GB 15193.14-2015_English: PDF (GB15193.14-2015)
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National food safety standard -- Teratogenicity test
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GB 15193.14-2015
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GB 15193.14-2003 | English | 239 |
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Teratogenicity study
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GB 15193.14-2003
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GB 15193.14-1994 | English | 199 |
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Teratogenicity test
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Standard ID | GB 15193.14-2015 (GB15193.14-2015) | Description (Translated English) | National food safety standard -- Teratogenicity test | Sector / Industry | National Standard | Classification of Chinese Standard | C53 | Classification of International Standard | 7.1 | Word Count Estimation | 10,119 | Date of Issue | 2015-08-07 | Date of Implementation | 2015-10-07 | Older Standard (superseded by this standard) | GB 15193.14-2003 | Administrative Organization | National Health and Family Planning Commission | Regulation (derived from) | National Food Safety Standard Announcement 2015 No.6 | Issuing agency(ies) | National Health and Family Planning Commission of the People's Republic of China | Summary | This standard specifies the test methods and technical requirements for animal teratogenic tests. This standard applies to the evaluation of teratogenicity of the test substance. | Standard ID | GB 15193.14-2003 (GB15193.14-2003) | Description (Translated English) | Teratogenicity study | Sector / Industry | National Standard | Classification of Chinese Standard | C53 | Classification of International Standard | 07.100 | Word Count Estimation | 6,662 | Date of Issue | 2003-09-24 | Date of Implementation | 2004-05-01 | Older Standard (superseded by this standard) | GB 15193.14-1994 | Drafting Organization | Zhejiang Medical University | Administrative Organization | Ministry of Health | Regulation (derived from) | Announcement of Newly Approved National Standards No. 11 of 2003 (77 national food hygiene standards) | Proposing organization | Ministry of Health of the People Republic of China | Issuing agency(ies) | The People Republic of China Ministry of Health, China National Standardization Management Committee | Summary | This Chinese standard specifies the prediction of environmental harmful substances, embryotoxicity and teratogenicity test methods. This standard applies to evaluation of food production, processing, preservation, transportation and sales process involves the possible health hazards of chemical, biological and physical factors teratogenicity test objects including food additives (including nutritional agent), food new resources and its components, the new resources of food, irradiated food, food containers and packaging materials, food tools, equipment, detergents, disinfectants, pesticides, veterinary drug residues, food industrial microorganisms. | Standard ID | GB 15193.14-1994 (GB15193.14-1994) | Description (Translated English) | Teratogenicity test | Sector / Industry | National Standard | Classification of Chinese Standard | C53 | Classification of International Standard | 07.100 | Word Count Estimation | 3,394 | Date of Issue | 1994/8/10 | Date of Implementation | 1994/8/10 | Regulation (derived from) | Announcement of Newly Approved National Standards 2003 No. 11 (77 food hygiene National Standards) | Summary | This Standard specifies the predicted environmental harmful substances produced embryo toxicity and teratogenicity test methods. This Standard is applicable to the evaluation of teratogenicity production and sale of all food ingredients and food products in. New resources such as food, food additives, food packaging materials, and washed with disinfectants. |
GB 15193.14-2015
(National food safety standards teratogenicity test)
National Standards of People's Republic of China
National Food Safety Standard
Teratogenicity test
Issued on.2015-08-07
2015-10-07 implementation
People's Republic of China
National Health and Family Planning Commission released
Foreword
This standard replaces GB 15193.14-2003 "teratogenicity test."
This standard compared with GB 15193.14-2003, the main changes are as follows.
--- Standard name was changed to "national food safety standards teratogenicity test";
--- Modify the scope;
--- Increasing the content of terms and definitions, test purposes, test report and interpretation;
--- Increasing the initial animal weight difference should not exceed 20% of the average weight requirement;
--- Increased animal feeding requirements;
--- Modify the number of pregnant mice when requested termination of the trial;
--- Adds a way to establish a positive control group "with cyclophosphamide (15mg/kg body weight) on day 12 of gestation intraperitoneal injection
1 time";
--- Increased maternal mortality rate shall not exceed 10% of the content;
--- Increased administration of the test substance;
--- Modify the traditional administration of the test substance teratogenicity test rats time;
--- Increased maternal performance observed during administration of the test substance, recording the amount of water, if necessary;
--- Increased for all pregnant mother visual examination;
--- Amend Table 2;
--- Delete the table "teratogenicity test record content," adds the need to organize the data content; statistics project and increased the net weight of
Other than that, remove the ovary weight statistics;
--- Increased content and information should be listed in the test report.
National Food Safety Standard
Teratogenicity test
1 Scope
This standard specifies the technical requirements and test methods for animal teratogenicity tests.
This standard applies to the evaluation of the test substance teratogenic effects.
2 Terms and definitions
2.1 Developmental Toxicity
Individuals prenatal exposure to the test substance, and development before becoming adult (including embryo, fetal and postnatal) harmful effects occur, table
Now the development of the organism structural abnormalities, altered growth, dysfunction and death.
2.2 teratogenicity
Test substance during organogenesis caused by structural abnormalities in the offspring of a permanent nature.
2.3 maternal toxicity
Effects of test substances cause health damage, direct or indirect parent females pregnant animals, expressed as weight reduction, dysfunction, and signs of poisoning,
even death.
3 test purposes and principles
The role of the parent subject to some hazardous substances through the placenta barrier during pregnancy, embryonic differentiation and organ development, leading to structural differences
Often, there fetal malformation. Therefore, in the conception of the animal embryo organogenesis of administration of the test substance, the substance can be detected in fetal teratogenicity
effect.
Teratogenic likelihood of detecting pregnant animals after contact with the test substance caused predict human potential teratogenicity.
4 instruments and reagents
4.1 Instruments and Equipment
Commonly used laboratory equipment, biological microscopes, stereo microscopes, calipers, analytical balance.
4.2 Reagents
4.2.1 Reagents
Formaldehyde, acetic acid, 2,4,6-nitro phenol, potassium hydroxide, glycerol, chloral hydrate, Alizarin Red.
4.2.2 The main method of reagent preparation
4.2.2.1 Alizarin Red stock solution
With 50% acetic acid as solvent alizarin red saturated liquid 5.0mL, glycerol 10.0mL, 1% chloral hydrate 60.0mL mixed, stored in a brown
Bottle.
4.2.2.2 Application of Alizarin Red solution
Stock solution 3mL ~ 5mL, diluted with 10g/L ~ 20g/L of potassium hydroxide solution to 1000mL, stored in a brown bottle.
4.2.2.3 Alizarin Red solution
Alizarin Red 0.1g, potassium hydroxide 10g, distilled water 1000mL, Extemporaneous (peeling method bone staining solution).
4.2.2.4 A transparent liquid
Glycerol 200mL, potassium hydroxide 10g, 790mL distilled water and mixed.
4.2.2.5 transparent liquid B
Distilled water mixed with glycerin and other volume.
4.2.2.6 fixative (Bouins liquid)
2,4,6-nitrophenol (saturated aqueous picric acid) 75 parts 20 parts 40% formaldehyde, 5 parts of glacial acetic acid.
5 Test methods
5.1 test substance
The test substance should be used in the original sample, if it can not use the original sample, the test should be treated in accordance with the principles of the test substance for proper disposal.
5.2 Experimental Animals
5.2.1 species Selection
Experimental animals should be selected in line with GB 14922.2 and regulations. The preferred rodent rat, rabbit preferred non-rodent. If the selection of its
He species should give reasons. Healthy, sexually mature males and females without mating animal weight difference when the trial began,
Should not exceed ± 20% of average weight. The animal should indicate species, strain, sex, weight and weeks of age.
5.2.2 Number and sex of animals
Sexually mature males and females usually in the ratio of 1 to 1 or 1.2 mated, if not mate within 5d, males should be replaced. in order to
Get enough litter to evaluate its teratogenic effects, the number of pregnant animals rats each dose level of at least 16 rabbits each dose level
Not less than 12 the number of pregnant animals.
5.2.3 Animal Preparation
Before the test, animals in the experimental animal room should be at least 3d ~ 5d acclimatization and quarantine observation.
5.2.4 Animal Feeding
Animal feeding conditions, drinking water, feed shall conform to GB 14925, GB 5749, GB 14924.3 and regulations. test
During free access to water and food animals, pregnant animals should be caged.
5.3 dose
The experiment included at least three dose groups, at the same time provided the vehicle control group, vehicle control group, in addition to not give the test substance, the rest were treated with dose group.
If necessary, positive control group, commonly used orally administered dose of positive control and reference enemy blight double (0.5mg/kg body weight ~ 1.0mg/kg
Weight), pentachlorophenol sodium (30mg/kg body weight), aspirin (250mg/kg body weight ~ 300mg/kg body weight) and vitamin A
(7500μg/kg body weight ~ 13000μg/kg body importance retinol equivalents), etc., or cyclophosphamide (15mg/kg body weight) on day 12 of gestation
Intraperitoneal injection of 1 times. Has been carried out with a teratogenicity test was positive, and in the experimental animals used in the germline have found positive results, the test set can be omitted
Positive control group.
High dose group principle should be part of the developmental toxicity of certain animals and (or) maternal toxicity, such as weight mild relief, but not to cause
Death or serious illness, if the mother animal has died, should not exceed 10% of the parent of the number of animals. There should not be any low-dose group
Observed maternal toxicity or developmental toxicity. We recommend decreasing the dose range of group spacing 2 to 4 times more appropriate. When compared with the gap between the two groups
Large (such as more than 10-fold) increase provided a test group.
The reference design test dose acute toxicity test dose 28-day oral toxicity test the 90-day oral toxicity test dose and the actual human body
Intake amount. For the LD50 test substance can be determined, based on LD50 value and the dose - response relationship curve slope design dose high dose group.
For not seeking LD50 test substance, if 28 days or 90 days oral toxicity test not observed adverse effects were not observed with the greatest harmful
Role as a high-dose dose; if the 28d or 90d oral test observed adverse effects, with minimal observed adverse effect level (LOA-
EL) for the high dose group, set up the following two-dose group. Reference should also be other information on toxicology test substance when setting the dose level.
5.4 Test procedure and outcome measures
5.4.1 "pregnant animals," inspection
For rats, male and female animals with cages every morning to check the female vaginal plug or vaginal smears to check for sperm, female isolated
Plug or sperm, that the animals mated, the same day as the "conception" zero days. For the latter rabbits, male and female rabbits caged rabbit vaginal smears to fine
Child day as "conception" zero days. The detection of "conception animals," randomly assigned to each group, and weighed and numbered.
5.4.2 administered test substance
The test substance is usually administered by oral gavage, if the choice of other means shall state the reasons. Generally, in the period of organogenesis administration of the test substance, (pregnant rats
6 days to 15 day period, rabbit gestation day 6 ~ 18 days). When the test was orally administered to the test substance is dissolved or suspended in an aggregate
Suitable solvents, the preferred solvent is water, water-insoluble test substance may be used vegetable oil (such as olive oil, corn oil, etc.), do not dissolve in water or oil by
Test composition may also be used carboxymethyl cellulose, starch paste or the like dubbed suspension. The test substance should be freshly prepared, our data show that its solution or
Except for the suspension storage stabilizer. Should be fed once daily at the same time, the volume of gavage maternal weight adjustment. Gavage volume is generally not
Than 10mL/kg body weight, such as aqueous solution, up to the maximum gavage volume 20mL/kg body weight; the case of an oily liquid, the volume should not be fed
More than 4mL/kg body weight; each group was given a consistent volume.
5.4.3 parent observed
Daily clinical observation of animals, including the case of skin, hair, eyes, mucous membranes, respiratory, nervous behavior, limb activities, timely recording
Intoxication signs, time, extent and duration of the performance including the occurrence and found weak or moribund animals should be isolated or killed, there is a parent
Miscarriage or premature signs should be timely autopsy.
In the day 0 of pregnancy, the test was administered on day 1, the test was administered during three days and killed every day, said maternal body weight. If transit through drinking water
Trail administration of the test substance, the amount of water should also be recorded.
5.4.4 pregnancy checks and maternal death
5.4.4.1 General inspection
1 day before delivery (generally the first 20 days pregnant rats, rabbits for the first 28 days of pregnancy) were sacrificed mother, abdominal examination parental pregnancy situation and the carcass
development. Quickly removed the uterus, called the uterine fetal weight even to arrive at the net weight of pregnant animals. Record number of corpora lutea, the number of early fetal death, stillbirth several nights, live
The number of births and the number of implantation.
All pregnant animals were sacrificed when the autopsy and visual inspection, found naked save changes in organs, in order to facilitate histology
Check, while preserving the corresponding organs of sufficient controls for comparison.
5.4.4.2 fetal visual inspection
Each record fetal sex, body weight, body length, check whether the appearance of fetal abnormality. Visual inspection includes at least 1 meter project.
Table 1 teratogenicity test fetal appearance inspection project
Site inspection items
head
brainless
Encephalocele
Top fractures
hydrocephalus
Microcephaly
Facial cleft
Small Eye Disease
Exophthalmos
No ear disease
Microtia
Ear low
No jaw disease
Site inspection items
head
Small jaw syndrome
Cleft chin
Mouth cleft lip
Trunk
Cleft sternum
Chest split
Spina bifida
Gastroschisis
Scoliosis
After the curved spine
Umbilical hernia
Hypospadias
No anal
Site inspection items
Trunk
Short-tailed capuchin
Tailless
Four limbs
Polyarthra
Amelia
Short limb
Semi-limb
Polydactyly
No toe
And toe
Short-toed
Missing toe
5.4.4.3 fetal skeleton specimen preparation and examination
A skeleton specimen preparation method. 1/2 live births per litter will put 95% ethanol fixed 2 weeks to 3 weeks, remove the litter several minutes under running water
After the clock into 10g/L ~ 20g/L potassium hydroxide solution within (at least 5 times the volume of litter) 8h ~ 72h, after the transparent application into Alizarin Red
Solution dyed 6h ~ 48h, and rocked 1 times/d ~ 2 times/d, to red skull appropriate. Then into a transparent liquid A 1d ~ 2d, into transparent
Liquid B 2d ~ 3d, red bone and soft tissue to be substantially faded.
Bone specimens Method II (peeling method). The fetal rat peeled, gutted and fat, Alizarin red solution into the stained glass that day shaking
Bottle 2 to 3 times, to be the bones dyed red so far. The fetal shift into a transparent liquid A 1d ~ 2d, change into a transparent liquid B 2d ~ 3d.
Fetal bone has to be red, and purple soft tissue basic fade, interchangeable set glycerol.
Fetal bone scan. A bone sample into a small dish, with a transmission source, the overall observed testimony in stereoscopic microscope, and then gradually inspection
skeleton. Measuring between parietal and occipital defect case, then check the number of missing or fused sternum (breastbone ossification centers 5, xiphoid
1; ossification insufficiency lack 5th sternum first, 2 times for lack sternum), ribs usually 12 pairs to 13 pairs, common deformity fusion rib bifurcation
Ribs, wavy ribs, short ribs, and more ribs (14 common rib), missing ribs, ribs break. Spine development and number of vertebrae (7 cervical, thoracic 12 ~
13, lumbar 5 to 6, the bottom four vertebrae, caudal three to five), with or without fusion, longitudinal and the like. Final inspection limbs.
Fetal skeletal examinations are shown in Table 2.
Table 2 teratogenicity test fetal skeletal examinations
Site inspection items
Missing occipital ossification center
The number of spinal bone, abnormal shape, fusion, longitudinal, partially split, lack of ossification centers, narrowing from
Pelvic ossification center is missing, abnormal shape, fusion, split, narrowing from
The number of limbs, abnormal shape
Carpal missing ossification center
Metacarpal abnormal shape
Abnormal toe shape
The number of ribs, abnormal shape, fusion, bifurcation defect
The number of the sternum, integration, lack of ossification center
5.4.4.4 fetal visceral examination
For rats, 1/2 of live fetuses per litter of liquid into Bouins fixed two weeks, for visceral examination. First washed with tap water to the fixative will
Yang rat on board paraffin, cut off limbs and tail, the transverse head with a blade or a knife slit 5, and then cut open the chest and abdominal cavity. Different parts
The cross-sectional observation organ size, shape and relative position. Normal section shown in Figure 1 to 5.
a) orally from the mouth to the tongue and two transverse occipital (section ①), the brain can be observed between the brain, the cerebellum, tongue and palate;
b) in front of the vertical plane for slitting (section ②), the nose can be observed;
c) from the head vertical longitudinal section through the eye as the central (section ③), the eye can be observed;
d) located along the head of the largest cross section through the brain as (section ④), can be observed ventricle, cut ① ~ ④ purpose is tongue were observed
Split, double fork tongue, palate, eye, nose deformities, abnormal brain and ventricles;
e) level by the middle of the neck along the jaw as a crosscutting (section ⑤), can be observed trachea, esophagus and extension of the brain or spinal cord.
After midline cut from the chest, abdomen, and then click to check the size of the heart, lungs, diaphragm, liver, stomach, intestines and other organs, and location, check its completion will be removed,
Then check the kidney, ureter, bladder, uterus or testis position and developmental conditions. The kidney is then cut to observe whether the hydronephrosis and expanded.
The internal structure of the heart needs to be checked if necessary. Teratogenicity test fetal viscera inspection table includes at least 3 projects.
Figure 1 a schematic view of the head of fetuses
Figure 2 illustrates the head of the first ① section --- palate Figure 3 illustrates the head of the first section ② --- meatus
Figure 4 illustrates the head of the first section ③ --- eye Figure 5 illustrates the head of the first section ④ --- ventricle
Table 3 teratogenicity test fetal visceral examinations
Site inspection items
head
(spinal cord)
Hypoplasia of the olfactory bulb
Ventricle dilatation
Third ventricle dilatation
No brain disease
No eye disease
Small eye disease
Corneal defects
Single eye
chest
Dextrocardia
Room septal defect
Ventricular septal defect
Site inspection items
chest
Aortic arch
Esophageal atresia
Tracheal stenosis
No pulmonary disease
More lung disease
Lobe fusion
Diaphragmatic hernia
Tracheoesophageal fistula
Heterotaxia
abdomen
Abnormal liver leaf
Adrenal missing
Site inspection items
abdomen
Polycystic kidney disease
Horseshoe kidney
Hydronephrosis
Kidney missing
Lack of bladder
Missing Testis
Ovarian missing
Ovarian ectopic
Missing uterus
Uterine hypoplasia
Hydrosalpinx
Non-rodents, such as rabbits, to deal with all of the fetal skeleton and internal organs are carried out to check its inspection procedures refer rats.
6 Data processing and evaluation of results
Finishing each animal data and test results list, including the beginning of the test weight, each test group the number of animals, the number of offspring of animals, test
Number of animals died during or human death, the number of pregnant animals, clinical signs of poisoning and poisoning of the number of animals. Fetal observation knot
Fruit, including the type of malformations and other relevant information.
A reasonable statistical methods for statistical analysis of the following indicators. maternal body weight, body weight gain (maternal body weight at sacrifice - 6d pregnant body
Weight), even fetal uterine weight, weight net weight (weight when maternal death - even fetal uterine weight - weight pregnant 6d), implantation number, number of corpora lutea, absorbed fetus
Number, number of live births, the number and percentage of stillbirths, fetal body weight and body length, deformity of litter (including appearance, skeletal and visceral malformations), deformity
The number and percentage of fetal litter, calculate the total animal teratogenic teratogenic rate and a single rate. Statistics related indicators should be based on fetal nest units.
7 Test report
The test report should include the following details.
a) Name of test, the test unit name and contact details, report number;
b) Test the requester name and contact information, sample acceptance date;
c) Test start and end dates, test project manager, technical director of the test unit, date of issue;
d) test summary;
e) the name of the test substance, dosage form, date of manufacture (batch), Appearance, preparation and method of the vehicle;
f) animal species, strain, level, quantity, weight, sex, origin (supplier name, animal production license number), animals
Quarantine, adaptation, breeding environment (temperature, relative humidity, using experimental animal facility license number), feed sources (supplier name
Said animal feed production license number);
g) dose and groups, including the selection of the dose based on the principle or the dose and constituencies animal groupings and the number of animals per group;
h) Test conditions and methods, including the mode of administration and duration of the test substance, the test period, observe the indicators;
i) Test results. The text description and itemized summary form, including maternal weight, pregnancy, number of corpus luteum, implantation, absorbed fetus
Number, number of live births, stillbirths and percentage number. Case fetuses (weight, length), teratogenic type (appearance, bones and internal organs), the number of
And percentages, given the statistical treatment of the results;
j) Test Conclusion. Based on the observed effect dose level evaluated and have an effect whether or teratogenic, and type of deformity. give
The teratogenic effects, and other developmental toxicity endpoints and maternal toxicity LOAEL not observed adverse effect level (NOAEL).
Explanation 8 trial
Teratogenicity test pregnancy test animals orally repeated exposure to offspring developmental toxicity and teratogenicity test was produced. Test results should be combined
Subchronic, reproductive toxicity, toxicokinetics and other test results of integrated interpretation. Due to the existence of species differences in animals and humans, so the test results outside
Push people to certain limitations.
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