Method for evaluating eye stimulation of infant cosmetics by using zebra fish

Abstract

The invention provides a method for evaluating eye stimulation of cosmetics for infants and children by using zebra fish. The method comprises the following steps: exposing zebrafish embryos developed for 3 days in an embryo culture solution containing baby cosmetics, and culturing the embryo at a specific temperature and in a periodic light and dark environment by adopting a semi-static culture method; and when the zebrafish embryos develop for 6 days, evaluating the eye stimulation of juvenile zebrafish, response ability to light stimulation and variation conditions of genes related to eye development. The model organism zebra fish used in the method has the advantages that the breeding cycle is short, the dosage of used medicine is small, the genetic level is similar to that of human beings, the size is small, embryos are transparent and micromanipulation is convenient. In addition, the visual system of the zebra fish is very similar to that of other vertebrates including the human beings, so that the zebra fish eyes can serve as a good visual model, and meanwhile, zebra fish embryos which are just bred and developed are selected as models, and evaluation on infant cosmetics is more representative.

Description

technical field

[0001] The invention belongs to the technical field of toxicity evaluation, and in particular relates to a method for evaluating eye irritation of baby cosmetics by using zebrafish. Background technique

[0002] Infants are essential to the use of infant and child cosmetics. However, the safety of such products must be considered during use, especially the eye irritation evaluation of infant and child cosmetics due to contact with eyes for bath and shampoo products It is an important item for safety evaluation. The eye irritation evaluation method for a long time is the rabbit eye Draize test. However, with the implementation of the "3R" principle, the Draize test has been questioned ethically and scientifically, causing great pain to animals, and the test data is subjective. is no longer recognized. At present, a variety of in vitro alternative methods have been approved by the European Chemicals Agency, the United States National Environmental Protection ...

Examples

Embodiment 1

[0038] The selection of the safety concentration of baby cosmetics to be tested in embodiment 1:

[0039] 1) The present invention selects the test model as the wild type AB;

[0040] 2) Using dimethyl sulfoxide (DMSO) as a solvent to prepare infant cosmetics containing 0.59% acyl glutamate into a high-concentration mother solution with a concentration of 120 mg / mL, and store at -20°C;

[0041]3) Dilute the prepared mother solution with embryo culture solution into working solutions of 10, 20, 40, 60, 80, 100 and 120 μg / mL. The solutions are prepared and used immediately, and the concentration of DMSO is kept less than one thousandth;

[0042] 4) Inject the culture solution containing the baby cosmetics to be tested and the culture solution not containing the sample to be tested into a 6cm petri dish, and control the volume of the culture solution at 8-9mL.;

[0043] 5) Select healthy and well-developed embryos reproduced and developed by zebrafish for exposure experiments, a...

Embodiment 2

[0047] Example 2 In Vivo Observation of Eye Damage of Cosmetic Zebrafish Containing 0.59% Acyl Glutamate Content

[0048] 1) Randomly select 3dpf zebrafish with normal development, 20 in each group;

[0049] 2) Select cosmetics containing 0.59% acyl glutamate content, and other components are mild and non-irritating, with a concentration of 10 μg / mL and 20 μg / mL as the experimental group, and 0.1% DMSO as the control group.

[0050] 3) Place the petri dish in a constant temperature light-controlled incubator, the temperature is controlled at 28±1°C, the light and dark cycle is 14 / 10h, the semi-static culture method is adopted, the culture medium is replaced every 24h, and the exposure time is 72h;

[0051] 4) Observe the morphological changes of the eyes of zebrafish juveniles under a stereomicroscope and take pictures to record;

[0052] 5) Measure iris bandwidth and pupil diameter with Image J software, and calculate the ratio of the two.

[0053] The result is as image ...

Embodiment 3

[0054] Example 3 Analysis of Behavioral Trajectories of Zebrafish After Exposure to Cosmetics Containing 0.59% Acyl Glutamate Content for 72h

[0055] 1) Randomly select 3dpf zebrafish with normal development, 20 in each group;

[0056] 2) Choose a cosmetic containing 0.59% of acyl glutamate, and other components are mild and non-irritating, with a concentration of 10 μg / mL and 20 μg / mL as the experimental group, and 0.1% DMSO as the control group;

[0057] 3) Place the petri dish in a constant temperature light-controlled incubator, the temperature is controlled at 28±1°C, the light and dark cycle is 14 / 10h, the semi-static culture method is adopted, the culture medium is replaced every 24h, and the exposure time is 72h;

[0058] 4) After 72 hours, wash 3 times, place in a 96-well plate, one fish per well, 15 fish per group, put them into the zebrafish behavior track tracking system (zebrafish behavior detector) and let stand for 10 minutes;

[0059] 5) Immediately give ligh...

technical field

[0001] The invention belongs to the technical field of toxicity evaluation, and in particular relates to a method for evaluating eye irritation of baby cosmetics by using zebrafish. Background technique

[0002] Infants are essential to the use of infant and child cosmetics. However, the safety of such products must be considered during use, especially the eye irritation evaluation of infant and child cosmetics due to contact with eyes for bath and shampoo products It is an important item for safety evaluation. The eye irritation evaluation method for a long time is the rabbit eye Draize test. However, with the implementation of the "3R" principle, the Draize test has been questioned ethically and scientifically, causing great pain to animals, and the test data is subjective. is no longer recognized. At present, a variety of in vitro alternative methods have been approved by the European Chemicals Agency, the United States National Environmental Protection ...

Examples

Embodiment 1

[0038] The selection of the safety concentration of baby cosmetics to be tested in embodiment 1:

[0039] 1) The present invention selects the test model as the wild type AB;

[0040] 2) Using dimethyl sulfoxide (DMSO) as a solvent to prepare infant cosmetics containing 0.59% acyl glutamate into a high-concentration mother solution with a concentration of 120 mg / mL, and store at -20°C;

[0041]3) Dilute the prepared mother solution with embryo culture solution into working solutions of 10, 20, 40, 60, 80, 100 and 120 μg / mL. The solutions are prepared and used immediately, and the concentration of DMSO is kept less than one thousandth;

[0042] 4) Inject the culture solution containing the baby cosmetics to be tested and the culture solution not containing the sample to be tested into a 6cm petri dish, and control the volume of the culture solution at 8-9mL.;

[0043] 5) Select healthy and well-developed embryos reproduced and developed by zebrafish for exposure experiments, a...

Embodiment 2

[0047] Example 2 In Vivo Observation of Eye Damage of Cosmetic Zebrafish Containing 0.59% Acyl Glutamate Content

[0048] 1) Randomly select 3dpf zebrafish with normal development, 20 in each group;

[0049] 2) Select cosmetics containing 0.59% acyl glutamate content, and other components are mild and non-irritating, with a concentration of 10 μg / mL and 20 μg / mL as the experimental group, and 0.1% DMSO as the control group.

[0050] 3) Place the petri dish in a constant temperature light-controlled incubator, the temperature is controlled at 28±1°C, the light and dark cycle is 14 / 10h, the semi-static culture method is adopted, the culture medium is replaced every 24h, and the exposure time is 72h;

[0051] 4) Observe the morphological changes of the eyes of zebrafish juveniles under a stereomicroscope and take pictures to record;

[0052] 5) Measure iris bandwidth and pupil diameter with Image J software, and calculate the ratio of the two.

[0053] The result is as image ...

Embodiment 3

[0054] Example 3 Analysis of Behavioral Trajectories of Zebrafish After Exposure to Cosmetics Containing 0.59% Acyl Glutamate Content for 72h

[0055] 1) Randomly select 3dpf zebrafish with normal development, 20 in each group;

[0056] 2) Choose a cosmetic containing 0.59% of acyl glutamate, and other components are mild and non-irritating, with a concentration of 10 μg / mL and 20 μg / mL as the experimental group, and 0.1% DMSO as the control group;

[0057] 3) Place the petri dish in a constant temperature light-controlled incubator, the temperature is controlled at 28±1°C, the light and dark cycle is 14 / 10h, the semi-static culture method is adopted, the culture medium is replaced every 24h, and the exposure time is 72h;

[0058] 4) After 72 hours, wash 3 times, place in a 96-well plate, one fish per well, 15 fish per group, put them into the zebrafish behavior track tracking system (zebrafish behavior detector) and let stand for 10 minutes;

[0059] 5) Immediately give ligh...

Images