GB/T 15670.6-2017Toxicological test methods for pesticides registration - Part 6: Acute inhalation toxicity test (English PDF)

6.1 Test preparation

6.1.1 Test substance The test substance shall be a sample with a stable preparation process and in compliance with the quality standards, with the name, source, batch number, content (or specification), storage conditions and preparation method, etc., and a test report attached. The excipients, solvents or other media used shall be marked with the batch number, specification and manufacturer.

6.1.2 Selection and preparation of test substance vaporization or aerosol preparation method

6.1.2.1 Gaseous test substance After mixing it with air to a certain concentration through a flow meter, directly input it into the toxicity cabinet.

6.1.2.2 Low boiling point volatile liquid test substance After volatilization through air bubbling or appropriate heating, mix it with air and pass into the toxicity cabinet.

6.1.2.3 High boiling point non-volatile liquid test substance According to the physical and chemical properties of the test object, a spray method can be selected, or an aerosol generating device can be used to prepare a liquid aerosol that meets the test requirements and then mix it with a certain amount of air and input it into the toxicity cabinet.

6.1.2.4 Powdered or solid test substances Prepare the test substance into solid aerosol using an aerosol generator; mix it with a certain amount of air and introduce it into the toxicity cabinet.

6.1.3 Dynamic inhalation toxicity method Use mechanical ventilation equipment to continuously and evenly deliver air containing a certain concentration of the test substance into the toxicity cabinet, with a ventilation volume of about 12 times/h ~ 15 times/h to maintain a relatively stable toxicity concentration. Use whole-body exposure or head/nose exposure to conduct acute inhalation toxicity on animals for 4 hours. During toxicity, keep the distribution of toxicity gas in the toxicity cabinet balanced, the oxygen content not less than 19%, the CO2 content not higher than 1%, the temperature at (22±3) °C, and the relative humidity at 30% ~ 70% (except for special circumstances, such as steam toxicity). If the toxicity not to interfere with the individual activities of the animals and the observed reactions. The animals shall be fed with regular feed and shall have free access to water.

6.1.5.3 Number and gender Each concentration group shall have at least 10 animals, 5 males and 5 females. Female animals shall be non-pregnant and nulliparous.

6.1.5.4 Inhaled dose and grouping At least 3 different concentrations of toxicity groups shall be set up, with appropriate concentration intervals between groups, so that a certain degree of toxic effect and death can occur in the test animals in each concentration group. At the same time, blank and/or corresponding excipient, solvent or other medium control groups shall be set up, and the data obtained shall be sufficient to draw a concentration-death curve and calculate the LC50 value. If the toxicity of the test substance is very low, 20 rats (half male and half female) can also be used to conduct a single dose acute inhalation toxicity test. The single acute inhalation toxicity concentration shall be 2×10-2 (gas test substance), 20 mg/L (liquid test substance vapor), 5 mg/L (solid or liquid test substance aerosol). If the concentration requirement cannot be reached due to the physical and chemical properties of the test substance, the test shall also be conducted at the highest concentration that the test substance can reach. When the test substance is explosive, care shall be taken to avoid using a concentration that may cause an explosion.

6.2.1 Implementation of inhalation toxicity

6.2.1.1 Balance and control of toxicity concentration Various appropriate conditions in the test preparation stage shall be used to control the toxicity concentration, inhalable diameter, etc.

6.2.1.2 Body weight measurement of toxicity animals and observation during toxicity Before the test begins, each animal shall be weighed. The time for animals to continuously inhale the test substance is 4 hours. During the toxicity process, the animals are usually in a state of fasting and water deprivation. During inhalation toxicity, attention shall be paid to observing the effects of the test substance on the respiratory system, such as respiratory irritation reactions, changes in respiratory rhythm, and changes in inhalation and exhalation patterns.

6.2.2 Test observation and inspection

6.2.2.1 Observation period After the inhalation toxicity, the test animals shall be carefully observed at least once a day for 14 consecutive days. However, the observation period is not fixed and can be adjusted according to the time of occurrence of toxic reactions and signs, as well as the length of the recovery period. If the animals show toxicity reactions and delayed death after exposure, the observation period shall be appropriately extended, and the maximum period shall not exceed 21 days.

6.2.2.2 Observation content Observe and record the clinical manifestations and signs of toxicity animals in detail, including the time of onset and recovery, the time of animal death, etc. The content of observation includes.

a) Central nervous system and neuromuscular system. abnormal posture, abnormal voice, restlessness, dullness, tremor, convulsion, paralysis, movement disorder, hypersensitivity or slowness in external response, abnormal behavior such as excessive and repeated scratching around the mouth, combing, circling, even self-mutilation, walking backwards, etc.;

b) Autonomic nervous system. pupil dilation or constriction, salivation or tearing;

c) Respiratory system. nasal discharge, epistaxis, flaring of the nostrils, deep and slow breathing, tachypnea, wasp waist;

d) Urogenital system. dirty perineum, discharge, vaginal swelling;

e) Skin and coat. skin congestion, cyanosis, fluffy coat or moist dirty;

f) Eyes. proptosis, conjunctival congestion, hemorrhagic secretions, corneal opacity;

g) Digestive system. diarrhea, anorexia. Record the appearance and disappearance of toxicity signs and the time of animal death. In particular, if there is a trend of delayed death, more careful observation and recording shall be made. Weak or dying animals shall be quickly isolated, humanely killed and autopsied, and treated as toxicity deaths. If an animal is found dead during the observation period, it shall be autopsied in time. If it cannot be examined immediately, it shall be refrigerated and autopsied within 24 hours.

6.2.2.3 Weighing The body weight shall be measured and recorded in detail before and after toxicity (at least once a week) and before the animals are killed.

6.2.2.4 Pathological examination combination with the results of gross anatomy and histopathological examinations. If relatively clear dose-effect and dose-response curves can be obtained, a preliminary evaluation can be made on the threshold of the test substance via respiratory inhalation toxicity. Calculate the LC50 value, to evaluate the acute inhalation toxicity level of the test substance, which is determined according to the corresponding standards in

3.3.2.8 of the "Provisions on Pesticide Registration Information". Summarize the safety range of the test substance, the severity of toxicity and recoverability. The results of acute inhalation toxicity tests can provide a reference for the selection of doses for related subsequent toxicology tests, and can also indicate some indicators that need to be observed in subsequent toxicity tests. When referring to or citing these test results, attention shall be paid to the organic connection between various data and information. The LC50 value shall be regarded as a relatively rough toxicological indicator, which is mainly used to identify the inhalation toxicity potency of a test substance and as a basis for grading its toxicity. When citing or referring to the LC50 value, in addition to combining the toxic effects of the test substance, histopathological examination results, etc., attention shall also be paid to specific issues such as the species of animals, the length of exposure time, and the method of calculating the LC50.