Some standard content:
National Standard of the People's Republic of China
Toxicological test methods of pesticides for registration
Toxicological test methods ofpesticides for registration
Subject content and scope of application
This standard specifies the basic requirements for the methods and conditions of toxicological tests for pesticide registration. This standard applies to toxicological tests conducted for pesticide registration. 2 Acute oral toxicity test
2.1: From
GB15670—1995
Determine the median lethal dose (LDs.) of the test pesticide on the test animal, observe the clinical manifestations of acute toxic effects, preliminarily estimate the target organs of toxic effects and possible toxic mechanisms; provide reference for the design of dose levels for subchronic, chronic and other toxicity tests; provide a basis for acute toxicity classification and the formulation of safety protection measures. 2.2 Test pesticides
Technical and formulations.
2.3 Experimental Animals
2.3.1 The main selection is rats of young adulthood with clear strains and genetic backgrounds. The weight difference of animals of the same sex in each dose group should be less than 10% of the average weight, and the weight difference of animals of the same sex between groups should be less than 5%. 2.3.2 There are 8 to 10 rats in each dose group (half male and half female). The animals should be observed for one week before the test and can only be used after they are confirmed to be healthy. 2.4 Dose Grouping
2.4.1 At least 4 to 5 dose groups should be set up, and there should be an appropriate dose interval between each dose group. So that each group will have different degrees of toxic effects (mortality) and obtain the dose-effect curve and LD50. 2.4.2 If the dose reaches 5000 mg/kg body weight or more and the animal still does not die, there is no need to conduct a higher dose test. 2.5 Dosage Method and Observation Time
2.5. 1 The animals should be fasted overnight before administration, but not water. After weighing, they should be gavaged once, and food should be fed at least 2 hours after administration. 2.5.2 Oral gavage volume
1 mL for rats based on 100 g body weight, 0.4 mL for mice based on 20 g body weight. Aqueous solution, oil solution or suspension can be used for oral gavage. 2.5.3 Immediately after administration, observe and record the poisoning symptoms of the animals, the time when the symptoms appear and disappear, and the time of death. Continuous observation should be made on the day of administration, and thereafter, observation should be made at least twice a day for a period of 14 days. If delayed new effects appear 96 hours after administration, the observation period should be extended to 3 weeks or 4 weeks.
2.6 Observation indicators
2.6.1 Symptoms of poisoning
Comprehensively observe the occurrence, development process and laws of poisoning, as well as the characteristics of poisoning and the target organs of poisoning. The systems to be observed include: central nervous system and neuromuscular system: abnormal posture, abnormal voice, restlessness, sluggishness, spasm, convulsion paralysis, movement disorder, allergic or slow reaction to external factors;
Approved by the State Administration of Technical Supervision on August 17, 1995 and implemented on January 1, 1996
GB15670-1995
Autonomic nervous system: pupil dilation or constriction, salivation or tearing; respiratory system: nasal discharge, nasal flaring, deep and slow breathing, tachypnea, wasp waist; urogenital system: dirty perineum, secretions, vaginal or breast swelling; skin and fur: congested and purple skin, fluffy and dirty fur; eyes: protruding eyeballs, conjunctival congestion, corneal turbidity; g. Digestive system: diarrhea, anorexia.
2.6.2 Body weight
Weigh the body weight once before administration and at the time of death, and once every 3 days during the observation period. 2.6.3 Generally, pathological histological examination and biochemical index test are not performed, but gross pathological observation should be performed on dead animals. If animals that survive for more than 24 hours have lesions in the naked eye, pathological histological examination should be performed. 2.7 Result evaluation
2.7.1 Calculate the LD5o value using statistical methods. The calculation method is shown in Appendix A (Supplement). 2.7.2 Evaluate according to the acute oral toxicity classification standard of pesticides (see Table 1). Table 1 Acute oral toxicity classification standards
3 Acute dermal toxicity test
3.1 Purpose
Oral LDsomg/kg
50~500
To understand the toxic effects of pesticides on the skin of test animals, such as local skin damage and systemic poisoning, and to find the median lethal dose (LD5.), so as to provide a basis for formulating protective measures in the production and application of pesticides. 3.2 Test pesticides
Original drugs and preparations.
3.3 Test animals
3.3.1 In order to have enough application area, adult rats are preferred, with a body weight requirement of 200~~300g. 3.3.2 8~10 rats (half male and half female) in each dose group. 3.4 Dose grouping
3.4.1 At least 4 dose groups should be set up, and each dose group should have a certain distance so that toxicity and death can occur in the drug administration group. 3.4.2 If there is still no death at the dose of 2000mg/kg, there is no need to conduct a high-dose test. 3.5 Dosage method and observation time
3.5.1 Remove the hair on the back 24 hours before administration. Be careful not to damage the skin. The application area should account for 10% of the body surface area, 4×5cm for rats, 12×14cm2 for rabbits, and 7×10cm2 for guinea pigs.
3.5.2 Use the original solution for liquid pesticides and dilute powdered pesticides with water or solvents. 3.5.3 Fix the animal first, apply a quantitative application in an area slightly smaller than the shaved area, then cover it with a box cover equivalent to the depilated area and fix it with tape. This is to prevent the animal from licking the test drug. 3.5.4 The application duration is 4 hours.
3.5.5 Remove the cover box 4 hours after application, wash off the test pesticide on the skin with warm water, and then conduct local observation. 3.5.6 The observation time is 2 weeks.
3.6 Observation indicators
3.6.1 Clinical manifestations of poisoning, refer to 2.6.1. 3.6.2 Time of death and mortality rate.
3.7 Result evaluation
GB15670—1995
3.7.1 Calculate LDso value by statistical method. See Appendix A (Supplement) for calculation method. 3.7.2
Evaluate according to the classification standard of acute dermal toxicity of pesticides (see Table 2). Table 2 Acute dermal toxicity classification standards
Acute inhalation toxicity test
4.1 Purpose
Dermal, LDso (4 h), mg/kg
20～200
200～2000
>2 000
To test the damage and degree of harm to the respiratory tract and the whole body caused by pesticides (especially highly volatile pesticides) that may enter the body through the respiratory tract. To calculate the median lethal concentration (I.Cso) of inhalation exposure, and provide a basis for the safety evaluation of pesticides and the formulation of protective measures in production and application. 4.2 Test pesticides
Technical and formulations.
4.3 Test Animals
One or more mammals, rats are the preferred animals. 4.3.1
4.3.2 The requirements for the sex, weight and number of animals in each group are the same as those in 2.3. 4.4 Dose Grouping
4.4.1 At least three dose groups should be set up for toxic effects until death. 4.4.2 If there is still no death after 2 hours of administration at a concentration of 2000 mg/m2, there is no need to conduct high-concentration tests. 4.5 Dosage Method and Observation Time
4.5.1 The administration methods include nose and head contact and whole body contact in the poison cabinet. 4.5.2
The air flow, pesticide concentration, temperature and humidity should be kept constant in the poison cabinet, and a slight negative pressure should be maintained in the cabinet to prevent pesticide leakage.
4.5.3 Inhalation administration is generally 2 hours.
4.5.4 Observe for at least 14 days after administration.
4.6 Observation index
4.6.1 Clinical manifestations of poisoning, refer to 2.6.1. 4.6.2 Time of death and mortality rate.
4.7 Result evaluation
4.7.1 Calculate LC5o value by statistical method. See Appendix A (Supplement) for calculation method. 4.7.2
Evaluate according to the classification standard of acute inhalation toxicity of pesticides (see Table 3). Table 3 Classification standard of acute inhalation toxicity
LCso(2 h),mg/m2
20～200
200~2000
>2 000
Acute skin irritation test
5.1 Purpose
GB15670—1995
To determine whether pesticides have irritation or corrosion effects on mammalian skin and estimate similar hazards that may occur when humans come into contact with the pesticides. 5.2 Test pesticides
Technical pesticides and formulations.
5.3 Test animals
5.3.1 Rabbits or guinea pigs, preferably white rabbits. 5.3.2 At least 4 healthy animals with intact skin. 5.4 Dosage
5.4.1 The amount of drug applied per time is 0.5mL or 0.5g. 5.4.2 The animal's own skin is used as a control.
5.5 Test steps
5.5.1 Cut the hair on the back 24 hours before the test, with an area of about 6cm2. 5.5.2 Apply the test pesticide, cover it with gauze, fix it with tape or use other closed covers to prevent the test animals from licking it. 5.5.3 The application duration is generally 4 hours. At the end of the test, wash off the residual pesticide with water or an appropriate solvent, but be careful not to damage the skin.
Further observation is carried out as needed to determine the reversibility of the reaction. The general observation period does not exceed 14 days. 5.5.4
5.6 Result evaluation
Each animal test result is scored for irritation reaction according to Table 4, and the average score is calculated. The intensity is evaluated according to Table 5. Table 4 Skin irritation reaction scoring
Symptoms and degree
A Erythema formation
No erythema
Barely visible
Obvious erythema
Moderate to severe erythema
Purple-red spots with focal formation
B Edema formation
No edema
Barely visible
Edema with clear outline
Edema with a protrusion of about 1 mm
Edema with a protrusion of more than 1 mm and a wider range
No irritation
Mild irritation
Moderate irritation
Strong irritation
GB 15670—1995
Table 5 Skin irritation intensity classification
Note: Pesticides with pH≤2 or pH≥11.5 do not need to undergo this test. 6 Eye irritation test
6.1 Purpose
0. 5～1. 9
To understand the irritation effect and degree of the test pesticide on the eyes, and to provide a basis for safety protection in the production and use of pesticides. 6.2 Test pesticides
Technical materials and preparations, but this test is not required for compounds with strong acid and strong base properties (pH<2, pH≥11.5). 6.3 Test animals
At least 4 adult white domestic rabbits. Check the eyes of the test rabbits 24 hours before the test, and do not use those with abnormalities. 6.4. Dosage
0.1mL of liquid pesticide or 100-fold dilution, no more than 100mg of granular pesticide, and the granules should be ground into fine powder first. 6.5 Dosage method and observation time
6.5.1 Gently pull down the lower eyelid of one side and drop the test pesticide into the conjunctival sac. To prevent the liquid from spilling out, immediately close the eyelid gently for about 1 minute. If the spraying method is used, separate the eyelids and spray quickly for 1 second at a distance of 10 cm in front of the eyes. 6.5.2 Do not wash the eyes within 24 hours after administration.
6.5.3 The contralateral eye is the control.
6.5.4 Check the eyes at 1, 24, 48, and 72 hours after administration. If there is still no irritation reaction after 72 hours, terminate the test. 6.5.5 If corneal damage or reactions occur in other parts of the eye, continue to observe the course of damage and its reversibility. The longest observation period shall not exceed 21 days.
6.5.6 If the irritation reaction does not subside after 72 hours, select at least 4 other domestic animals to observe the effect of eye washing. That is, wash the eyes with saline for 5 minutes at 4s and 30s after the drug is dripped, and observe the eye reaction after washing. 6.6 Observation indicators
In addition to observing the conjunctiva, cornea, and iris, attention should also be paid to damage to other parts. 6.7 Result evaluation
Add the irritation response scores of the cornea, iris, and conjunctiva of each animal according to Table 6, which is the total score of the eye irritation response of an animal. The sum of the irritation response scores of each animal divided by the number of animals is the final score of the eye irritation of the test pesticide. Then evaluate the degree of eye irritation according to Table 7.
Table 6 Scoring of eye damage degree
Location and degree
A Opacity (based on the densest part)
No turbidity
Dispersed or diffuse turbidity, iris is clearly visible Points
GB15670-1995
Continued Table 6
Location and degree
Translucent area is easy to distinguish, iris is blurred and gray-white translucent area appears, iris details are unclear, pupil size can barely be seen Cornea is opaque, due to turbidity, iris cannot be identified Corneal damage range
1/4~1/2
1/2~3/4
Highest score
Wrinkles are obviously deepened, congestion, swelling, mild congestion around the cornea, pupil still reacts to light Bleeding, visible damage to the naked eye, no reaction to light (or one of the reactions appears ) Highest score
Congestion state (refers to the blood vessels in the conjunctiva and bulbar conjunctiva of the face)Normal congestion of blood vessels
B. Congestion of blood vessels is bright red
B. Congestion of blood vessels is dark red, and the blood vessels are difficult to distinguish. Diffuse congestion is purple-red
B. Edema
Mild edema (including nictitating membrane)
Obvious edema, accompanied by partial eversion of eyelids
Edema to the point where the eyelids are nearly half closed| |tt||Edema to the eyelid more than half closed
C Secretion
Secretion makes the eyelid and eyelashes moist or sticky. Secretion makes the entire eye area moist or sticky.
Maximum score
The maximum cumulative score of corneal, iris, and conjunctival reactions is 212
80 (score A×B×5)
10 (score×5)
20[(A+B+C)X 2]
110(80+10+20）
Non-irritating
Mild irritating
Irritation intensity
Mild to moderate irritating
Moderate irritating
Moderate to severe irritating
Severe irritating
Skin allergy (sensitization) test
7.1 Purpose
GB15670---1995Www.bzxZ.net
Eye irritation classification
Eye irritation integral index
(1. A.0.1. )
15～30
80~110
Average index of gingival irritation
(M. 1.0. 1. )
0 after 48h
less than 5 after 48h
less than 5 after 4d
less than 20 after 7d
less than 40 after 7d
The possibility of immune-mediated skin reaction in the test animal after repeated contact with pesticides, thereby determining the allergic reactivity (sensitization) of the pesticide.
7.2 Test pesticide
Preparation.
7.3 Test animal
Guinea pig, 10~~20 animals per group.
7.4 Dosage
The sensitizing (inducing) concentration is allowed to cause mild skin irritation (i.e., the highest tolerable concentration). The irritation concentration should generally be lower than the sensitizing concentration and should not cause primary irritant inflammatory reaction.
7.5 Test steps
7.5.1 The pathogenesis of contact sensitization includes two stages: sensitization and irritation. Sensitization contact
24 h before the experiment, remove 3 cm×3 cm of hair on the left side of the animal's back. Apply 0.1~~0.2 mL of the test pesticide on a 2 cm×2 cm filter paper and apply it to the hair-removed area, cover it with two layers of gauze, one layer of oil paper or impermeable plastic film, and then seal and fix it with non-irritating adhesive tape for 6 h. Repeat the same method once on the 7th and 14th days. b. Provocative contact
24 h before the experiment, remove 2 cm×2 cm of hair on the right side of the animal's back. 14~28 days after the last sensitization, apply 0.1~0.2 ml of the test pesticide or a concentration lower than the induction concentration on the right hair-removed area, and cover it in the same way as 7.5.1a. Continue for 6 h, remove the patch and the test pesticide, and observe every day until the 12th day. If there is no mutation, it can be stimulated again to determine whether the test pesticide can cause allergic reactions. 7.5.2 The test must have a positive or negative control group. The negative control group only receives provocative contact. 7.6 Result evaluation
The number of animals with skin erythema or edema (regardless of severity) is divided by the total number of animals to calculate the animal sensitization rate, and then the sensitization rate intensity is evaluated according to Table 8.
Sensitization rate intensity classification
Sensitization rate
Intensity classification
Weak sensitizer
Mild sensitizer
Sensitization rate
81～100
8Subacute oral toxicity test
8.1 Objective
GB 15670-1995
Continued Table 8
Intensity Classification
Moderate Sensitizer
Strong Sensitizer
Extremely Strong Sensitizer
Administer different doses of the test pesticide repeatedly through the oral route over a period of 1 to 4 weeks to observe the adverse reactions caused by the cumulative effect of the test pesticide, determine the subacute no-effect dose and target organs, and provide a basis for setting the dose of subchronic or chronic toxicity tests.
8.2 Test Pesticide
Technical Material.
8.3 Test Animals
8.3.1 Rats are preferred. When used as a pre-test for long-term tests, animals of the same genus and strain should be used for both tests. 8.3.2 The age of the selected rats is generally 6 to 8 weeks, and the body weight variation should not exceed 10% of the average body weight. 8.3.3 Each dose group shall have at least 10 animals, half male and half female. If some animals need to be killed in advance for examination during the test, or an additional group is needed to observe the reversibility of toxic reactions, the number of animals should be increased accordingly at the beginning of the test. 8.4 Dose grouping
Generally, three dose groups and one control group are set up. According to the data of similar compounds, if it is expected that oral doses exceeding 1000 mg/kg will not produce toxicity, two dose groups can be set up. 8.4.1 The high dose group should produce more obvious toxic effects in animals. 8.4.2 The medium dose group should produce slight observable toxic effects. 8.4.3 The low dose group should not show any toxic effects, but must exceed the possible exposure dose for humans. 8.4.4 Except for not being exposed to the test pesticide, the control group should be treated in the same way as the test group. 8.5 Dosage method
The test pesticide is mixed into the feed or dissolved in the drinking water, and fed at a fixed concentration or gavaged through a stomach tube at a fixed dose according to the animal's body weight. When used as a preparatory test for long-term toxicity tests, the same administration method should be used for both tests. Administration by feeding should be continuous. For tube feeding, the drug is administered at the same time every day, and the dosage is adjusted according to body weight at a certain interval (weekly). 8.6 Administration time
Administer continuously for 7 to 28 days or 5 days/week. In order to observe the reversibility of toxic reactions, the animals in the additional group are fed for another 14 days after stopping administration, and then the toxic effect test is performed.
8.7 Clinical observation and examination
8.7.1 Observe the toxic effects, occurrence time, duration and degree of the test animals during and after administration. If dead animals are found, they should be dissected in time. Measure the food intake (or water intake) and body weight at least once a week. 8.7.2 Hematological examination
The test items include hemoglobin content, red blood cell count, white blood cell count and its classification, etc. 8.7.3 Blood biochemical examination
Test items should generally include liver and kidney function, such as serum aspartate aminotransferase, alanine aminotransferase, urea nitrogen, creatinine, etc. If necessary, total protein, albumin, blood sugar, total cholesterol, total bilirubin, methemoglobin, cholinesterase, potassium, sodium, calcium, phosphorus, etc. should also be tested. 8.8 Pathological examination
8.8.1 Systematic dissection
GB 15670—1995
All experimental animals, including those that died during the experiment, should be completely dissected and carefully observed with the naked eye. The visible or suspected injury sites should be sampled and fixed for further histological examination. 8.8.2 Organ weight
Weigh the weight of organs such as spleen, liver, kidney, adrenal gland, testicle, etc. and calculate their organ coefficient (organ weight/body weight×100%). 8.8.3 Histological examination
The animals in the control group and high-dose group, as well as the abnormal tissues found during the system dissection, shall be subjected to detailed histological examination. Other dose groups are generally only conducted when abnormalities are found in the high-dose group. The animals in the additional groups shall focus on the tissues and organs that have been confirmed to have sexual effects in the test group. The organs to be examined generally include the brain, liver, spleen, kidney, adrenal gland, testis, ovary and other organs that are damaged or have changed in size by naked eye observation. Generally, the liver, kidney and suspected target organs are the main targets. In addition, tissues or organs that are in direct contact with the pesticide, such as the stomach, shall also be included. 9 Subacute percutaneous toxicity test
9.1 Purpose
Administer the drug repeatedly percutaneously for 21 days, observe the adverse reactions caused by it, and find the no-effect dose. 9.2 Test pesticide
Original drug.
9.3 Test animals
Perform as in 8.3.
9.4 Dosage Grouping
9.4.1 Three dose groups and one control group are usually set up. If necessary, a solvent control group should also be set up. 9.4.2 The highest dose group should show obvious toxic effects, but the number of dead animals should not hinder effective evaluation. 9.4.3 If no toxic effects are seen after administration of a dose above 1000 mg/kg body weight, it is not necessary to set up other higher dose groups.
9.5 Dosage Method
9.5.1 Shave the hair on the back of the animal, apply the test pesticide evenly on the intact part, and cover it with non-irritating plastic film and gauze, and fix it with adhesive tape to keep the pesticide in good contact with the skin and prevent it from being licked by the animal. 9.5.2 The application area is generally not less than 10% of the animal's body surface area (about 20-40 cm for rats). The application area of highly toxic pesticides can be slightly reduced, and the application should be as thin and even as possible.
9.5.3 If severe skin irritation and damage occur to animals after applying pesticides, the concentration of the test pesticide should be reduced and the test should be repeated. 9.6 Administration time
~ times a day, 4-6 hours each time, or 5 days/week, for a total of 21 days. In order to observe the reversibility of toxic effects, the animals in the additional group are kept for another 14 days after stopping administration, and then the toxic effect reaction is tested. 9.7 Clinical observation and examination
Perform as in 8.7.
9.8 Pathological examination
In addition to the procedures in 8.8, it is necessary to add skin examinations that are in direct contact with the pesticide. 10 Subacute inhalation toxicity test
10.1 Purpose
In the prescribed period of 3-4 weeks, the drug is repeatedly administered by inhalation to observe the adverse reactions caused by continuous administration through the respiratory tract and to determine the no-effect dose.
10.2 Test pesticide
Original drug.
10.3 Experimental animals
Perform in accordance with 8.3.
10.4 Dosage grouping
Perform in accordance with 8.4.
10.5 Dosage method
GB15670-1995
10.5.1 The distribution of the tested pesticide in the poisoning cabinet should be maintained uniform, and the oxygen content should not be less than 19%. 10.5.2 Concentration monitoring
The concentration of the tested pesticide in the poisoning cabinet should be monitored regularly or continuously and maintained as constant as possible. 10.6 Exposure time
Once a day, 4 hours each time, or 5 days/week. Continuous exposure for 21 to 28 days. After stopping the exposure, the additional group of animals are kept for another 14 days to observe the reversibility of the toxic reaction.
10.7 Clinical observation and examination
Perform in accordance with 8.7.
10.8 Pathological examination
In addition to the procedures in 8.8, it is also necessary to add the examination of the respiratory system that is in direct contact with the pesticide, such as the lungs. 11 Subchronic oral toxicity test
11.1 Purpose
To clarify the manifestation of the subchronic oral toxicity effect of the test pesticide, obtain the maximum no-effect dose parameters of the subchronic oral test pesticide, and provide dose reference data for the chronic toxicity test of the test pesticide. 11.2 Test pesticide
Original drug.
11.3 Test animals
11.3.1 Rats are preferred, 4 to 6 weeks old, half male and half female, and the difference in the mean weight of animals of the same sex between groups should be less than 10% of the average weight. 11.3.2 Number of animals
Each group should have no less than 10 male rats. When the rats are killed in the middle of the test, the number should be increased accordingly. 11.4 Test period
3 to 6 months.
11.5 Dosage grouping
Generally, there are three dose groups and one control group. 11.5.1 The high dose group should produce more obvious toxic effects in animals, but the death rate of animals should not exceed 10%. 11.5.2 The medium dose group should produce mild toxic effects in animals. If multiple medium dose groups are set up, they should produce different degrees of toxic effects. 11.5.3 The low dose group should not cause any toxic effects and is a no-effect dose. 11.5.4 The control group should be the same as the test group except that it does not come into contact with the test pesticide. When the test pesticide is a preparation and the biological activity of its adjuvants and additives is unclear, a corresponding adjuvant control group should also be set up. 11.6 Administration route
11.6.1 Administration method
Mix in feed or dissolve in drinking water. Give to animals continuously for 7 days a week. If the test pesticide causes poor palatability of feed and drinking water, affecting the normal intake of animals, tube feeding can be used. 11.6.2Except for nutrients, the test pesticides mixed into feed should generally not exceed 5%. The test pesticide concentration in feed or drinking water should be monitored regularly to observe its uniformity and stability.
11.7 Clinical observation and examination
11.7.1 Clinical observation
GB 15670-1995
Observe the physical signs, behavioral activities and feces shape every day. Record the time of occurrence and changes of toxic effects in detail. If dead or dying animals are found, they should be dismembered in time. Body weight and food consumption are measured once a week. 11.7.2 Hematological examination
The examination indicators include red blood cell count, white blood cell count and its classification, hemoglobin content, etc. The test time is at the beginning of the test, 3 months later and at the end. At least 6 male and female rats in each dose group are examined each time, and all non-rodents are examined. 11.7.3 Urine examination
The examination items include appearance, pH value, protein, sugar, occult blood (semi-quantitative) and sediment microscopy (semi-quantitative). The test time and quantity are the same as blood test, and the same animal should be used for the test as much as possible. 11.7.4 Blood biochemical examination
The test items include serum aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, urea nitrogen, creatinine, total protein, albumin, blood sugar, total cholesterol, total bilirubin, cholinesterase, etc. 11.8 Pathological examination
11.8.1 Systematic dissection
All experimental animals, including those that died during the experiment, should be completely dissected and carefully observed with the naked eye. All lesions or suspected lesions visible to the naked eye should be fixed for further pathological histological examination. 11.8.2 Organ weight
Weigh the weight of organs such as brain, heart, lung, liver, spleen, kidney, adrenal gland, testicle and calculate their organ coefficient (organ weight/body weight×100%). 11.8.3 Pathological histological examination
Abnormal tissues found during the autopsy of animals in the control group and high-dose group and the system should be subjected to detailed histological examination. Histological examination is generally performed on other dose groups only when abnormalities are found in the high-dose group. The organs examined generally include brain, spinal cord, heart, lung, liver, spleen, kidney, pancreas, stomach, duodenum, jejunum, ileum, colon, rectum, bladder, adrenal gland, pituitary gland, thyroid gland (including parathyroid gland), thymus gland, testicle, epididymis, prostate gland, ovary, uterus, breast gland, skin, muscle, bone, lymph node, eyeball, etc. In case of inhalation exposure, the respiratory tract should also be included. 11.9 Result evaluation
After carefully summarizing and studying the test results, and paying attention to the statistical differences between the control group and the dose group and the dose-response relationship between the doses, the following contents are evaluated
11.9.1 The main manifestations of the animal oral toxicity effect of the test pesticide. 11.9.2 The subchronic oral maximum no-effect dose of the test pesticide. 11.9.3 The appropriate dose range of the animal oral chronic toxicity test of the test pesticide for reference. 12 Accumulative toxicity test
12.1 Purpose
Understand the strength of the cumulative toxicity of the test pesticide and provide a reference for the dose selection of the chronic toxicity test and other related toxicity tests. 12.2 Test pesticide
Generally, the original drug is used. When the rat oral LDso is greater than 5000mg/kg, or a metabolism test has been conducted with half-life (t1/2) data, this test can be exempted.
12.3 Experimental animals
Newly grown mammals, rats or mice, half male and half female. If there is no oral LD50 data, its oral LD50 needs to be predicted. 12.4 Administration
Oral administration is adopted. In order to accurately grasp the dosage, oral gavage method must be used. 12.5 Fixed-dose 20-day accumulation method
12.5.1 Dose grouping
The experimental animals are randomly divided into four dose groups, the low dose is 1/20LD50, and the four dose groups are 1/10LD50, 1/5LD50 and 1/2LD50 in sequence. A negative (no drug) control group is also set up, each group has 10 animals, half male and half female. 217
12.5.2 Administration time
GB 15670—1995
The animals in the dose group are given a fixed dose once a day for 20 consecutive days. The control group was not given the test pesticide, and other conditions were the same as those of the dose group. 12.5.3 Observation index
The total number of male and female animal deaths in each dose group. 12.5.4 Result evaluation
No death occurred in each dose group, which means that the accumulation was not obvious; if only the high dose (1/2LD5o) group had death, and no death occurred in other groups, it was weak accumulation; if there was no death in the low dose group, and the number of deaths in other groups had a dose-response relationship, it was moderate accumulation; if there was death in the low dose group, and there was a dose-response relationship, it was strong accumulation. 12.6 Dose-increasing accumulation coefficient method
12.6.1 Dose grouping
The experimental animals were randomly divided into two groups of male and female, with 20 animals in each group. 12.6.2 Administration time and dose
The administration was continued for 4 days. The first period of each animal is 0.1LD50, and the subsequent periods are increased by 50%, that is, the second period is 0.15LD50, the third period is 0.225LD50, and so on, until half of the male and female groups die (20 animals), or the administration is continued until the cumulative dose reaches 5.0LDs or more (20 days). 12.6.3 During the administration period (4 days), the absolute amount of administration shall be calculated according to the actual animal weight. 12.6.4 Result calculation
After stopping the administration, the accumulation coefficient K is calculated according to formula (1): LDso(n)
LDso(1)
Where: LDs(n)-
LDso(1)
12.6.5 Result evaluation
The number of LDso equivalent to the test pesticide ingested by each animal during the administration period; the oral LDso dose of the test pesticide to the test animal. The cumulative toxicity of the tested pesticides shall be evaluated according to the following standards: K<1 highly cumulative;
K≥1 obviously cumulative;
K≥3 moderately cumulative;
K≥5 slightly cumulative.
13 Delayed neurotoxicity test
13.1 Purpose
To understand the delayed neurotoxicity of the tested pesticides and to find the delayed neurotoxicity no-effect dose. 13.2 Test pesticide
Original material.
13.3 Test animals
13.3.1 Select hens with clear genetic background, healthy and normal gait, aged 8~~14 months and weighing 1.5~~2kg. 13.3.2 Quantity
(1)
The number of hens in each dose group should ensure that at least 6 are alive at the end of the observation. If the animals are to be sacrificed at maturity, the number of animals for extended observation should be increased at the beginning of the experiment if the recovery is to be observed.3 Pathological histological examination
The animals in the control group and the high-dose group and the abnormal tissues found during the system dissection need to be subjected to detailed histological examination. The other dose groups are generally only subjected to histological examination when abnormalities are found in the high-dose group. The organs examined generally include the brain, spinal cord, heart, lung, liver, spleen, kidney, pancreas, stomach, duodenum, jejunum, ileum, colon, rectum, bladder, adrenal gland, pituitary gland, thyroid gland (including parathyroid gland), thymus, testicles, epididymis, prostate gland, ovary, uterus, breast, skin, muscle, bone, lymph node, eyeball, etc. In case of inhalation poisoning, the respiratory tract should also be included. 11.9 Evaluation of results
On the basis of carefully summarizing and studying the test results, and paying attention to the statistical differences between the control group and the dose group and the dose-response relationship between the doses, the following contents are evaluated
11.9.1 The main manifestations of the animal oral toxic effects of the test pesticide. 11.9.2 The subchronic oral maximum no-effect dose of the test pesticide. 11.9.3 Suitable dosage range for reference of experimental pesticide animal oral chronic toxicity test. 12 Accumulative toxicity test
12.1 Purpose
To understand the strength of the experimental pesticide's cumulative toxicity and provide reference for the dosage selection of chronic toxicity test and other related toxicity tests. 12.2 Experimental pesticide
Generally, the original drug is used. When the rat oral LDso is greater than 5000mg/kg, or a metabolism test has been conducted with half-life (t1/2) data, this test can be exempted.
12.3 Experimental animals
Newly-adult mammals, rats or mice, half male and half female. For those without oral LDso data, it is necessary to predict their oral LD50p12.4 Administration
Oral administration is adopted. In order to accurately grasp the dosage, gavage method must be used. 12.5 Fixed-dose 20-day bud accumulation method
12.5.1 Dose grouping
The experimental animals were randomly divided into four dose groups, the low dose was 1/20LD50, followed by 1/10LDso, 1/5LD5o and 1/2LDso. A negative (no-dose) control group was set up, each group had 10 animals, half of them were male and half were female. 217
12.5.2 Time of administration
GB 15670—1995
The animals in the dose group were administered once a day at a fixed time, and the administration lasted for 20 days. Except that the control group was not given the test pesticide, other conditions were the same as those of the dose group. 12.5.3 Observation index
The total number of male and female animals in each dose group died. 12.5.4 Result evaluation
No death occurred in each dose group, which means that the accumulation was not obvious; if there was death only in the high dose (1/2LD5o) group and no death occurred in other groups, it was weak accumulation; if there was no death in the low dose group and the number of deaths in other groups had a dose-response relationship, it was moderate accumulation; if there was death in the low dose group and there was a dose-response relationship, it was strong accumulation. 12.6 Dose-escalation accumulation coefficient method
12.6.1 Dose grouping
The experimental animals were randomly divided into two groups of male and female, with 20 animals in each group. 12.6.2 Administration time and dose
The administration was continued for 4 days. The first period of each animal is 0.1LD50, and the subsequent periods are increased by 50%, that is, the second period is 0.15LD50, the third period is 0.225LD50, and so on, until half of the male and female groups die (20 animals), or the administration is continued until the cumulative dose reaches 5.0LDs or more (20 days). 12.6.3 During the administration period (4 days), the absolute amount of administration shall be calculated according to the actual animal weight. 12.6.4 Result calculation
After stopping the administration, the accumulation coefficient K is calculated according to formula (1): LDso(n)
LDso(1)
Where: LDs(n)-
LDso(1)
12.6.5 Result evaluation
The number of LDso equivalent to the test pesticide ingested by each animal during the administration period; the oral LDso dose of the test pesticide to the test animal. The cumulative toxicity of the tested pesticides shall be evaluated according to the following standards: K<1 highly cumulative;
K≥1 obviously cumulative;
K≥3 moderately cumulative;
K≥5 slightly cumulative.
13 Delayed neurotoxicity test
13.1 Purpose
To understand the delayed neurotoxicity of the tested pesticides and to find the delayed neurotoxicity no-effect dose. 13.2 Test pesticide
Original material.
13.3 Test animals
13.3.1 Select hens with clear genetic background, healthy and normal gait, aged 8~~14 months and weighing 1.5~~2kg. 13.3.2 Quantity
(1)
The number of hens in each dose group should ensure that at least 6 are alive at the end of the observation. If the animals are to be sacrificed at maturity, the number of animals for extended observation should be increased at the beginning of the experiment if the recovery is to be observed.3 Pathological histological examination
The animals in the control group and the high-dose group and the abnormal tissues found during the system dissection need to be subjected to detailed histological examination. The other dose groups are generally only subjected to histological examination when abnormalities are found in the high-dose group. The organs examined generally include the brain, spinal cord, heart, lung, liver, spleen, kidney, pancreas, stomach, duodenum, jejunum, ileum, colon, rectum, bladder, adrenal gland, pituitary gland, thyroid gland (including parathyroid gland), thymus, testicles, epididymis, prostate gland, ovary, uterus, breast, skin, muscle, bone, lymph node, eyeball, etc. In case of inhalation poisoning, the respiratory tract should also be included. 11.9 Evaluation of results
On the basis of carefully summarizing and studying the test results, and paying attention to the statistical differences between the control group and the dose group and the dose-response relationship between the doses, the following contents are evaluated
11.9.1 The main manifestations of the animal oral toxic effects of the test pesticide. 11.9.2 The subchronic oral maximum no-effect dose of the test pesticide. 11.9.3 Suitable dosage range for reference of experimental pesticide animal oral chronic toxicity test. 12 Accumulative toxicity test
12.1 Purpose
To understand the strength of the experimental pesticide's cumulative toxicity and provide reference for the dosage selection of chronic toxicity test and other related toxicity tests. 12.2 Experimental pesticide
Generally, the original drug is used. When the rat oral LDso is greater than 5000mg/kg, or a metabolism test has been conducted with half-life (t1/2) data, this test can be exempted.
12.3 Experimental animals
Newly-adult mammals, rats or mice, half male and half female. For those without oral LDso data, it is necessary to predict their oral LD50p12.4 Administration
Oral administration is adopted. In order to accurately grasp the dosage, gavage method must be used. 12.5 Fixed-dose 20-day bud accumulation method
12.5.1 Dose grouping
The experimental animals were randomly divided into four dose groups, the low dose was 1/20LD50, followed by 1/10LDso, 1/5LD5o and 1/2LDso. A negative (no-dose) control group was set up, each group had 10 animals, half of them were male and half were female. 217
12.5.2 Time of administration
GB 15670—1995
The animals in the dose group were administered once a day at a fixed time, and the administration lasted for 20 days. Except that the control group was not given the test pesticide, other conditions were the same as those of the dose group. 12.5.3 Observation index
The total number of male and female animals in each dose group died. 12.5.4 Result evaluation
No death occurred in each dose group, which means that the accumulation was not obvious; if there was death only in the high dose (1/2LD5o) group and no death occurred in other groups, it was weak accumulation; if there was no death in the low dose group and the number of deaths in other groups had a dose-response relationship, it was moderate accumulation; if there was death in the low dose group and there was a dose-response relationship, it was strong accumulation. 12.6 Dose-escalation accumulation coefficient method
12.6.1 Dose grouping
The experimental animals were randomly divided into two groups of male and female, with 20 animals in each group. 12.6.2 Administration time and dose
The administration was continued for 4 days. The first period of each animal is 0.1LD50, and the subsequent periods are increased by 50%, that is, the second period is 0.15LD50, the third period is 0.225LD50, and so on, until half of the male and female groups die (20 animals), or the administration is continued until the cumulative dose reaches 5.0LDs or more (20 days). 12.6.3 During the administration period (4 days), the absolute amount of administration shall be calculated according to the actual animal weight. 12.6.4 Result calculation
After stopping the administration, the accumulation coefficient K is calculated according to formula (1): LDso(n)
LDso(1)
Where: LDs(n)-
LDso(1)
12.6.5 Result evaluation
The number of LDso equivalent to the test pesticide ingested by each animal during the administration period; the oral LDso dose of the test pesticide to the test animal. The cumulative toxicity of the tested pesticides shall be evaluated according to the following standards: K<1 highly cumulative;
K≥1 obviously cumulative;
K≥3 moderately cumulative;
K≥5 slightly cumulative.
13 Delayed neurotoxicity test
13.1 Purpose
To understand the delayed neurotoxicity of the tested pesticides and to find the delayed neurotoxicity no-effect dose. 13.2 Test pesticide
Original material.
13.3 Test animals
13.3.1 Select hens with clear genetic background, healthy and normal gait, aged 8~~14 months and weighing 1.5~~2kg. 13.3.2 Quantity
(1)
The number of hens in each dose group should ensure that at least 6 are alive at the end of the observation. If the animals are to be sacrificed at maturity, the number of animals for extended observation should be increased at the beginning of the experiment if the recovery is to be observed.2 Test pesticides
Original pesticide.
13.3 Test animals
13.3.1 Select hens with clear genetic background, healthy and normal gait, aged 8~~14 months, weighing 1.5~~2kg. 13.3.2 Quantity
(1)
The number of hens in each dose group should ensure that at least 6 survive at the end of the observation. The hens will be killed at the end of the observation period. If the recovery needs to be observed, the number of animals for the extended observation period should be increased at the beginning of the experiment. 2182 Test pesticides
Original pesticide.
13.3 Test animals
13.3.1 Select hens with clear genetic background, healthy and normal gait, aged 8~~14 months, weighing 1.5~~2kg. 13.3.2 Quantity
(1)
The number of hens in each dose group should ensure that at least 6 survive at the end of the observation. The hens will be killed at the end of the observation period. If the recovery needs to be observed, the number of animals for the extended observation period should be increased at the beginning of the experiment. 218
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