GB 15193.9-2014National Food Safety Standard -- Rodent Dominant Lethal Test (English PDF)

2.1 Dominant lethal mutation

One kind of chromosome aberrations in the germ cells, structure, or change this number does not cause genetic germ cells (sperm or egg Sub) dysfunction, but the cause of death or developing embryo fertilized egg directly.

4.1 Test substance

4.1.1 test substance formulation. the test substance should be dissolved or suspended in a suitable solvent, the solvent should be non-toxic substances, the chemical does not occur with the test substance reaction. The preferred solvent is water, the test substance may be used liposoluble edible vegetable oils (such as olive oil, corn oil, etc.), insoluble in water or oil tested may be made using carboxymethyl cellulose, starch suspension was dubbed, not formulated as a suspension, other forms may also be formulated as a paste.

In general test substances should now use the existing, data show that except for its storage solution or suspension stabilizer.

4.1.2 give way. should gavage, or feeding method, commonly used mice fed volume of 10mL/kg body weight ~ 20mL/kg body weight, large Common rat gavage volume of 10mL/kg body weight. The method may also be used a positive control intraperitoneal injection, the injection volume of 10mL/kg of body weight -20mL/kg of body weight.

4.1.3 The administration of the test substance. Normal use once daily, continuous 5d to give way. If you think otherwise reasonably also be used, the disposable or three consecutive months of administration of the test substance.

4.2 Experimental Animals

4.2.1 Level and animal strains. Animals dominant lethal strain should choose the background value is low, the pregnancy rate and the implantation of a few more, the pre-test conception fertility Rate of more than 70% who recommended using mouse or rat. Experimental animals should meet the requirements of GB 14922.1 and GB 14922.2 and quality Certificate, healthy adult male mice (sexual maturity, 6 weeks to 8 weeks old, weighing 30g or more), or rat (sexual maturity, 8 weeks to 10 weeks Age, weight 200g or above). Mating with a female adult, it should never have been mating and reproductive history, different female mating cycle of weeks, Weight should phase approximation.

4.2.2 Number of animals. You should use the appropriate number of male animals, the number of males should be sufficient so that each mating cycle each produce 30 ~ 50 Pregnant females. Each male should be at least 15.

4.2.3 Animal Feeding. animal breeding conditions should be consistent with GB 14925, drinking water should be consistent with GB 5749, feed shall comply with GB 14924 the relevant provisions.

4.3 Dose

The experiment included at least three doses of the test substance group, high dose group animals should be able to cause some toxic symptoms, such as a slight drop in fertility, high Dose of test substance dose can be between 1/10LD50 ~ 1/3LD50. Acute toxicity test was the maximum tolerated dose administration of the test (Maximum Maximum capacity of concentration and intragastric) LD50 not find time, places 10g/kg body weight or body may intake of 100 times, or to the maximum test substance the highest dose to dose, then this dose and then set up two dose groups. General should also make positive and negative (vehicle) control group, if the same A laboratory in the last 12 months, the positive control group Pat obtain positive results, and laboratory environmental conditions and animal strains did not change, you can not Then positive control group. The positive control was often used cyclophosphamide (cyclophosphamide), 30mg/kg body weight ~ 40mg/kg body weight, Intraperitoneal injection once a day for 5d; but also use other triethylene melamine (triethylenemelamine), methane sulfonic acid ethyl ester (ethyl methanesulfonate), methyl methanesulfonate (methylmethanesulfonate) and the like.

4.4 Test procedure and outcome measures

4.4.1 Mating. The male subjects were given the following day (once exposure method), or after the last administration of the test substance in male rats following day (multiple exposure method), male Press 1 to 1 or the female mated with 1.2 ratio after 5d, or sperm based on the vagina, check to make sure the female plug mating success, remove the other female Line feeding. After the interval 2d, then the proportion of males in the same with another batch of females mated with mice so batches for 5 to 6 batches, batch ~ 8 rats 10 batches.

4.4.2 embryos check. In male and female rats with cage date of 15 days to 17 days after the females were sacrificed, caesarean section to remove the uterus, inspect and record Recorded live embryos each female, the early death of embryos and embryos late deaths.

Embryo survival or death of identification as follows.

--- Live births. a complete shape, bright red color, natural movement, in sports after the reaction to mechanical stimulation.

--- Early death embryo. Embryo smaller body, the shape is not complete, the placenta is small or inconspicuous. The earliest embryonic death in utero Film uplift a small tumor. As has been completely absorbed, leaving a dark brown ridge point was only in the endometrium.

--- Late embryonic death. forming, dark color, no natural motion, no motor response after mechanical stimulation.

5.1 Data Processing

5.1.1 Statistical Analysis

Check the results of dominant lethal test in the test group were calculated in units of the following metrics for each mating cycle. Press the test group and control the indicators group of animals were used appropriate statistical analysis methods, such as chi2 test, ANOVA or rank sum test, in order to assess by Mutagenicity test substance. Pregnancy rates available chi2 test, the average number of implantation, the average number of available embryos death ANOVA or rank sum test France, embryonic mortality by rank sum test anyway analysis or ANOVA with post-string conversion.

5.1.2 Fertility index

Fertility index according to equation (1) to (3) Calculated.

Pregnancy rate = number of pregnant females/number of females mated × 100% (1) = Total number of implanted embryos died early deaths late the number of viable embryos (2) Average = total number of implantation implantation/pregnancy female number (3)

5.1.3 Dominant lethal index

Dominant lethal metrics by formula (4) to (6) Calculated.

Death = number of embryos died early embryonic deaths late number of embryos (4) Embryonic mortality = death embryo/total number of implantation × 100% (5) Death = average number of embryos embryos/number of pregnant females (6) Death

5.2 Evaluation Results

The main index based on the results of dominant lethal determination.

Number of embryos a) test group and the control group compared to embryonic mortality (%) and (or) average mortality was significantly higher, statistically significant Righteousness and a dose - response relationship when, to confirm a positive result.

b) if the difference was statistically significant, but there was no dose-response relationship, the test should be repeated, the results can be determined by repeated Positive. At the same time, we should make the final assessment in considering biological significance and statistical basis.