SlideShare a Scribd company logo

Inhalation Toxicity Studies- OECD guidelines

Cerin Philip, profile picture
Cerin Philip

This document summarizes guidelines for inhalation toxicity studies including acute, subacute, and subchronic toxicity studies. It describes the objectives, principles, procedures, observations, and reporting requirements for these studies. Key aspects covered include selecting animal species and exposure concentrations, monitoring exposure conditions, conducting traditional and CxT exposure protocols, observing animals for signs of toxicity, and reporting study results including toxicity estimates. The purpose of these studies is to evaluate the toxic effects of inhaled substances over different exposure durations.

Healthcare
1 of 54
1
2
3
4
5
6
7
Most read
8
9
10
11
12
13
14
15
16
17
Most read
18
Most read
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
SEMINAR 2 PHARMACOLOGICALAND TOXICOLOGICAL SCREENING METHODS SUBMITTED BY : CERIN MAREENA PHILIP SECOND SEMESTER M.PHARM DEPARTMENT OF PHARMACOLOGY KMCH COLLEGE OF PHARMACY SUBMITTED TO :DR.G.VENKATESH ASSOCIATE PROFESSOR DEPARTMENT OF PHARMACOLOGY KMCH COLLEGE OF PHARMACY DATE:1/08/2019
INHALATION TOXICITY STUDIES(1981) ACUTE TOXICITY {433(2017),403(2009),39} SUBACUTE TOXICITY(412) SUBCHRONIC TOXICITY (413)
CONTENT ACUTE TOXICITY STUDY FOR INHALATION • INTRODUCTION • PRINCIPLE • DESCRIPTION OF METHOD • EXPOSURE CONDITIONS AND MONITORING • PROCEDURE • OBSERVATION • DATAAND REPORT • INTERPRETATION SUBACUTE TOXICITY STUDY FOR INHALATION SUBCHRONIC TOXICITY STUDY FOR INHALATION REFERENCE
Inhalation Toxicity Studies- OECD guidelines
INTRODUCTION • Acute toxicity study for inhalation was documented as document no. 39 as OECD GUIDELINE FOR THE TESTING OF CHEMICALS • This revised Test Guideline 403 (TG 403) has been designed to be more flexible, to reduce animal usage, and to fulfill regulatory needs. • The revised TG 403 features two study types: • 1. A Traditional LC50 protocol and • 2. A Concentration * Time (C x t) protocol.
• SELECTION  of the most appropriate species,strain,sex,mode of exposure,appropriate test concentrations include • the identity, • chemical structure, and physicochemical properties of the test article; • results of any in vitro or in vivo toxicity tests; • anticipated uses and potential for human exposure; • available (Q)SAR data and toxicological data on structurally related substance. • The targeted concentrations should not induce severe irritation/corrosive effects, yet reach scientific objective of the test.
• to provide lethality data. • (e.g. LC50, LC01 and slope) for one or both sexes as needed for quantitative risk assessments.This Guideline offers two test methods.  Traditional protocol • groups of animals are exposed to a limit concentration (limit test) • stepwise procedure • predetermined duration of usually 4 hours.  (C x t) protocol • groups of animals are exposed to one (limit concentration) • multiple concentrations • multiple durations. • OECD Guidance Document No. 19 PRINCILPE
DESCRIPTION OF THE METHOD 1. Selection of animal species:- 2. Preparation of animals 3. Animal husbandry 4. Inhalation chambers  Selection of animal species:- • Healthy young adult animals of commonly used laboratory strains should be used. • The preferred species is the rat and justification should be provided if other species are used. • both sex
 Preparation of animals 1. Females should be nulliparous and nonpregnant. 2. On the exposure day, animals should be young adults 8 to 12 weeks of age, and body weights should be within ±20% of the mean weight for each sex of any previously exposed animals of the same age. 3. The animals are randomly selected and marked for individual identification. The animals are kept in their cages for at least 5 days prior to the start of the test.
• Animal husbandry • The temperature should be 22±3°C. • The relative humidity should ideally be maintained in the range of 30 to 70%, • Before and after exposures, animals generally should be caged in groups by sex and concentration, • When animals are to be exposed nose-only, to be acclimated to the restraining tubes. • Animals exposed whole-body to an aerosol should be housed individually during exposure • Lighting should be artificial, the sequence being 12 hours light/12 hours dark.
 Inhalation chambers • The nature of the test article and the objective of the test should be considered when Selecting an inhalation chamber. • The preferred mode of exposure snout-only. • preferred for studies of liquid or solid aerosols and for vapours that may condense to form aerosols. • Toensure atmosphere stability when using a whole-body chamber.
EXPOSURE CONDITIONS: Administration of concentrations Particle-size distribution Test article preparation in a vehicle Control animals
Administration of Concentration Nose-only exposures may be any duration up to 6 hours in rats. If mice are exposed nose- only, exposures generally should not exceed 4 hours. Particle-size distribution Particle sizing should be performed for all aerosols and for vapours that may condense to form aerosols. ( metal fumes may be smaller than this standard, and charged particles, fibres, and hygroscopic materials ) Test article preparation in a vehicle vehicle may be used to generate an appropriate concentration and particle size of the test in atmosphere (water). Adequate care should be taken to not contaminate the test material. Control animals A concurrent negative (air) control group is not necessary.
MONITORING OF EXPOSURE CONDITIONS 1. Chamber airflow 2. Chamber temperature and relative humidity 3. Test article: Nominal concentration 4. Test article: Actual concentration 5. Test article: Particle size distribution
1. Chamber Airflow The flow of air should be carefully Controlled,Continuously monitored,Recorded( at least hourly during each exposure). Oxygen concentration should be at least 19% and carbon dioxide concentration should not exceed 1%.(if not measured) 2. Chamber temperature and relative humidity • Chamber temperature should be maintained at 22±3°C. • Relative humidity should be monitored and recorded at least three times for durations of up to 4 hrs, and hourly for shorter durations. • The relative humidity maintained in the range of 30 to 70%. 3. Test article: Nominal concentration • mass of generated test • the total volume of air passed through the chamber system. • The nominal exposure chamber concentration should be calculated and recorded.
• 4.Test article:Actual concentration The actual concentration is the test article concentration at the animals breathing zone in an inhalation chamber. Actual concentrations can be obtained by specific methods (e.g. direct sampling, adsorptive or chemical reactive methods, & analytical characterisation) or by nonspecific methods such as gravimetric filter analysis. the test sample should be stored under conditions that maintain its purity, homogeneity, and stability. 5. Test article: Particle size distribution determined at least twice during each 4 hour exposure by using a cascade impactor or an aerodynamic particle sizer. A second device, such as a gravimetric filter or an impinger/gas bubbler, should be used in parallel . Particle sizing should be performed for vapours if vapour condensation may result in the formation of an aerosol,
PROCEDURE- Traditional General considerations: In a Traditional study, groups of animals are exposed to a test article for a fixed period of time (generally 4 hours) in either a nose-only or whole-body exposure chamber. Animals are exposed to either a limit concentration (limit test), or to at least three concentrations in a stepwise procedure (main study). A sighting study may precede a main study unless some information about the test article already exists, such as a previously performed TG 436. 2. Sighting study A sighting study is used to estimate test article potency, identify sex differences in susceptibility, and assist in selecting exposure concentration levels for the main study or limit test. When selecting concentration levels for the sighting study, all available information should be used (3 animals/sex may be needed to establish a sex difference). A sighting study may consist of a single concentration, but more concentrations may be tested if necessary. 3. Limit test A limit test is used when the test article is known or expected to be virtually non-toxic, Three animals of both sex each. In those situations where there is little or no information about its toxicity, or the test material is expected to be toxic, the main test should be performed. When the GHS Classification System is used, the limit concentrations for gases, vapours, and aerosols are 20000 ppm, 20 mg/L, and 5 mg/L, respectively (or the maximum attainable concentration) 4. Main Test A main study is typically performed using five males and five females (or 5 animals of the susceptible sex, if known) per concentration level, with at least three concentration levels. The time interval between exposure groups is determined by the onset, duration, and severity of toxic signs.
C X T PROTOCOL General considerations: 2. Sighting study 3 animal of per sex is used for test and exposure of animal is done for single duration mainly 240 min. When selecting the initial target concentration , the study director should consider the mortality patterns observed in any available TG 436 studies 3. Initial Concentration Group of 1 animal/sex is expose to the test article initial conc for time interval of 15, 30, 60 , 120 and 240 min Total 10 animals are used in this test. When the GHS Classification System is used, the limit concentrations for gases, vapours, and aerosols are 20000 ppm, 20 mg/L and 5 mg/L, respectively. If less than 50% lethality occurs at the maximum attainable concentration, no further testing is necessary. Main study
Inhalation Toxicity Studies- OECD guidelines
MAIN STUDY Exposure Session I –Testing at the limit concentration 1 animal/sex per concentration/time point; 10 animals in total a Target concentration = limit concentration. Expose five groups of animals at this target concentration for durations of 15, 30, 60, 120 and 240 minutes, respectively Exposure Session II – Main Study 1 animal/sex per concentration/time point; 10 animals in total. Expose five groups of animals at a lower concentration d (1/2L) with slightly lower duration of exposure.
Exposure Session III – Main Study 1 animal/sex per concentration/time point; 10 animals total. Expose five groups of animals at a lower concentration d (1/4L) with slightly longer exposure durations. Exposure Session IV′ – Main Study 1 animal/sex per concentration/time point; 10 animals total. Expose five groups of animals at a lower concentration d (1/8L) with slightly longer exposure durations Exposure Session IV – Main Study 1 animal/sex per concentration/time point; 10 animals total. Expose five groups of animals at a higher concentration e (2L) with slightly shorter exposure durations.
OBSERVATION Once daily for 14 days. The length of the observation period is not fixed, The times at which signs of toxicity appear and disappear are important, individual records being maintained for each animal Animals found in a moribund condition should be humanely killed for animal welfare reasons. Cage-side observations should include changes in the skin and fur, eyes and mucous membranes, and also respiratory, circulatory, autonomic and central nervous systems, and somatomotor activity and behaviour patterns Attention should be directed to observations of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.
• Body weights • Individual animal weights should be recorded once during the acclimatization period, on the day of exposure prior to exposure (day 0), and at least on days 1, 3 and 7 (and weekly thereafter), and at the time of death or euthanasia • Pathology • All test animals, including those which die during the test or are euthanized and removed from the study for animal welfare reasons, should be subjected to gross necropsy.
DATA AND REPORTING • Data : Individual animal data on body weights and necropsy findings should be provided. • summarized in tabular form, showing for each test group the number of animals used, the number of animals displaying specific signs of toxicity, the number and time of animals found dead during the test or killed for humane reasons, , a description and time course of toxic effect.
TEST REPORT • The test report should include the following information, as appropriate: • Description of caging conditions, including: number (or change in number) of animals per cage, bedding material, ambient temperature and relative humidity, photoperiod, and identification of diet • - Species/strain used and justification for using a species other than the rat • - Number, age and sex of animals • - Method of randomization • - Details of food and water quality (including diet type/source, water source); • - Description of any pre-test conditioning including diet, quarantine, and treatment for disease
RESULTS • Tabulation of chamber temperature, humidity, and airflow; - Tabulation of chamber nominal and actual concentration data • Tabulation of particle size data • Individual body weights of animals collected on study; • date and time of death if prior to scheduled euthanasia, • time course of onset of signs of toxicity and whether these were reversible for each animal • Necropsy findings and histopathological findings for each animal, if available • Lethality estimates (e.g. LC50, LD01) including 95% confidence limits, and slope (if provided by the evaluation method) • Statistical relation, including estimate for the exponent n (C x t protocol). The name of the statistical software used should be provided.
SUBACUTE TOXICITY TESTING AS PER OECD GUIDELINES (412)
OECD Guidelines No. 412: 28-day inhalation Toxicity Study in Rodents  Toxico-kinetics and systemic toxicity study is also studied.  Provide robust data for quantitative inhalation risk assessments. Subacute inhalation toxicity studies are primarily used to derive regulatory concentrations for assessing worker risk in occupational settings. (Q)SAR data and toxicological data on structurally related chemicals. The data derived from subacute inhalation toxicity studies can be used for quantitative risk assessments and for the selection of concentrations for chronic studies.
PRINCIPLE Accommodate the testing of nanomaterials as well as to reflect the evolving state-of-the-science for the testing of inhaled gases, vapours, and aerosols. Broncho-alveolar lavage fluid (BALF) to be performed for all test chemicals POE-BALF analysis and lung burden measurements are performed. for all test chemicals( 24h) after exposure termination When testing a solid aerosol, it is useful to have information on its retention and kinetics in the lung. Dilutions of corrosive or irritating test chemicals may be tested at concentrations that will yield the desired degree of toxicity.
Inhalation Toxicity Studies- OECD guidelines
1. Selection of Animal Species Healthy young adult rodents of commonly used laboratory strains should be employed. The preferred species is the rat. Justification should be provided if other species are used. 2. Preparation of Animals 5 males and females should be exposed at each concentration. On the day of randomization, animals should be young adults 7 to 9 weeks of age. Body weights should be within ± 20% of the mean weight for each sex. allow for acclimatization to laboratory conditions. 3Inhalation Chambers Subacute inhalation toxicity studies are always performed in dynamic inhalation chambers. The use of a static inhalation chamber, which has no airflow, is not acceptable. 1Limit Concentrations The maximum concentration tested should consider: attainable the need to maintain an adequate oxygen supply, and/or animal welfare considerations. Up to a maximum concentration of 5 mg/L for aerosols,20 mg/L for vapors, and 20,000 ppm for gases DESCRIPTION OF THE METHOD
Dynamic Inhalation Chamber & Static Inhalation Chamber
2 Range-Finding Study  To inform the selection of concentration levels for a main study.  No Observed Adverse Effects Concentration (NOAEC), Lowest Observed Adverse Effects Concentration (LOAEC), Maximum Tolerated Concentration (MTC), and/or the benchmark concentration (BMC) in a main study. The study director should use a range-finding study to identify the upper concentration that is tolerated without undue stress to the animals. A range-finding study should last a minimum of 5 days and generally no more than 14 days, and may include a post-exposure period and animal numbers should be adjusted accordingly.
When testing poorly soluble particles, it may be necessary for a range-finding study to be longer than 14 days to allow for a robust assessment of test chemical solubility and lung burden. The rationale for the selection of concentrations for the main study should be provided in the study report. 3 Main Study The main study consists of at least three test chemical concentration levels and concurrent negative (air) or vehicle controls. This guideline differentiates two study designs depending on the nature of the test chemical. Option A, which is generally used for test chemicals (as gas,vapour, aerosol, or a mixture thereof), option B is used when testing chemicals that are likely to be retained in the lungs.
Inhalation Toxicity Studies- OECD guidelines
Inhalation Toxicity Studies- OECD guidelines
OBSERVATIONS Bronchoalveolar Lavage • BAL should be performed at PEO-1 (within 24h at termination of exposure) and at one post-exposure interval (PEO-2) • When testing a poorly soluble aerosol, BAL should be performed at PEO-1 (within 24 hours of exposure termination) in both sexes and in female animals at PEO-2 if a recovery group is scheduled. • The right lung is generally preferred for lavage. The duration of the post-exposure period and the timing of the PEOs are determined by the study director based on findings in the range-finding study and other available, relevant information. • The mandatory BALF analysis encompasses the following parameters: Lactate dehydrogenase (LDH) Total protein or albumin Cell counts and differentials for alveolar macrophages, lymphocytes, neutrophils, and eosinophils
•Lung Burden • When testing poorly soluble solid aerosols, measurements of lung burden can provide clarity on the retained dose. • Males are used because they have a higher minute volume than females and may thus have greater lung burdens. • To obtain clear information on lung clearance kinetics the same lung (the right lung is recommended) should be measured for lung burden at all post-exposure time points.  Ophthalmological Examination • Refractive media, iris, and conjunctivae. Gross Pathology And Organ Weights
DATA AND REPORTING • Individual animal data on body weights, food consumption, clinical pathology, BALF analysis, gross pathology, organ weights, lung burden (when evaluated) and histopathology should be provided for both the range-finding and main study. • Clinical observation data should be summarized in tabular form showing for each test group the number of animals used, the numberof animals displaying specific signs of toxicity, • the number of animals found dead during the test or killed for humane reasons, time of death of individual animals, a description and time course of toxic effects and reversibility, and necropsy findings.
DISCUSSION & INTERPRETATION OF RESULTS • The respirability of aerosol particles in light of the overall findings should be addressed, especially if the MMAD standard could not be met. • The consistency of methods used to determine analytical and nominal concentrations, and the relation of analytical concentrations to nominal concentrations should be included in the overall assessment of the study.  The test report must include the following information: - species/strain used & toxic response data by sex and dose; - time of death during the study or whether animals survived or termination; - Toxic effects and the time of observation of each abnormal sign and its subsequent course & food and body weight data.
90-DAY (SUBCHRONIC) INHALATION TOXICITY STUDY
•INTRODUCTION •The original subchronic inhalation Test Guideline 413 (TG 413) was adopted in 1981 (2). •This revision requires specific measurements of bronchoalveolar lavage fluid (BALF), Measurements of lung burden, should be done when a range-finding study or other relevant information suggests that inhaled test particles are poorly soluble and likely to be retained in the lung. •A range-finding study (or studies), which primarily is (are) performed to assess concentration levels for the main study should also include BALF analysis, and may also include lung burden measurements •Subchronic inhalation toxicity studies are primarily used to derive regulatory concentrations for assessing worker risk in occupational settings. •This guideline enables the characterization of adverse effects following repeated daily inhalation exposure to a test chemical for 90 days. •Definitions of technical terms used in this Test Guideline can be found in GD 39 (1). •revising this test guideline was to accommodate the testing of nanomaterials as well as to reflect the evolving state-of-the-science for the testing of inhaled gases, vapours, and aerosols.
INITIAL CONSIDERATIONS •All available information on the test chemical should be considered by the testing laboratory prior to conducting the main study in order to enhance the quality of the study, minimize animal usage, and avoid the need to repeat the study. •The respirable (or alveolar) fraction of poorly soluble particles that are slowly cleared can accumulate with each consecutive exposure period. •Dilutions of corrosive or irritating test chemicals may be tested at concentrations that will yield the desired degree of toxicity. •Animals that are moribund obviously in pain or showing signs of severe and enduring distress should be humanely sacrificed. •DESCRIPTION OF THE METHOD • Selection of animal species:- • Preparation of animals • Animal husbandry • Inhalation chambers
TOXICITY STUDIES Limit Concentrations Range-Finding Study • A range-finding study should be performed unless sufficient information already exists to perform a robust main study. • A range-finding study may, for example, provide information regarding analytical methods, particle size distribution, systemic toxicity, toxicokinetics, test chemical solubility in the lung,BALF data, and estimates of what may be the No Observed Adverse Effects Concentration (NOAEC), Lowest Observed Adverse Effects Concentration (LOAEC), Maximum Tolerated Concentration (MTC), and/or the benchmark concentration (BMC) in a main study. • A range-finding study may consist of one or more test chemical concentration levels and a control group. Depending on the endpoints chosen, typically no more than 5 males and 5 females should be exposed at each concentration level. • should last a minimum of 5 days and generally no more than 28 days,
The main study •The main study consists of at least three test chemical concentration levels and concurrent negative (air) or vehicle controls •Each group consists of a minimum of 10 male and 10 female rodents that are exposed to the test chemical for 6 hours per day on a 5 day per week basis for a period of 13 weeks (total study duration of at least 90 days). •Animals may also be exposed 7 days per week. •If rodent species other than rats are exposed nose-only, •A rationale should be provided when using exposure duration less than 6 hours/day, or when it is necessary to conduct a long duration (e.g. 22 hours/day) whole-body exposure study. •Feed should be withheld during the exposure period unless exposure exceeds 6 hours. Water may be provided throughout a whole-body exposure. Control Animals
EXPOSURE CONDITIONS Administration of Concentrations Test Chemical Preparation in a Vehicle Control animals Test Chemical: Aerosol Particle Size Distribution To ensure sufficient exposure of the lower respiratory tract, an aerosol should meet the following standard whenever possible: Mass Median Aerodynamic Diameter (MMAD) ≤2 μm with a geometric standard deviation (σg or GSD) of 1-3 for rats (see GD 39). MONITORING 1. Chamber airflow 2. Chamber temperature and relative humidity 3. Test article: Nominal concentration 4. Test article: Actual concentration 5. Test article: Particle size distribution
OBSERVATION •FOOD AND WATER CONSUMPTION To ensure sufficient exposure of the lower respiratory tract, an aerosol should meet the following standard whenever possible: Mass Median Aerodynamic Diameter (MMAD) ≤2 μm with a geometric standard deviation (σg or GSD) of 1-3 for rats (see GD 39). •OPHTHALMOLOGICAL EXAMINATION Using an ophthalmoscope or an equivalent device, ophthalmological examinations of the fundus, refractive media, iris, and conjunctivae should be performed for all animals prior to the administration of the test chemical, •GROSS PATHOLOGY AND ORGAN WEIGHTS All test animals, including those which die during the test or are removed from the study for humane reasons should be subjected to complete exsanguination (if feasible) and gross necropsy •HISTOPATHOLOGY A histopathological evaluation of all the organs and tissues listed in Table 2 should be performed for the control and high concentration groups, and for all animals which die or are sacrificed during the study.
Inhalation Toxicity Studies- OECD guidelines
DATA AND REPORTING •Individual animal data on body weights, food consumption, clinical pathology, BALF analysis, gross pathology, organ weights, lung burden (when evaluated) and histopathology should be provided for both the range- finding and main study. •Clinical observation data should be summarized in tabular form showing for each test group the number of animals used, the number of animals displaying specific signs of toxicity, the number of animals found dead during the test or killed for humane reasons, time of death of individual animals, a description and time course of toxic effects and reversibility, and necropsy findings. • Any generally accepted statistical method may be used and the statistical methods should be selected during the design of the study.
TEST REPORT Test animals and husbandry Test chemical Vehicle Exposure data Test conditions
RESULTS Tabulation of chamber temperature, humidity, and airflow. Tabulation of chamber target, analytical, and nominal concentration data. For aerosols atmospheres with an MMAD >1μm (i.e., fines), information on the aerodynamic diameter size distribution For aerosols predominantly <100 nm, information on the distribution of thermodynamic equivalent diameter For aerosols with significant components in both the nano-size range and larger a combination of the above data Tabulation of response data and concentration level for each animal Tabulation of individual animal weights. Tabulation of food consumption Tabulation of clinical pathology data Tabulation of bronchoalveolar lavage fluid (BALF) data Tabulation of lung burden, when measured. Tabulation of pulmonary function data and body temperatures, when measured. Organ weights, gross pathology, and histopathological findings for each animal, if available. Report findings from optional health assessments of systemic toxicity (e.g. immunotoxicity, neurotoxicity, hepatotoxicity, cardiovascular effects).
DISCUSSION AND INTERPRETATION OF RESULTS Particular emphasis should be given to the description of methods used to meet the criteria of this test guideline. The respirability of aerosol particles in light of the overall findings should be addressed, especially if the MMAD standard could not be met. The consistency of methods used to determine analytical and nominal concentrations, and the relation of analytical concentrations to nominal concentrations should be included in the overall assessment of the study. The likely cause of death and predominant mode-of-action (systemic versus local) should be addressed. An explanation should be provided if there was a need to humanely sacrifice animals in pain or showing signs of severe and enduring distress, based on the criteria in the OECD Guidance Document on Humane Endpoints (5). The target organ(s) should be identified. The BMC or NOAEC and LOAEC should be determined. Descriptions of any complications that occurred in the main study that might have an impact on the results of the study. If applicable, detailed justification for the inclusion, design and methodology of post-observation lung burden determinations
REFERENCE •https://www.piscltd.org.uk/wp-content/uploads/2018/10/Anna-Maione_In- Vitro-Acute-Inhal-Tox.pdf •https://www.oecd.org/india/ •"Test No. 413: Subchronic Inhalation Toxicity: 90-day Study." OECD Guidelines for the Testing of Chemicals, Section 4, 2018. doi:10.1787/9789264070806-en. •"Test No. 412: Subchronic Inhalation Toxicity: 90-day Study." OECD Guidelines for the Testing of Chemicals, Section 4, 2018. doi:10.1787/9789264070806-en. •"Test No. 403: Subchronic Inhalation Toxicity: 90-day Study." OECD Guidelines for the Testing of Chemicals, Section 4, 2018. doi:10.1787/9789264070806-en.
Inhalation Toxicity Studies- OECD guidelines

More Related Content

PPTX
Genotoxicity studies according to oecd guildline.
Diana Lou
 
PPTX
test item characterization of regulatory of toxicological studies
SonaliJain736101
 
PPTX
ACUTE, SUB ACUTE & CHRONIC TOXICOLOGICAL STUDIES
Dr. Sindhu K., Asst. Prof., Dept. of VPT, VCG.
 
PPTX
REPRODUCTIVE TOXICITY STUDIES
Raghavendra institute of pharmaceutical education and research .
 
PPTX
Applications of proteomic sciences
sukanyakk
 
PPTX
Screening models for acute eye irritation &amp; skin sentization
pradnya Jagtap
 
PPTX
PPT on SMART city
Manjusha Manchala
 
PPTX
Informed consent process and procedures
Siddu K M
 
Genotoxicity studies according to oecd guildline.
Diana Lou
 
test item characterization of regulatory of toxicological studies
SonaliJain736101
 
ACUTE, SUB ACUTE & CHRONIC TOXICOLOGICAL STUDIES
Dr. Sindhu K., Asst. Prof., Dept. of VPT, VCG.
 
REPRODUCTIVE TOXICITY STUDIES
Raghavendra institute of pharmaceutical education and research .
 
Applications of proteomic sciences
sukanyakk
 
Screening models for acute eye irritation &amp; skin sentization
pradnya Jagtap
 
PPT on SMART city
Manjusha Manchala
 
Informed consent process and procedures
Siddu K M
 

What's hot (20)

PPTX
Invivo Carcinogenecity Studies
Raghavendra institute of pharmaceutical education and research .
 
PPTX
Acute dermal toxicity studies
jeshicabulsara
 
PPTX
Toxicology & Regulatory Guidelines for Conducting Toxicity Study
Janhavi Burade
 
PPTX
Male and female reproductive toxicology
Khadga Raj
 
PPTX
Toxicity Studies : Acute Eye Irritation, Dermal Irritation
Shubhu20
 
PPTX
alternative methods of animal toxicity.pptx
ashharnomani
 
PPTX
Female reproductive toxicity studies.pptx
ashharnomani
 
PPTX
dermal irritation and dermal toxicity studies.pptx
yashvikhankar1
 
PDF
Ind enabling studies.
Shikha Choudhary
 
PPTX
IND Enabling Studies by Kashikant Yadav
Kashikant Yadav
 
PDF
Pharmacology of free radicals
Shivam Diwaker
 
PPTX
Female reproductive toxicity studies acoording to SECHDULE Y AND ICH S5R3
SONALPANDE5
 
PPTX
toxicokinetics and saturation kinetics
pharmacistnitish
 
PPTX
Acute inhalational toxicity studies.pptx
Tanuj Silori
 
PPTX
Genotoxicity_studies M pharmacy Pharmacology.pptx
Ayodhya Paradhe
 
PPTX
TEST ITEM CHARACTERIZATION IN REGULATORY TOXICOLOGY STUDIES.pptx
ashharnomani
 
PPTX
IN-VIVO CARCINOGENICITY STUDIES.pptx TG451
NikitaBankoti2
 
PPTX
MALE REPRODUCTIVE TOXICITY STUDIES(Toxicokinetics).pptx
KhanSabit
 
PPTX
Acute dermal toxicity-402
Dr Ajay Mandal
 
PPTX
Alternative methods to animal toxicity testing
Sanchit Dhankhar
 
Invivo Carcinogenecity Studies
Raghavendra institute of pharmaceutical education and research .
 
Acute dermal toxicity studies
jeshicabulsara
 
Toxicology & Regulatory Guidelines for Conducting Toxicity Study
Janhavi Burade
 
Male and female reproductive toxicology
Khadga Raj
 
Toxicity Studies : Acute Eye Irritation, Dermal Irritation
Shubhu20
 
alternative methods of animal toxicity.pptx
ashharnomani
 
Female reproductive toxicity studies.pptx
ashharnomani
 
dermal irritation and dermal toxicity studies.pptx
yashvikhankar1
 
Ind enabling studies.
Shikha Choudhary
 
IND Enabling Studies by Kashikant Yadav
Kashikant Yadav
 
Pharmacology of free radicals
Shivam Diwaker
 
Female reproductive toxicity studies acoording to SECHDULE Y AND ICH S5R3
SONALPANDE5
 
toxicokinetics and saturation kinetics
pharmacistnitish
 
Acute inhalational toxicity studies.pptx
Tanuj Silori
 
Genotoxicity_studies M pharmacy Pharmacology.pptx
Ayodhya Paradhe
 
TEST ITEM CHARACTERIZATION IN REGULATORY TOXICOLOGY STUDIES.pptx
ashharnomani
 
IN-VIVO CARCINOGENICITY STUDIES.pptx TG451
NikitaBankoti2
 
MALE REPRODUCTIVE TOXICITY STUDIES(Toxicokinetics).pptx
KhanSabit
 
Acute dermal toxicity-402
Dr Ajay Mandal
 
Alternative methods to animal toxicity testing
Sanchit Dhankhar
 
Ad

Similar to Inhalation Toxicity Studies- OECD guidelines (20)

PPTX
inhational studies PPT.pptx
adityamalan2
 
PPTX
Acute toxicity study for inhalation
DRx Priya Shukla
 
PPTX
INHALATIONAL TOXICITY STUDIES.M pharmacy
AartiPal23
 
PPTX
OECD Guideline of Acute Inhalation toxicity
Central University haryana
 
PPTX
OECD Dermal testing By Priya.pptx
Priya818982
 
PPTX
Oecd guidelines for the testing of chemicals
kanchangupta66
 
PPTX
Chronic toxicity studies a brief outlook
RxVichuZ
 
PPTX
Oecd guidelines
Umangi Thakkar
 
PPTX
Chronic Toxicity Study (OECD TG-452).pptx
SIRAJUDDIN MOLLA
 
PPTX
Oecd guide line2
drkamalsahil
 
PPTX
Oecd guide line2
drkamalsahil
 
PPTX
OECD 423 GUIDELINES AND COMPARISON WITH THE 420 AND 425.
SanjaySinghrajwar
 
PPTX
Oecd acute,subacte, sub chronic dermal toxicity studies(402, 410, 411).
helasri gummadi
 
PPTX
Updates onAcute and Chronic Studies.pptx
Department of Pharmacy, MLN Medical College Prayagraj
 
PDF
420-Acute oral toxicity -Fixed dose method.pdf
Dheeraj Saini
 
PPTX
ptsm2 , unit 2 ( acute dermal toxicity) By; priyashree.pptx
PriyashreeK1
 
PPTX
chronic dermal and inhalational studies as per OECD
Sohil Shah
 
PPTX
Oecd for chronic toxicity
pavanreddy292
 
PPTX
Skin sensitisation, OECD Test guideline 406 .pptx
NikitaBankoti2
 
PPTX
dermal toxicity.pptx
VaishnaviPatel76
 
inhational studies PPT.pptx
adityamalan2
 
Acute toxicity study for inhalation
DRx Priya Shukla
 
INHALATIONAL TOXICITY STUDIES.M pharmacy
AartiPal23
 
OECD Guideline of Acute Inhalation toxicity
Central University haryana
 
OECD Dermal testing By Priya.pptx
Priya818982
 
Oecd guidelines for the testing of chemicals
kanchangupta66
 
Chronic toxicity studies a brief outlook
RxVichuZ
 
Oecd guidelines
Umangi Thakkar
 
Chronic Toxicity Study (OECD TG-452).pptx
SIRAJUDDIN MOLLA
 
Oecd guide line2
drkamalsahil
 
Oecd guide line2
drkamalsahil
 
OECD 423 GUIDELINES AND COMPARISON WITH THE 420 AND 425.
SanjaySinghrajwar
 
Oecd acute,subacte, sub chronic dermal toxicity studies(402, 410, 411).
helasri gummadi
 
Updates onAcute and Chronic Studies.pptx
Department of Pharmacy, MLN Medical College Prayagraj
 
420-Acute oral toxicity -Fixed dose method.pdf
Dheeraj Saini
 
ptsm2 , unit 2 ( acute dermal toxicity) By; priyashree.pptx
PriyashreeK1
 
chronic dermal and inhalational studies as per OECD
Sohil Shah
 
Oecd for chronic toxicity
pavanreddy292
 
Skin sensitisation, OECD Test guideline 406 .pptx
NikitaBankoti2
 
dermal toxicity.pptx
VaishnaviPatel76
 
Ad

Recently uploaded (20)

PDF
01. Histology New Classification of histo is clear calssification
voxapo4201
 
PPTX
Nancy Caroline Emergency Paramedic Chapter 16
djorgenmorris
 
PPTX
Understanding The Self : 1Sexual health
GMEVCCGarchitorenaLi
 
PDF
CHAPTER 9 MEETING SAFETY NEEDS FOR OLDER ADULTS.pdf
ANNIELOURRIECASABUEN1
 
PPTX
Nancy Caroline Emergency Paramedic Chapter 17
djorgenmorris
 
PPTX
Nancy Caroline Emergency Paramedic Chapter 4
djorgenmorris
 
PPTX
PEDIATRIC OSCE, MBBS, by Dr. Sangit Chhantyal(IOM)..pptx
SangeetChhantyal
 
PPTX
Nancy Caroline Emergency Paramedic Chapter 1
djorgenmorris
 
PDF
chapter 14.pdf Ch+12+SGOB.docx hilighted important stuff on exa,
mikolwarschaw
 
PPTX
DeployedMedicineMedical EquipmentTCCC.pptx
chucomgloco
 
PPTX
Nancy Caroline Emergency Paramedic Chapter 15
djorgenmorris
 
DOCX
ch 9 botes for OB aka Pregnant women eww
mikolwarschaw
 
PPTX
Nancy Caroline Emergency Paramedic Chapter 13
djorgenmorris
 
PPTX
Acute renal failure.pptx for BNs 2nd year
neelamjoshi0922
 
PDF
health promotion and maintenance of elderly
prasana8
 
PDF
NURSING INFORMATICS AND NURSE ENTREPRENEURSHIP
ANNIELOURRIECASABUEN1
 
PDF
Assessment of Complications in Patients Maltreated with Fixed Self Cure Acryl...
Aun Haider
 
PPTX
FUNCTIONS OF BLOOD PART I AND PART 2 WHOLE
SANJANASHARMA999053
 
PPTX
Nancy Caroline Emergency Paramedic Chapter 7
djorgenmorris
 
PPTX
Newer Technologies in medical field.pptx
advocatehiteshbhatt
 
01. Histology New Classification of histo is clear calssification
voxapo4201
 
Nancy Caroline Emergency Paramedic Chapter 16
djorgenmorris
 
Understanding The Self : 1Sexual health
GMEVCCGarchitorenaLi
 
CHAPTER 9 MEETING SAFETY NEEDS FOR OLDER ADULTS.pdf
ANNIELOURRIECASABUEN1
 
Nancy Caroline Emergency Paramedic Chapter 17
djorgenmorris
 
Nancy Caroline Emergency Paramedic Chapter 4
djorgenmorris
 
PEDIATRIC OSCE, MBBS, by Dr. Sangit Chhantyal(IOM)..pptx
SangeetChhantyal
 
Nancy Caroline Emergency Paramedic Chapter 1
djorgenmorris
 
chapter 14.pdf Ch+12+SGOB.docx hilighted important stuff on exa,
mikolwarschaw
 
DeployedMedicineMedical EquipmentTCCC.pptx
chucomgloco
 
Nancy Caroline Emergency Paramedic Chapter 15
djorgenmorris
 
ch 9 botes for OB aka Pregnant women eww
mikolwarschaw
 
Nancy Caroline Emergency Paramedic Chapter 13
djorgenmorris
 
Acute renal failure.pptx for BNs 2nd year
neelamjoshi0922
 
health promotion and maintenance of elderly
prasana8
 
NURSING INFORMATICS AND NURSE ENTREPRENEURSHIP
ANNIELOURRIECASABUEN1
 
Assessment of Complications in Patients Maltreated with Fixed Self Cure Acryl...
Aun Haider
 
FUNCTIONS OF BLOOD PART I AND PART 2 WHOLE
SANJANASHARMA999053
 
Nancy Caroline Emergency Paramedic Chapter 7
djorgenmorris
 
Newer Technologies in medical field.pptx
advocatehiteshbhatt
 

Inhalation Toxicity Studies- OECD guidelines

  • 1. SEMINAR 2 PHARMACOLOGICALAND TOXICOLOGICAL SCREENING METHODS SUBMITTED BY : CERIN MAREENA PHILIP SECOND SEMESTER M.PHARM DEPARTMENT OF PHARMACOLOGY KMCH COLLEGE OF PHARMACY SUBMITTED TO :DR.G.VENKATESH ASSOCIATE PROFESSOR DEPARTMENT OF PHARMACOLOGY KMCH COLLEGE OF PHARMACY DATE:1/08/2019
  • 3. CONTENT ACUTE TOXICITY STUDY FOR INHALATION • INTRODUCTION • PRINCIPLE • DESCRIPTION OF METHOD • EXPOSURE CONDITIONS AND MONITORING • PROCEDURE • OBSERVATION • DATAAND REPORT • INTERPRETATION SUBACUTE TOXICITY STUDY FOR INHALATION SUBCHRONIC TOXICITY STUDY FOR INHALATION REFERENCE
  • 5. INTRODUCTION • Acute toxicity study for inhalation was documented as document no. 39 as OECD GUIDELINE FOR THE TESTING OF CHEMICALS • This revised Test Guideline 403 (TG 403) has been designed to be more flexible, to reduce animal usage, and to fulfill regulatory needs. • The revised TG 403 features two study types: • 1. A Traditional LC50 protocol and • 2. A Concentration * Time (C x t) protocol.
  • 6. • SELECTION  of the most appropriate species,strain,sex,mode of exposure,appropriate test concentrations include • the identity, • chemical structure, and physicochemical properties of the test article; • results of any in vitro or in vivo toxicity tests; • anticipated uses and potential for human exposure; • available (Q)SAR data and toxicological data on structurally related substance. • The targeted concentrations should not induce severe irritation/corrosive effects, yet reach scientific objective of the test.
  • 7. • to provide lethality data. • (e.g. LC50, LC01 and slope) for one or both sexes as needed for quantitative risk assessments.This Guideline offers two test methods.  Traditional protocol • groups of animals are exposed to a limit concentration (limit test) • stepwise procedure • predetermined duration of usually 4 hours.  (C x t) protocol • groups of animals are exposed to one (limit concentration) • multiple concentrations • multiple durations. • OECD Guidance Document No. 19 PRINCILPE
  • 8. DESCRIPTION OF THE METHOD 1. Selection of animal species:- 2. Preparation of animals 3. Animal husbandry 4. Inhalation chambers  Selection of animal species:- • Healthy young adult animals of commonly used laboratory strains should be used. • The preferred species is the rat and justification should be provided if other species are used. • both sex
  • 9.  Preparation of animals 1. Females should be nulliparous and nonpregnant. 2. On the exposure day, animals should be young adults 8 to 12 weeks of age, and body weights should be within ±20% of the mean weight for each sex of any previously exposed animals of the same age. 3. The animals are randomly selected and marked for individual identification. The animals are kept in their cages for at least 5 days prior to the start of the test.
  • 10. • Animal husbandry • The temperature should be 22±3°C. • The relative humidity should ideally be maintained in the range of 30 to 70%, • Before and after exposures, animals generally should be caged in groups by sex and concentration, • When animals are to be exposed nose-only, to be acclimated to the restraining tubes. • Animals exposed whole-body to an aerosol should be housed individually during exposure • Lighting should be artificial, the sequence being 12 hours light/12 hours dark.
  • 11.  Inhalation chambers • The nature of the test article and the objective of the test should be considered when Selecting an inhalation chamber. • The preferred mode of exposure snout-only. • preferred for studies of liquid or solid aerosols and for vapours that may condense to form aerosols. • Toensure atmosphere stability when using a whole-body chamber.
  • 12. EXPOSURE CONDITIONS: Administration of concentrations Particle-size distribution Test article preparation in a vehicle Control animals
  • 13. Administration of Concentration Nose-only exposures may be any duration up to 6 hours in rats. If mice are exposed nose- only, exposures generally should not exceed 4 hours. Particle-size distribution Particle sizing should be performed for all aerosols and for vapours that may condense to form aerosols. ( metal fumes may be smaller than this standard, and charged particles, fibres, and hygroscopic materials ) Test article preparation in a vehicle vehicle may be used to generate an appropriate concentration and particle size of the test in atmosphere (water). Adequate care should be taken to not contaminate the test material. Control animals A concurrent negative (air) control group is not necessary.
  • 14. MONITORING OF EXPOSURE CONDITIONS 1. Chamber airflow 2. Chamber temperature and relative humidity 3. Test article: Nominal concentration 4. Test article: Actual concentration 5. Test article: Particle size distribution
  • 15. 1. Chamber Airflow The flow of air should be carefully Controlled,Continuously monitored,Recorded( at least hourly during each exposure). Oxygen concentration should be at least 19% and carbon dioxide concentration should not exceed 1%.(if not measured) 2. Chamber temperature and relative humidity • Chamber temperature should be maintained at 22±3°C. • Relative humidity should be monitored and recorded at least three times for durations of up to 4 hrs, and hourly for shorter durations. • The relative humidity maintained in the range of 30 to 70%. 3. Test article: Nominal concentration • mass of generated test • the total volume of air passed through the chamber system. • The nominal exposure chamber concentration should be calculated and recorded.
  • 16. • 4.Test article:Actual concentration The actual concentration is the test article concentration at the animals breathing zone in an inhalation chamber. Actual concentrations can be obtained by specific methods (e.g. direct sampling, adsorptive or chemical reactive methods, & analytical characterisation) or by nonspecific methods such as gravimetric filter analysis. the test sample should be stored under conditions that maintain its purity, homogeneity, and stability. 5. Test article: Particle size distribution determined at least twice during each 4 hour exposure by using a cascade impactor or an aerodynamic particle sizer. A second device, such as a gravimetric filter or an impinger/gas bubbler, should be used in parallel . Particle sizing should be performed for vapours if vapour condensation may result in the formation of an aerosol,
  • 17. PROCEDURE- Traditional General considerations: In a Traditional study, groups of animals are exposed to a test article for a fixed period of time (generally 4 hours) in either a nose-only or whole-body exposure chamber. Animals are exposed to either a limit concentration (limit test), or to at least three concentrations in a stepwise procedure (main study). A sighting study may precede a main study unless some information about the test article already exists, such as a previously performed TG 436. 2. Sighting study A sighting study is used to estimate test article potency, identify sex differences in susceptibility, and assist in selecting exposure concentration levels for the main study or limit test. When selecting concentration levels for the sighting study, all available information should be used (3 animals/sex may be needed to establish a sex difference). A sighting study may consist of a single concentration, but more concentrations may be tested if necessary. 3. Limit test A limit test is used when the test article is known or expected to be virtually non-toxic, Three animals of both sex each. In those situations where there is little or no information about its toxicity, or the test material is expected to be toxic, the main test should be performed. When the GHS Classification System is used, the limit concentrations for gases, vapours, and aerosols are 20000 ppm, 20 mg/L, and 5 mg/L, respectively (or the maximum attainable concentration) 4. Main Test A main study is typically performed using five males and five females (or 5 animals of the susceptible sex, if known) per concentration level, with at least three concentration levels. The time interval between exposure groups is determined by the onset, duration, and severity of toxic signs.
  • 18. C X T PROTOCOL General considerations: 2. Sighting study 3 animal of per sex is used for test and exposure of animal is done for single duration mainly 240 min. When selecting the initial target concentration , the study director should consider the mortality patterns observed in any available TG 436 studies 3. Initial Concentration Group of 1 animal/sex is expose to the test article initial conc for time interval of 15, 30, 60 , 120 and 240 min Total 10 animals are used in this test. When the GHS Classification System is used, the limit concentrations for gases, vapours, and aerosols are 20000 ppm, 20 mg/L and 5 mg/L, respectively. If less than 50% lethality occurs at the maximum attainable concentration, no further testing is necessary. Main study
  • 20. MAIN STUDY Exposure Session I –Testing at the limit concentration 1 animal/sex per concentration/time point; 10 animals in total a Target concentration = limit concentration. Expose five groups of animals at this target concentration for durations of 15, 30, 60, 120 and 240 minutes, respectively Exposure Session II – Main Study 1 animal/sex per concentration/time point; 10 animals in total. Expose five groups of animals at a lower concentration d (1/2L) with slightly lower duration of exposure.
  • 21. Exposure Session III – Main Study 1 animal/sex per concentration/time point; 10 animals total. Expose five groups of animals at a lower concentration d (1/4L) with slightly longer exposure durations. Exposure Session IV′ – Main Study 1 animal/sex per concentration/time point; 10 animals total. Expose five groups of animals at a lower concentration d (1/8L) with slightly longer exposure durations Exposure Session IV – Main Study 1 animal/sex per concentration/time point; 10 animals total. Expose five groups of animals at a higher concentration e (2L) with slightly shorter exposure durations.
  • 22. OBSERVATION Once daily for 14 days. The length of the observation period is not fixed, The times at which signs of toxicity appear and disappear are important, individual records being maintained for each animal Animals found in a moribund condition should be humanely killed for animal welfare reasons. Cage-side observations should include changes in the skin and fur, eyes and mucous membranes, and also respiratory, circulatory, autonomic and central nervous systems, and somatomotor activity and behaviour patterns Attention should be directed to observations of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.
  • 23. • Body weights • Individual animal weights should be recorded once during the acclimatization period, on the day of exposure prior to exposure (day 0), and at least on days 1, 3 and 7 (and weekly thereafter), and at the time of death or euthanasia • Pathology • All test animals, including those which die during the test or are euthanized and removed from the study for animal welfare reasons, should be subjected to gross necropsy.
  • 24. DATA AND REPORTING • Data : Individual animal data on body weights and necropsy findings should be provided. • summarized in tabular form, showing for each test group the number of animals used, the number of animals displaying specific signs of toxicity, the number and time of animals found dead during the test or killed for humane reasons, , a description and time course of toxic effect.
  • 25. TEST REPORT • The test report should include the following information, as appropriate: • Description of caging conditions, including: number (or change in number) of animals per cage, bedding material, ambient temperature and relative humidity, photoperiod, and identification of diet • - Species/strain used and justification for using a species other than the rat • - Number, age and sex of animals • - Method of randomization • - Details of food and water quality (including diet type/source, water source); • - Description of any pre-test conditioning including diet, quarantine, and treatment for disease
  • 26. RESULTS • Tabulation of chamber temperature, humidity, and airflow; - Tabulation of chamber nominal and actual concentration data • Tabulation of particle size data • Individual body weights of animals collected on study; • date and time of death if prior to scheduled euthanasia, • time course of onset of signs of toxicity and whether these were reversible for each animal • Necropsy findings and histopathological findings for each animal, if available • Lethality estimates (e.g. LC50, LD01) including 95% confidence limits, and slope (if provided by the evaluation method) • Statistical relation, including estimate for the exponent n (C x t protocol). The name of the statistical software used should be provided.
  • 27. SUBACUTE TOXICITY TESTING AS PER OECD GUIDELINES (412)
  • 28. OECD Guidelines No. 412: 28-day inhalation Toxicity Study in Rodents  Toxico-kinetics and systemic toxicity study is also studied.  Provide robust data for quantitative inhalation risk assessments. Subacute inhalation toxicity studies are primarily used to derive regulatory concentrations for assessing worker risk in occupational settings. (Q)SAR data and toxicological data on structurally related chemicals. The data derived from subacute inhalation toxicity studies can be used for quantitative risk assessments and for the selection of concentrations for chronic studies.
  • 29. PRINCIPLE Accommodate the testing of nanomaterials as well as to reflect the evolving state-of-the-science for the testing of inhaled gases, vapours, and aerosols. Broncho-alveolar lavage fluid (BALF) to be performed for all test chemicals POE-BALF analysis and lung burden measurements are performed. for all test chemicals( 24h) after exposure termination When testing a solid aerosol, it is useful to have information on its retention and kinetics in the lung. Dilutions of corrosive or irritating test chemicals may be tested at concentrations that will yield the desired degree of toxicity.
  • 31. 1. Selection of Animal Species Healthy young adult rodents of commonly used laboratory strains should be employed. The preferred species is the rat. Justification should be provided if other species are used. 2. Preparation of Animals 5 males and females should be exposed at each concentration. On the day of randomization, animals should be young adults 7 to 9 weeks of age. Body weights should be within ± 20% of the mean weight for each sex. allow for acclimatization to laboratory conditions. 3Inhalation Chambers Subacute inhalation toxicity studies are always performed in dynamic inhalation chambers. The use of a static inhalation chamber, which has no airflow, is not acceptable. 1Limit Concentrations The maximum concentration tested should consider: attainable the need to maintain an adequate oxygen supply, and/or animal welfare considerations. Up to a maximum concentration of 5 mg/L for aerosols,20 mg/L for vapors, and 20,000 ppm for gases DESCRIPTION OF THE METHOD
  • 32. Dynamic Inhalation Chamber & Static Inhalation Chamber
  • 33. 2 Range-Finding Study  To inform the selection of concentration levels for a main study.  No Observed Adverse Effects Concentration (NOAEC), Lowest Observed Adverse Effects Concentration (LOAEC), Maximum Tolerated Concentration (MTC), and/or the benchmark concentration (BMC) in a main study. The study director should use a range-finding study to identify the upper concentration that is tolerated without undue stress to the animals. A range-finding study should last a minimum of 5 days and generally no more than 14 days, and may include a post-exposure period and animal numbers should be adjusted accordingly.
  • 34. When testing poorly soluble particles, it may be necessary for a range-finding study to be longer than 14 days to allow for a robust assessment of test chemical solubility and lung burden. The rationale for the selection of concentrations for the main study should be provided in the study report. 3 Main Study The main study consists of at least three test chemical concentration levels and concurrent negative (air) or vehicle controls. This guideline differentiates two study designs depending on the nature of the test chemical. Option A, which is generally used for test chemicals (as gas,vapour, aerosol, or a mixture thereof), option B is used when testing chemicals that are likely to be retained in the lungs.
  • 37. OBSERVATIONS Bronchoalveolar Lavage • BAL should be performed at PEO-1 (within 24h at termination of exposure) and at one post-exposure interval (PEO-2) • When testing a poorly soluble aerosol, BAL should be performed at PEO-1 (within 24 hours of exposure termination) in both sexes and in female animals at PEO-2 if a recovery group is scheduled. • The right lung is generally preferred for lavage. The duration of the post-exposure period and the timing of the PEOs are determined by the study director based on findings in the range-finding study and other available, relevant information. • The mandatory BALF analysis encompasses the following parameters: Lactate dehydrogenase (LDH) Total protein or albumin Cell counts and differentials for alveolar macrophages, lymphocytes, neutrophils, and eosinophils
  • 38. •Lung Burden • When testing poorly soluble solid aerosols, measurements of lung burden can provide clarity on the retained dose. • Males are used because they have a higher minute volume than females and may thus have greater lung burdens. • To obtain clear information on lung clearance kinetics the same lung (the right lung is recommended) should be measured for lung burden at all post-exposure time points.  Ophthalmological Examination • Refractive media, iris, and conjunctivae. Gross Pathology And Organ Weights
  • 39. DATA AND REPORTING • Individual animal data on body weights, food consumption, clinical pathology, BALF analysis, gross pathology, organ weights, lung burden (when evaluated) and histopathology should be provided for both the range-finding and main study. • Clinical observation data should be summarized in tabular form showing for each test group the number of animals used, the numberof animals displaying specific signs of toxicity, • the number of animals found dead during the test or killed for humane reasons, time of death of individual animals, a description and time course of toxic effects and reversibility, and necropsy findings.
  • 40. DISCUSSION & INTERPRETATION OF RESULTS • The respirability of aerosol particles in light of the overall findings should be addressed, especially if the MMAD standard could not be met. • The consistency of methods used to determine analytical and nominal concentrations, and the relation of analytical concentrations to nominal concentrations should be included in the overall assessment of the study.  The test report must include the following information: - species/strain used & toxic response data by sex and dose; - time of death during the study or whether animals survived or termination; - Toxic effects and the time of observation of each abnormal sign and its subsequent course & food and body weight data.
  • 42. •INTRODUCTION •The original subchronic inhalation Test Guideline 413 (TG 413) was adopted in 1981 (2). •This revision requires specific measurements of bronchoalveolar lavage fluid (BALF), Measurements of lung burden, should be done when a range-finding study or other relevant information suggests that inhaled test particles are poorly soluble and likely to be retained in the lung. •A range-finding study (or studies), which primarily is (are) performed to assess concentration levels for the main study should also include BALF analysis, and may also include lung burden measurements •Subchronic inhalation toxicity studies are primarily used to derive regulatory concentrations for assessing worker risk in occupational settings. •This guideline enables the characterization of adverse effects following repeated daily inhalation exposure to a test chemical for 90 days. •Definitions of technical terms used in this Test Guideline can be found in GD 39 (1). •revising this test guideline was to accommodate the testing of nanomaterials as well as to reflect the evolving state-of-the-science for the testing of inhaled gases, vapours, and aerosols.
  • 43. INITIAL CONSIDERATIONS •All available information on the test chemical should be considered by the testing laboratory prior to conducting the main study in order to enhance the quality of the study, minimize animal usage, and avoid the need to repeat the study. •The respirable (or alveolar) fraction of poorly soluble particles that are slowly cleared can accumulate with each consecutive exposure period. •Dilutions of corrosive or irritating test chemicals may be tested at concentrations that will yield the desired degree of toxicity. •Animals that are moribund obviously in pain or showing signs of severe and enduring distress should be humanely sacrificed. •DESCRIPTION OF THE METHOD • Selection of animal species:- • Preparation of animals • Animal husbandry • Inhalation chambers
  • 44. TOXICITY STUDIES Limit Concentrations Range-Finding Study • A range-finding study should be performed unless sufficient information already exists to perform a robust main study. • A range-finding study may, for example, provide information regarding analytical methods, particle size distribution, systemic toxicity, toxicokinetics, test chemical solubility in the lung,BALF data, and estimates of what may be the No Observed Adverse Effects Concentration (NOAEC), Lowest Observed Adverse Effects Concentration (LOAEC), Maximum Tolerated Concentration (MTC), and/or the benchmark concentration (BMC) in a main study. • A range-finding study may consist of one or more test chemical concentration levels and a control group. Depending on the endpoints chosen, typically no more than 5 males and 5 females should be exposed at each concentration level. • should last a minimum of 5 days and generally no more than 28 days,
  • 45. The main study •The main study consists of at least three test chemical concentration levels and concurrent negative (air) or vehicle controls •Each group consists of a minimum of 10 male and 10 female rodents that are exposed to the test chemical for 6 hours per day on a 5 day per week basis for a period of 13 weeks (total study duration of at least 90 days). •Animals may also be exposed 7 days per week. •If rodent species other than rats are exposed nose-only, •A rationale should be provided when using exposure duration less than 6 hours/day, or when it is necessary to conduct a long duration (e.g. 22 hours/day) whole-body exposure study. •Feed should be withheld during the exposure period unless exposure exceeds 6 hours. Water may be provided throughout a whole-body exposure. Control Animals
  • 46. EXPOSURE CONDITIONS Administration of Concentrations Test Chemical Preparation in a Vehicle Control animals Test Chemical: Aerosol Particle Size Distribution To ensure sufficient exposure of the lower respiratory tract, an aerosol should meet the following standard whenever possible: Mass Median Aerodynamic Diameter (MMAD) ≤2 μm with a geometric standard deviation (σg or GSD) of 1-3 for rats (see GD 39). MONITORING 1. Chamber airflow 2. Chamber temperature and relative humidity 3. Test article: Nominal concentration 4. Test article: Actual concentration 5. Test article: Particle size distribution
  • 47. OBSERVATION •FOOD AND WATER CONSUMPTION To ensure sufficient exposure of the lower respiratory tract, an aerosol should meet the following standard whenever possible: Mass Median Aerodynamic Diameter (MMAD) ≤2 μm with a geometric standard deviation (σg or GSD) of 1-3 for rats (see GD 39). •OPHTHALMOLOGICAL EXAMINATION Using an ophthalmoscope or an equivalent device, ophthalmological examinations of the fundus, refractive media, iris, and conjunctivae should be performed for all animals prior to the administration of the test chemical, •GROSS PATHOLOGY AND ORGAN WEIGHTS All test animals, including those which die during the test or are removed from the study for humane reasons should be subjected to complete exsanguination (if feasible) and gross necropsy •HISTOPATHOLOGY A histopathological evaluation of all the organs and tissues listed in Table 2 should be performed for the control and high concentration groups, and for all animals which die or are sacrificed during the study.
  • 49. DATA AND REPORTING •Individual animal data on body weights, food consumption, clinical pathology, BALF analysis, gross pathology, organ weights, lung burden (when evaluated) and histopathology should be provided for both the range- finding and main study. •Clinical observation data should be summarized in tabular form showing for each test group the number of animals used, the number of animals displaying specific signs of toxicity, the number of animals found dead during the test or killed for humane reasons, time of death of individual animals, a description and time course of toxic effects and reversibility, and necropsy findings. • Any generally accepted statistical method may be used and the statistical methods should be selected during the design of the study.
  • 51. RESULTS Tabulation of chamber temperature, humidity, and airflow. Tabulation of chamber target, analytical, and nominal concentration data. For aerosols atmospheres with an MMAD >1μm (i.e., fines), information on the aerodynamic diameter size distribution For aerosols predominantly <100 nm, information on the distribution of thermodynamic equivalent diameter For aerosols with significant components in both the nano-size range and larger a combination of the above data Tabulation of response data and concentration level for each animal Tabulation of individual animal weights. Tabulation of food consumption Tabulation of clinical pathology data Tabulation of bronchoalveolar lavage fluid (BALF) data Tabulation of lung burden, when measured. Tabulation of pulmonary function data and body temperatures, when measured. Organ weights, gross pathology, and histopathological findings for each animal, if available. Report findings from optional health assessments of systemic toxicity (e.g. immunotoxicity, neurotoxicity, hepatotoxicity, cardiovascular effects).
  • 52. DISCUSSION AND INTERPRETATION OF RESULTS Particular emphasis should be given to the description of methods used to meet the criteria of this test guideline. The respirability of aerosol particles in light of the overall findings should be addressed, especially if the MMAD standard could not be met. The consistency of methods used to determine analytical and nominal concentrations, and the relation of analytical concentrations to nominal concentrations should be included in the overall assessment of the study. The likely cause of death and predominant mode-of-action (systemic versus local) should be addressed. An explanation should be provided if there was a need to humanely sacrifice animals in pain or showing signs of severe and enduring distress, based on the criteria in the OECD Guidance Document on Humane Endpoints (5). The target organ(s) should be identified. The BMC or NOAEC and LOAEC should be determined. Descriptions of any complications that occurred in the main study that might have an impact on the results of the study. If applicable, detailed justification for the inclusion, design and methodology of post-observation lung burden determinations
  • 53. REFERENCE •https://www.piscltd.org.uk/wp-content/uploads/2018/10/Anna-Maione_In- Vitro-Acute-Inhal-Tox.pdf •https://www.oecd.org/india/ •"Test No. 413: Subchronic Inhalation Toxicity: 90-day Study." OECD Guidelines for the Testing of Chemicals, Section 4, 2018. doi:10.1787/9789264070806-en. •"Test No. 412: Subchronic Inhalation Toxicity: 90-day Study." OECD Guidelines for the Testing of Chemicals, Section 4, 2018. doi:10.1787/9789264070806-en. •"Test No. 403: Subchronic Inhalation Toxicity: 90-day Study." OECD Guidelines for the Testing of Chemicals, Section 4, 2018. doi:10.1787/9789264070806-en.