AST (U/L) | 238.2 (±78.13) | 263.4 (±82.12) | 192.3 (±84.08) | 181.8 (±23.10) | 207.5 (±96.51) | 195.8 (±70.16) | 265.6 (±26.8) | 167.8 (±30.81) | 196.9 (±60.23) | 168.3 (±18.03) |
ALT (U/L) | 61.52 (±19.55) | 90.14 (±84.82) | 44.37 (±21.49) | 83.43 (±17.10) | 52.26 (±13.00) | 60.64 (±32.21) | 74.84 (±58.61) | 38.33 (±12.79) | 72.70 (±15.67) | 63.04 (±15.46) |
LDH (U/L) | 924.2 (±285.6) | 908.8 (±279.8) | 506.0 (±241.0) | 547.0 (±74.75) | 609.0 (±249.0) | 739.5 (±334.1) | 942.4 (±310.5) | 441.0 (±146.5) | 694.0 (±387.2) | 607.8 (±143.7) |
GGT (U/L) | 1.71 (±2.28) | — | 1.29 (±1.99) | 1.56 (±0.36) | 1.62 (±1.57) | 0.34 (±0.25) | 2.24 (±3.14) | 6.29 (±4.82)* | 2.10 (±1.58) | 6.59 (±5.35)* |
CHE (U/L) | 953.9 (±526.7) | 1505 (±348.6) | 984.6 (±210.9) | 1754 (±838.4) | 892.8 (±317.8) | 344.3 (±111.1) | 546.4 (±308.4) | 385.0 (±71.40) | 716.5 (±488.1) | 528.0 (±94.73) |
Creatinin (mg/dL) | 0.44 (±0.20) | 0.43 (±0.07) | 0.44 (±0.02) | 0.38 (±0.05) | 0.49 (±0.10) | 0.54 (±0.12) | 0.50 (±0.12) | 0.60 (±0.10) | 0.52 (±0.09) | 0.54 (±0.17) |
Total protein (g/dL) | 5.83 (±1.07) | 6.92 (±1.23) | 5.88 (±1.12) | 6.98 (±0.06) | 5.93 (±0.08) | 5.52 (±0.76) | 5.96 (±0.39) | 5.95 (±0.37) | 6.26 (±0.71) | 6.38 (±0.72) |
Albumin (g/dL) | 2.36 (±1.05) | 2.40 (±1.31) | 1.83 (±1.51) | 2.04 (±1.09) | 1.87 (±1.04) | 1.72 (±0.86) | 2.18 (±0.72) | 2.16 (±0.92) | 2.40 (±0.20) | 2.01 (±0.81) |
Globulin (g/dL) | 3.47 (±0.89) | 4.52 (±1.53) | 4.05 (±1.36) | 4.94 (±1.12) | 4.06 (±1.01) | 3.80 (±0.27) | 3.79 (±0.84) | 3.79 (±0.55) | 3.86 (±0.84) | 4.38 (±1.43) |
Reticulocytes (%) | 4.21 (±0.35) | 8.82 (±1.61)* | 10.06 (±2.15)* | 2.34 (±0.25) | 9.92 (±3.42)* | 3.93 (±0.77) | 5.23 (±1.44)* | 6.65 (±1.15)* | 3.38 (±0.37) | 6.60 (±0.97)* |
Hematocrit (%) | 36.62 (±2.92) | 38.00 (±6.97) | 34.4 (±9.52) | 35.00 (±11.00) | 36.80 (±8.49) | 35.70 (±3.89) | 44.25 (±3.20)* | 37.30 (±6.41) | 41.33 (±2.51) | 43.60 (±4.72)* |
A/G | 0.74 (±0.39) | 0.64 (±0.46) | 0.56 (±0.63) | 0.47 (±0.34) | 0.53 (±0.37) | 0.46 (±0.24) | 0.62 (±0.31) | 0.60 (±0.30) | 0.65 (±0.21) | 0.55 (±0.36) |
AST: aspartate aminotransferase, ALT: alanine aminotransferase, LDH: lactate dehydrogenase, GGT: gamma-glutamyltransferase, CHE: cholinesterase, A/G: albumin and globulin, Sg: satellite group. Values are presented as the means ± the SDs.
The males that were exposed to 50 mg/kg of either of the carbamates exhibited elevated hematocrit (
The histopathological changes observed in the groups that were exposed to 25 or 50 mg/kg of either carbamate, including the satellite groups, were similar. Moderate albuminous degeneration, slight periacinar vacuolar degeneration, moderate diffuse congestion, and some hepatocytes with highly euchromatic nuclei or grayish cytoplasm were observed in the livers. The kidneys exhibited slight interstitial cell infiltration. Furthermore, hemosiderin deposits were observed in the spleen, kidneys, and ovaries (Figure
Histopathological findings from the rats that were subchronically exposed to the carbamate LQM 919. (A) Liver section (10x) of an unexposed rat (control). (B) Liver section (20x) of a rat that was exposed to 50 mg/kg/day of LQM 919 that exhibits highly euchromatic nuclei (hen), hepatocytes with two nuclei (dn), and colestasis (c). (C) Liver section (10x) that exhibits a degenerated area and congestion. (D) Kidney section (10x) of an unexposed rat (control). (E) Kidney section (10x) of a rat that was exposed to 50 mg/kg/day of carbamate that shows hemosiderin deposits (h). (F) Ovary section (20x) of an exposed rat (50 mg/kg/day) showing hemosiderin deposits (h).
Figure
Thiobarbituric reactive substances (TBARS) in the livers of female and male Wistar rats that were subchronically exposed to the carbamates LQM 919 or LQM 996. Sg = satellite group. Different letters indicate significant differences between the means (
Subchronic oral toxicity studies evaluate the adverse effects caused by prolonged exposures of animals to substances and provide information about the negative effects of the substances on target organs and the cumulative effects of the substances. Furthermore, these studies determine the dosage at which no observable adverse effects are present. In this study, exposure to the carbamates LQM 919 and LQM 996 was well-tolerated, and the absence of deaths or clinical signs of toxicity related to exposure indicates that prolonged exposure to the evaluated carbamates is relatively harmless.
It has been proposed that compounds with toxic potential affect the consumption of food, metabolic processes, and, ultimately, weight gain. Studies that have evaluated the long-term effects of compounds typically consider reductions in weight gain that exceed 10% to be detrimental to the animals [
Adequate water consumption is essential for the normal physiological processes of an animal. Some substances have been observed to modify water consumption and therefore affect animals’ metabolisms [
Detoxification processes primarily occur in the liver; together with the kidneys, the liver experiences the greatest exposure to xenobiotics and/or their metabolites. The susceptibilities of the liver and renal tissues to pesticide exposure-induced stress depend on the general balance between the degree of oxidative stress and antioxidant capacities [
Susceptibility to xenobiotics can vary depending on sex, and the differences in liver susceptibilities to the evaluated carbamates between the males and females observed in this study may have been due to the greater liver metabolism and chemical breakdown capacities of the males (males possess greater quantities of cytochrome P450), which also make males more or less susceptible to the toxicity of a compound depending on whether that compound is metabolized in a bioactivation or detoxification pathway [
Some pesticide products have been observed to have hemolytic activities. In this study, we did not directly evaluate such activity. Nevertheless, some findings, such as splenomegaly and the presence of hemosiderin in various organs (spleen, kidneys, and ovaries) suggest that the rate of erythrocyte destruction was increased. Moreover, the observed increases in reticulocytes and hematocrit in the groups exposed to the highest dosages may be related to a compensatory response of the bone marrow.
The evaluated carbamates have inhibitory effects on the development of ovary cells in
When a new chemical is evaluated, it is important to determine its nonobserved adverse effect level (NOAEL) to assess the risks associated with its use. Based on the results of this study, the evaluated carbamates exhibited a NOAEL of 12.5 mg/kg for female and male Wistar rats. Nevertheless, the results obtained at the 25 and 50 mg/kg dosages of the evaluated carbamates suggest caution and the need for complementary studies that evaluate the
The exposure to evaluated carbamates produced alterations in water consumption, hematocrit, percentages of reticulocytes, plasma proteins, some biochemical parameters, thiobarbituric acid reactive substances, the relative weight of the spleen, and slight pathological alterations in the liver. The low severity and the reversibility of these alterations suggest that the evaluated carbamates have low subchronic toxicity. The nonobserved adverse effect levels (NOAELs) of the evaluated carbamates were 12.5 mg/kg/day for both the female and male rats.
The authors declare that there is no conflict of interests regarding the publication of this paper.
This study was supported by the PAPIIT/UNAM Projects no. IT223311, IN216913, IG200813, and IT202412. This work was conducted as part of the Projects Cátedra: Diseño de Sustancias Bioactivas and Cátedra: Epidemiología de las Enfermedades Parasitarias of FESC-UNAM-2012. The authors would like to thank Cuenca-Verde C., Romero-Sánchez Y., Jiménez D., and Hernández M., from FESC-UNAM, for their technical assistance.