A method for detecting a substance toxicity threshold based on a rat drinking water experiment
By utilizing spontaneous drinking preference behavior in rat drinking experiments, this study resolves the ethical controversies and human intervention issues associated with existing toxicity detection methods, achieving accurate and convenient toxicity threshold assessment applicable to the toxicity evaluation of environmental pollutants, chemical substances, and drugs.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- PEKING UNIV
- Filing Date
- 2026-01-20
- Publication Date
- 2026-06-09
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of environmental toxicology technology and relates to a method for detecting the toxicity threshold of a substance based on a rat drinking water experiment. Background Technology
[0002] Among the existing technical problems, traditional toxicity detection methods (such as half-maximal inhibitory concentration determination) rely on animal death or pathological damage as an endpoint, which raises ethical controversies and fails to reflect the behavioral toxicity of low-dose exposures. Existing behavioral detection methods (such as conditioned taste aversion) require human intervention and cannot reflect the animal's ability to spontaneously recognize toxicity. Currently, there is a lack of a simple and easy-to-operate detection method that can quantitatively assess the toxicity threshold of a substance. Summary of the Invention
[0003] The purpose of this invention is to provide a method for detecting the toxicity threshold of substances based on a rat drinking experiment. This method achieves accurate determination of the toxicity threshold through spontaneous drinking preference behavior and is used to assess the toxic effects of environmental pollutants, chemicals, or drugs on organisms and their thresholds.
[0004] The present invention provides a method for detecting the toxicity threshold of a substance based on a rat drinking experiment, comprising the following steps: (1) drinking preference experiment 1) Adaptation period: Rats were allowed free access to water and food, with two bottles of purified water of equal volume, so that there was no significant difference in the amount of purified water consumed by the rats from the two bottles; 2) Observation period: In step 1), each rat was given an equal volume of water containing the substance being studied and a bottle of purified water. The amount of water consumed and the weight of the rat were recorded at regular intervals. 3) Recovery period: In step 2), each rat was provided with two bottles of purified water so that there was no significant difference in the amount of purified water consumed by the rats from the two bottles; (2) Determination of toxicity threshold Based on the experimental data in steps (1)-2), the amount of drinking water and pure water was compared to determine the time when the rats began to significantly avoid the water, the amount of toxic substance in the rats at that time was calculated, and the toxicity threshold of the rats was calculated. (3) Dosage conversion from rat to human Based on interspecies dose conversion using body surface area, the toxicity threshold of the rats is first converted into the equivalent dose for humans to obtain the acute exposure dose. Then, the acute exposure dose is converted into the chronic exposure dose for humans to obtain the substance toxicity threshold.
[0005] In the above method, in step (1)-1), before the rats have free access to water and food, the rats adapt to the experimental environment for 5 to 7 days, specifically 5 days.
[0006] In the above method, step (1)-1) also includes the following steps: record the amount of water consumed in the two bottles during the adaptation period, and analyze whether there is a significant difference in the amount of water consumed in the two bottles through a paired T test. If there is no significant difference, then proceed to the observation period. The main purpose of this step is to ensure that the difference in water consumption during the observation period comes from the difference in water quality.
[0007] In the above method, steps (1)-2) also include the following steps: weigh the water bottle at the beginning and record the initial volume of water, weigh the water bottle every day and record the volume of water consumed in a day, record the amount of water consumed, record for at least 7 days, and weigh the rat's body weight every day. The substance under study is selected from at least one of environmental pollutants, chemical substances, and pharmaceuticals; When the substance under study is an environmental pollutant, the mass percentage concentration of the substance under study in the water is 0.05% to 0.15%; when the substance under study is a chemical substance, the mass percentage concentration of the substance under study in the water is 0.01% to 0.10%; when the substance under study is a drug, the mass percentage concentration of the substance under study in the water is 0.02% to 0.20%, and the drug concentration is determined in conjunction with preliminary experiments to verify animal tolerance and avoid abnormal drinking behavior.
[0008] In the above method, steps (1)-3) also include the following steps: record the amount of water consumed in the two bottles during the recovery period, and analyze whether there is a significant difference in the amount of water consumed in the two bottles through a paired T test. If there is no significant difference for three consecutive days, the data is considered reliable and the difference in the amount of water consumed by rats during the observation period is due to the difference in water quality. If there is a significant difference, the experimental data is not reliable and step (1) needs to be repeated.
[0009] In the above method, the comparison of the amount of drinking water and pure water in step (2) is handled as follows: Based on the experimental data in steps (1)-2), the amount of drinking water and pure water is compared by paired T test to find the time when the rats begin to significantly avoid the water. When the P of the T test is less than 0.05, the amount of drinking water is significantly less than the amount of drinking pure water. The mass of toxic substance in the rat's body at that time is calculated, and the toxicity threshold is calculated according to the following formula I. Formula I: Toxicity threshold = Total cumulative toxicity / Body weight, mg / kg.
[0010] In steps (1)-2), the total amount of toxic substance is equal to the volume of the drinking water containing the substance multiplied by the mass percentage concentration of the water containing the substance. The volume of the drinking water containing the substance is the time when the rat began to significantly avoid the water containing the substance and the volume of the water containing the substance that it drank before that.
[0011] In the above method, step (3) calculates the toxicity threshold of the rats according to the following formula II, converting it into the equivalent dose for humans: Formula II: Human equivalent dose = Rat dose × (Rat body weight / Human body weight) 0.33 .
[0012] In the above method, when the human chronic exposure dose in step (3) is a 70-year chronic exposure dose, it is calculated according to the following formula III: Chronic threshold = Acute threshold / (t) 慢性 / t 急性 ) 0.25 .
[0013] In the above method, step (3) also includes the following step: when the exposure route of the substance under study is different from that of oral administration, exposure route correction needs to be added.
[0014] In the above method, when the substance under study is atmospheric particulate matter and the exposure route is inhalation, the difference in bioavailability between the two routes needs to be considered and calculated according to Equation IV: Formula IV: Inhaled equivalent dose = Human equivalent dose × (Oral bioavailability / Inhaled bioavailability).
[0015] The present invention has the following beneficial effects: 1. Accurate determination of toxicity thresholds: Toxicity assessment of low-dose exposures is achieved through spontaneous behavioral identification.
[0016] 2. Easy to operate: No complicated equipment or human intervention is required, making it suitable for large-scale screening.
[0017] 3. Wide range of applications: Applicable to the toxicity assessment and determination of non-destructive thresholds for various substances such as environmental pollutants, chemical substances, and pharmaceuticals. Attached Figure Description
[0018] Figure 1 Data on water intake (two bottles of purified water) for rats during the adaptation period.
[0019] Figure 2 This data represents drinking water data for Hohhot from day 1 to day 4 (data for drinking water that significantly avoids particulate matter during the drinking water period).
[0020] Figure 3 The relative percentage of bacteria in particulate matter in five cities.
[0021] Figure 4 The relative proportion of fungi in particulate matter in five cities.
[0022] Figure 5 The relative proportions of metallic elements (alkali metals, alkaline earth metals, and metalloids) in particulate matter from five cities.
[0023] Figure 6 The relative proportions of metallic elements (transition metals) in particulate matter from five cities.
[0024] Figure 7 The relative proportions of metallic elements (main group metals and lanthanides) in particulate matter from five cities.
[0025] Figure 8 The study identified neurotoxicity markers, inflammatory factor markers, and miRNA markers caused by particulate matter in five cities. Detailed Implementation
[0026] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.
[0027] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.
[0028] This invention provides a method for detecting the toxicity threshold of a substance based on a rat drinking water experiment: Step 1: Drinking Preference Experiment Adaptation period: Rats adapt to the experimental environment for 5 days with free access to water and food. Two bottles of purified water are provided to ensure that the rats adapt to the choice between the two bottles. The amount of water consumed by the two bottles during the adaptation period is recorded. Paired T test is used to analyze whether there is a significant difference in the amount of water consumed by the two bottles. If there is no significant difference, the observation period can be started. The main purpose of this step is to ensure that the difference in water consumption during the observation period comes from the difference in water quality. Observation period: Each rat was given two water bottles, one containing raw water and the other containing purified water. The weight of the water bottles was measured at the beginning, and then every 24 hours. The amount of water consumed was recorded for at least 7 days. The rat's body weight was measured daily. Recovery period: Each rat was provided with two bottles of purified water. The amount of water consumed by the rats in the two bottles was recorded during the recovery period. A paired T-test was used to analyze whether there was a significant difference in the amount of water consumed by the two bottles. If there was no significant difference for three consecutive days, the data was considered reliable and the difference in water consumption by the rats during the observation period was due to the difference in water quality. If there was a significant difference, the experimental data was not reliable and the experiment needed to be repeated. Step 2: Toxicity threshold determination Based on the experimental data, the amount of drinking water containing substances and pure water was compared using a paired T-test to find the time when rats began to significantly avoid substances. The mass of toxin in the rats' bodies at that time was calculated, and the toxicity threshold was calculated: Toxicity threshold = Total cumulative toxin / Body weight (mg / kg). Step 3: Dosage conversion from rats to humans First, based on interspecies dose conversion using body surface area, the toxicity threshold of rats was converted into the equivalent dose for humans using the following formula: Human equivalent dose = Rat dose × (Rat body weight / Human body weight) 0.33 Secondly, the acute exposure dose is converted into the human chronic exposure dose. Taking 70 years as an example, the chronic threshold = acute threshold / (t)慢性 / t 急性 ) 0.25 Finally, if the exposure route of the substance under study is different from that of oral administration, an exposure route correction must be added. For example, if the main exposure route of atmospheric particulate matter is inhalation, the difference in bioavailability between the two routes must be added. Inhalation equivalent dose = human equivalent dose × (oral bioavailability / inhalation bioavailability).
[0029] Example: Toxicity threshold detection of atmospheric particulate matter in four cities Animal grouping: 36 10-week-old Wistar rats were selected and divided into 4 groups of 9 rats each, based on similar body weight.
[0030] Experiment content: During the adaptation period, rats had free access to water and food, and were provided with two bottles of purified water. To ensure there was no significant difference in the amount of water consumed from the two bottles, the rats entered the observation period. During this period, each rat was provided with one bottle of particulate water corresponding to the city and one bottle of purified water. The amount of particulate water and purified water consumed by the rats was recorded over 7 days. Figure 1 As shown.
[0031] Particulate matter collection method: Environmental particulate matter samples were collected using automotive air conditioning filters. From December 2020 to April 2021, samples were collected from 31 major cities in China, with 15 samples collected from each city. The 15 samples from each city were combined into one composite sample. This composite sample was then passed through a 10 μm filter to obtain particles <10 μm. The <10 μm particles were then dissolved in purified water to prepare a 1 g / L water-soluble suspension for subsequent drinking water experiments (see non-patent literature: Lu Zhang, Maosheng Yao, Ambient particle composition and toxicity in 31 major cities in China, Fundamental Research, Volume 4, Issue 3, 2024; the 31 cities mentioned above are the same as those in the aforementioned non-patent literature). The specific biological composition information of the particulate matter is as follows: Figure 3 , Figure 4 As shown, the heavy metal composition information of particulate matter is as follows: Figure 5 , 6 As shown in Figures 7 and 8, the neurotoxic changes caused by particulate matter are as follows: Figure 8 As shown.
[0032] Experimental results: Rats in the Beijing, Hohhot, Guangzhou, Taiyuan, and Xi'an groups began to show significant avoidance of particulate water on days 7, 4, 4, 2, and 2, respectively (taking Hohhot as an example, the specific measurement data are as follows).Figure 2 As shown in the figure, the cumulative particulate matter levels in the five groups of rats at that time were 215.61 mg, 110 mg, 114.92 mg, 18.63 mg, and 29.78 mg, respectively. The calculation process is shown below using the Beijing group as an example: 1. Dosage conversion from rats to humans (oral → inhalation) (1) Interspecies dose conversion based on body surface area (converting oral doses in rats to equivalent oral doses in humans) Using the body surface area correction method: Human Equivalent Dose (HED) = Rat Dose × (Rat Body Weight / Human Body Weight) 0.33 The average weight of a rat is 0.38 kg, and the average weight of a human is 70 kg: HED = 57.03 mg / kg × (0.38 / 70) 0.33 ≈57.03 ×0.179≈10.21 mg / kg (2) Acute → Chronic (7 days → Lifetime 70 years): Chronic threshold = Acute threshold / (t) 慢性 / t 急性 ) 0.25 =10.21 / (25550 / 7) 0.25 = 1.31 mg / kg Considering population sensitivity and cumulative toxicity, an additional safety factor of 100× can be added: the chronic threshold is 0.0131 mg / kg. (3) Correction of exposure route (oral → inhalation) Difference in bioavailability between the two pathways: f 口服 =10%; f 吸入 =Respiratory tract deposition rate × absorption efficiency = 15% × 30% = 4.5% a. Respiratory tract deposition rate: The particulate matter in this study corresponds to a particle size of <100μm. This study only discusses inhalable particulate matter. The resting minute ventilation rate for adults is 6L (ventilation rate = tidal volume × frequency = 500 ml × 12 breaths / min). The total deposition rate is approximately 60% for <10μm particles in all respiratory tracts, with the majority in the upper respiratory tract. The alveolar deposition rate is only 15% for <10μm particles in the alveolar region.
[0033] b. Absorption efficiency: The proportion of particulate matter deposited in the alveoli that can be absorbed into the bloodstream. The alveolar absorption rate of particulate matter <10μm is 30%.
[0034] Inhaled equivalent dose = HED × (f 口服 / f 吸入 =0.0131×2.22≈0.03 mg / kg 2. Calculation of human inhalation dose (1) Deposition of particulate matter in the respiratory tract If the concentration of particulate matter in the air is C mg / m³, and the daily air intake is 17 m³, the daily alveolar deposition volume = C × 17 × 0.15 = 2.55 × C mg (Calculated based on a body weight of 70 kg: 2.55 × C / 70 = 0.036 C mg / kg) (2) Derivation of safety limits The threshold for long-term human exposure is 0.03 mg / kg (from step 1), based on a daily exposure threshold of no more than 1% (chronic toxicity prevention principle): 0.036×C ≤ 0.01×0.03 mg / kg C ≤ 0.0083 mg / m 3 Based on the calculation formula, the equivalent inhalation dose (toxicity threshold) of particulate matter in Beijing, Hohhot, Guangzhou, Taiyuan, and Xi'an are 0.03, 0.024, 0.021, and 0.0091 mg / kg, respectively. Further derivation of human safety limits follows. If the concentration of particulate matter in the air is C mg / m³, the daily inhaled air volume is 17 m³, and the deposition rate of particles smaller than 10 μm in the alveolar region is only 15%, then the daily alveolar deposition of particulate matter can be calculated as C × 17 × 0.15 = 2.55 × C mg. Assuming a human weight of 70 kg, the daily deposition per kilogram of body weight is 0.036 × C mg / kg. According to the principle of chronic toxicity prevention, the daily particulate matter exposure should not exceed the 1% threshold, and 0.036 × C ≤ 0.01 × toxicity threshold. Therefore, the safe concentrations of particulate matter for long-term human exposure in Beijing, Guangzhou, Hohhot, and Taiyuan can be calculated to be 8.3, 6.84, 5.8, and 2.54 μg / m³, respectively. 3 .
Claims
1. A method for detecting the toxicity threshold of a substance based on a rat drinking experiment, comprising the following steps: (1) drinking preference experiment 1) Adaptation period: Rats were allowed free access to water and food, with two bottles of purified water of equal volume, so that there was no significant difference in the amount of purified water consumed by the rats from the two bottles; 2) Observation period: In step 1), each rat was given an equal volume of water containing the substance being studied and a bottle of purified water. The amount of water consumed and the weight of the rat were recorded at regular intervals. 3) Recovery period: In step 2), each rat was provided with two bottles of purified water so that there was no significant difference in the amount of purified water consumed by the rats from the two bottles; (2) Determination of toxicity threshold Based on the experimental data in steps (1)-2), the amount of drinking water and pure water was compared to determine the time when the rats began to significantly avoid the water, the amount of toxic substance in the rats at that time was calculated, and the toxicity threshold of the rats was calculated. (3) Dosage conversion from rat to human Based on interspecies dose conversion using body surface area, the toxicity threshold of the rats is first converted into the equivalent dose for humans to obtain the acute exposure dose. Then, the acute exposure dose is converted into the chronic exposure dose for humans to obtain the substance toxicity threshold.
2. The method according to claim 1, characterized in that, In step (1)-1), the rats are allowed to acclimatize to the experimental environment for 5-7 days before they are allowed to drink water and eat freely.
3. The method according to claim 1 or 2, characterized in that, Step (1)-1) also includes the following steps: record the amount of water consumed in the two bottles during the adaptation period, and analyze whether there is a significant difference in the amount of water consumed in the two bottles through a paired T test. If there is no significant difference, then proceed to the observation period.
4. The method according to claim 1 or 2, characterized in that, Steps (1)-2) also include the following steps: weigh the water bottle at the beginning and record the initial volume of water. Weigh the water bottle every day and record the volume of water consumed in a day. Record the amount of water consumed for at least 7 days. Weigh the rat's body weight every day. The substance under study is selected from at least one of environmental pollutants, chemical substances, and pharmaceuticals; When the substance under study is an environmental pollutant, the mass percentage concentration of the substance under study in the water is 0.05% to 0.15%; when the substance under study is a chemical substance, the mass percentage concentration of the substance under study in the water is 0.01% to 0.10%; when the substance under study is a drug, the mass percentage concentration of the substance under study in the water is 0.02% to 0.20%, and the drug concentration is determined in conjunction with preliminary experiments to verify animal tolerance.
5. The method according to claim 1 or 2, characterized in that, Steps (1)-3) also include the following steps: record the amount of water consumed in the two bottles during the recovery period, and analyze whether there is a significant difference in the amount of water consumed in the two bottles through a paired T test. If there is no significant difference for three consecutive days, the data is considered reliable; if there is a significant difference, the experimental data is not reliable and step (1) needs to be repeated.
6. The method according to claim 4, characterized in that, The comparison of the amount of drinking water and purified water in step (2) is handled as follows: Based on the experimental data in steps (1)-2), the amount of drinking water and purified water is compared by paired T test to find the time when the rats begin to significantly avoid the water. When the P of the T test is less than 0.05, the amount of drinking water is significantly less than the amount of drinking purified water. The mass of toxic substance in the rats at that time is calculated, and the toxicity threshold is calculated according to the following formula I. Formula I: Toxicity threshold = Total cumulative toxicity / Body weight, mg / kg; In steps (1)-2), the total amount of toxic substance is equal to the volume of the drinking water containing the substance multiplied by the mass percentage concentration of the water containing the substance. The volume of the drinking water containing the substance is the time when the rat began to significantly avoid the water containing the substance and the volume of the water containing the substance that it drank before that.
7. The method according to claim 1 or 6, characterized in that, In step (3), the toxicity threshold of the rats is converted into the equivalent dose for humans according to the following formula II: Formula II: Human equivalent dose = Rat dose × (Rat body weight / Human body weight) 0.33 .
8. The method according to claim 7, characterized in that, In step (3), when the human chronic exposure dose is the chronic exposure dose over 70 years, it is calculated according to the following formula III: Chronic threshold = Acute threshold / (t) 慢性 / t 急性 ) 0.25 .
9. The method according to claim 1 or 8, characterized in that, Step (3) also includes the following step: when the exposure route of the substance under study is different from oral administration, exposure route correction needs to be added.
10. The method according to claim 9, characterized in that, When the substance under study is atmospheric particulate matter, and the exposure route is inhalation, the difference in bioavailability between the two routes needs to be factored in, calculated according to Equation IV: Formula IV: Inhaled equivalent dose = Human equivalent dose × (Oral bioavailability / Inhaled bioavailability).