natgillin/translations-raw huggingface dataset:
Coagulation of the embryo:
795–801.
Under bright field illumination, coagulated zebrafish embryos show a variety of intransparent inclusions.
Asakura, M. et al.
Figure 2
(2008), „An improved system for exposure of cultured mammalian cells to gaseous compounds in the chromosomal aberration assay“, Mutation Research, Vol.
Lack of somite formation:
652/2, pp.
Although retarded in development by approx.
122–130.
10 hrs, the 24 hrs old zebrafish embryo in (a) shows well-developed somites (→), whereas the embryo in (b) does not show any sign of somite formation (→).
Galloway, S. et al.
Although showing a pronounced yolk sac oedema (*), the 48 hrs old zebrafish embryo in (c) shows distinct formation of somites (→), whereas the 96 hrs old zebrafish embryo depicted in (d) does not show any sign of somite formation (→).
(2011), „Workshop summary: Top concentration for in vitro mammalian cell genotoxicity assays; and Report from working group on toxicity measures and top concentration for in vitro cytogenetics assays (chromosome aberrations and micronucleus)“, Mutation Research/Genetic Toxicology and Environmental Mutagenesis, Vol.
Note also the spinal curvature (scoliosis) and the pericardial oedema (*) in the embryo shown in (d).
723/2, pp.
Bud in lateral view (a: →; 96 hrs old zebrafish embryo).
77–83.
Note also the lack of the eye bud (*).
Honma, M. (2011), „Cytotoxicity measurement in in vitro chromosome aberration test and micronucleus test“, Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Vol.
Figure 4
724/1–2, pp. 86–87.
Lack of heartbeat is, by definition, difficult to illustrate in a micrograph.
Richardson, C. et al.
Lack of heartbeat is indicated by non-convulsion of the heart (double arrow).
(1989), „Analysis of Data from In Vitro Cytogenetic Assays“, väljaandes Statistical Evaluation of Mutagenicity Test Data, Kirkland, D.J.
Immobility of blood cells in, e.g. the aorta abdominalis (→ in insert) is not an indicator for lack of heartbeat.
OECD (2014), „Document supporting the WNT decision to implement revised criteria for the selection of the top concentration in the in vitro mammalian cell assays on genotoxicity (Test Guidelines 473, 476 and 487)“, ENV/JM/TG(2014)17.
Note also the lack of somite formation in this embryo (*, homogenous rather than segmental appearance of muscular tissues).
Morita, T., M. Honma, K. Morikawa (2012), „Effect of reducing the top concentration used in the in vitro chromosomal aberration test in CHL cells on the evaluation of industrial chemical genotoxicity“, Mutation Research/Genetic Toxicology and Environmental Mutagenesis, Vol.
The observation time to record an absence of heartbeat should be at least of one minute with a minimum magnification of 80×.
741/1–2, pp. 32–56.
C.50 Sediment-free Myriophyllum spicatum toxicity test
Levy (2013), „Evaluation of the Highest Concentrations Used in the In Vitro Chromosome Aberrations Assay“, Environmental and Molecular Muagenesis, Vol.
This test method is equivalent to OECD test guideline 238 (2014).
54/1, pp. 36–43.
It is designed to assess the toxicity of chemicals to Myriophyllum spicatum, a submersed aquatic dicotyledon, a species of the water milfoils family.
ISCN (2013), An International System for Human Cytogenetic Nomenclature, Schaffer, L.G., J. MacGowan-Gordon, M. Schmid (eds.), Karger Publishers Inc., Connecticut.
It is based on an ASTM existing test method (1) modified as a sediment-free test system (2) to estimate the intrinsic ecotoxicity of test chemicals (independent of the distribution-behaviour of the test chemical between water and sediment).
Hayashi, M. et al.
A test system without sediment has a low analytical complexity (only in the water phase) and the results can be analysed in parallel and/or comparison with those obtained in Lemna sp. test (3); in addition, the required sterile conditions allow to keep the effects of microorganisms and algae (chemical uptake/ degradation, etc.) as low as possible.
(2011), „Compilation and use of genetic toxicity historical control data“, Mutation Research, Vol.
This test does not replace other aquatic toxicity tests; it should rather complement them so that a more complete aquatic plant hazard and risk assessment is possible.
723/2, pp.
The test method has been validated by a ring-test (4).
87–90.
Details of testing with renewal (semi-static) and without renewal (static) of the test solution are described.
Parry (1993), „A comparison of two in vitro mammalian cell cytogenetic assays for the detection of mitotic aneuploidy using 10 known or suspected aneugens“, Mutation Research, Vol.
Depending on the objectives of the test and the regulatory requirements, the use of semi-static method is recommended, e.g. for substances that are rapidly lost from solution as a result of volatilisation, adsorption, photodegradation, hydrolysis, precipitation or biodegradation.
287/1, pp. 29–46.
Further guidance is given in (5).
Locke-Huhle, C. (1983), „Endoreduplication in Chinese hamster cells during alpha-radiation induced G2 arrest“, Mutation Research, Vol.
This test method applies to substances, for which the test method has been validated, (see details in the ring-test report (4)) or to formulations, or known mixtures; if a mixture is tested, its constituents should be as far as possible identified and quantified.
119/3, pp. 403–413.
The sediment-free Myriophyllum spicatum test method complements the water-sediment Myriophyllum spicatum Toxicity Test (6).
Trosko (1983), „Aphidicolin – induced endoreduplication in Chinese hamster cells“, Cancer Research, Vol.
Before use of the test method for the testing of a mixture intended for a regulatory purpose, it should be considered whether, and if so why, it may provide adequate results for that purpose.
43/3, pp. 1362–1364.
Continuously growing plant cultures of Myriophyllum spicatum (only in modified Andrews' medium, see Appendix 2) are allowed to grow as monocultures in different concentrations of the test chemical over a period of 14 days in a sediment-free test system.
Galloway (1994), „Cytotoxicity measurement in in vitro chromosome aberration test and micronucleus test“, Mutation Research, Vol.
The objective of the test is to quantify chemical-related effects on vegetative growth over this period based on assessments of selected measurement variables.
312, pp. 139–149.
Growth of shoot length, of lateral branches and roots as well as development of fresh and dry weight and increase of whorls are the measurement variables.
lühiajaliste uuringute puhul: iga looma kehamass katse alguses ja lõpus; ühest nädalast pikemate uuringute puhul: iga looma kehamass uuringu vältel ja sööda tarbimine.
In addition, account is taken of distinctive qualitative changes in test organisms, such as disfigurement or chlorosis and necrosis indicated by yellowing or white and brown colouring.
Tuleks lisada kehamassi vahemik, keskväärtus ja standardhälve iga rühma kohta.
To quantify chemical-related effects, growth in the test solutions is compared with that of the controls and the concentration bringing about a specified x % inhibition of growth is determined and expressed as the ECx; “x” can be any value depending on the regulatory requirements, e.g. EC10, EC20, EC50.
157–165.
It should be noted that estimates of EC10 and EC20 values are only reliable and appropriate in tests where coefficients of variation in control plants fall below the effect level being estimated, i.e. coefficients of variation should be < 20 % for robust estimation of an EC20.
Tice, R.R. et al.
Both average specific growth rate (estimated from assessments of main shoot length and three additional measurement variables) and yield (estimated from the increase in main shoot length and three additional measurement variables) of untreated and treated plants should be determined.
(2000), „Single cell gel/Comet assay: guidelines for in vitro and in vivo genetic toxicology testing“, Environmental and Molecular Mutagenesis, Vol.
Specific growth rate (r) and yield (y) are subsequently used to determine the ErCx (e.g. ErC10, ErC20, ErC50) and EyCx (e.g. EyC10, EyC20, EyC50), respectively.
35/3, pp.
In addition, the lowest observed effect concentration (LOEC) and the no observed effect concentration (NOEC) may be statistically determined.
305–312.
An analytical method, with adequate sensitivity for quantification of the test chemical in the test medium, should be available.
Adler, I.D. et al.
Information on the test chemical which may be useful in establishing the test conditions includes the structural formula, purity and impurities, water solubility, stability in water and light, acid dissociation constant (pKa), partition coefficient octanol-water (Kow), vapour pressure and biodegradability.
(1998), „Recommendations for statistical designs of in vivo mutagenicity tests with regard to subsequent statistical analysis“, Mutation Research/Genetic Toxicology and Environmental Mutagenesis, Vol. 417/1, pp.
Water solubility and vapour pressure can be used to calculate Henry's Law constant, which will indicate if significant losses of the test chemical during the test period are likely.
19–30.
This will help indicate whether particular steps to control such losses should be taken.
87–90.
Where information on the solubility and stability of the test chemical are uncertain, it is recommended that these be assessed under the conditions of the test, i.e. growth medium, temperature, lighting regime to be used in the test.
293–304.
The pH control of the test medium is particularly important, e.g. when testing metals or substances which are hydrolytically unstable.
313–319.
Further guidance for testing chemicals with physical-chemical properties that make them difficult to test is provided in a OECD Guidance Document (5).
Pacchierotti, F., V. Stocchi (2013), „Analysis of chromosome aberrations in somatic and germ cells of the mouse“, Methods in Molecular Biology, Vol. 1044, pp. 147–163.
For the test to be valid, the doubling time of main shoot length in the control must be less than 14 days.
lühiajaliste uuringute puhul: iga looma kehamass katse alguses ja lõpus; ühest nädalast kauem kestva uuringu korral: iga looma kehamass uuringu vältel ja toidu tarbimine.
Using the media and test conditions described in this test method, this criterion can be attained using a static or semi-static test regime.
10–30.
The mean coefficient of variation for yield based on measurements of shoot fresh weight (i.e. from test initiation to test termination) and the additional measurement variables (see paragraph 37) in the control cultures do not exceed 35 % between replicates.
MacGregor, J.T. et al.
More than 50 % of the replicates of the control group are kept sterile over the exposure period of 14 days, which means visibly free of contamination by other organisms such as algae, fungi and bacteria (clear solution).
(1990), „The in vivo erythrocyte micronucleus test: measurement at steady state increases assay efficiency and permits integration with toxicity studies“, Fundamental and Applied Toxicology, Vol.
Note: Guidance on how to assess sterility is provided in the ring-test report (4).
14/3, pp.
REFERENCE CHEMICAL
92–107.
Reference chemical(s), such as 3,5-dichlorophenol used in the ring test (4), may be tested as a mean of checking the test procedure; from the ring test data, the mean EC50-values of 3,5-DCP for the different response variables (see paragraphs 37-41 of this test method) are between 3,2 mg/l and 6,9 mg/l (see ring test report for details about confidence interval for these values).
Dertinger, S.D. et al.
It is advisable to test a reference chemical at least twice a year or, where testing is carried out at a lower frequency, in parallel to the determination of the toxicity of a test chemical.
(2011), „Flow cytometric scoring of micronucleated erythrocytes: an efficient platform for assessing in vivo cytogenetic damage“, Mutagenesis, Vol.
All equipment in contact with the test media should be made of glass or other chemically inert material.
26/1, pp.
Glassware used for culturing and testing purposes should be cleaned of chemical contaminants that might leach into the test medium and should be sterile.
83–91.
The test vessels should be long enough for the shoot in the control vessels to grow in the water phase without reaching the surface of the test medium at the end of the test.
139–145.
Thick-walled borosilicate glass test tubes without lip, inner diameter approximately 20 mm, length approximately 250 mm, with aluminium caps are recommended.
Garriott (1996), „Validation of an automated image analysis micronucleus scoring system“, Mutation Research, Vol.
Since the modified Andrews' medium contains sucrose (which stimulates the growth of fungi and bacteria), the test solutions have to be prepared under sterile conditions.
370/1, pp. 65–73.
All liquids as well as equipment are sterilised before use.
Asano, N. et al.
Sterilisation is carried out via heated air treatment (210 °C) for 4 hours or autoclaving for 20 minutes at 121 °C.
(1998), „An automated new technique for scoring the rodent micronucleus assay: computerized image analysis of acridine orange supravitally stained peripheral blood cells“, Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Vol.
In addition, all flasks, dishes, bowls etc. and other equipment undergo flame treatment at a sterile workbench just prior to use.
404/1-2, pp.
The cultures and test vessels should not be kept together.
149–154.
This is best achieved using separate environmental growth chambers, incubators, or rooms.
Styles, J.A. et al.
Illumination and temperature should be controllable and maintained at a constant level.
(2001), „Automation of mouse micronucleus genotoxicity assay by laser scanning cytometry“, Cytometry, Vol.
Test organism
44/2, pp.
Myriophyllum spicatum — a submersed aquatic dicotyledon — is a species of the water milfoils family.
153–155.
Between June and August, inconspicuous pink-white flowers protrude above the water surface.
, „A rapid in vivo test for chromosomal damage“, Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Vol.
The plants are rooted in the ground by a system of robust rhizomes and can be found in the entire northern hemisphere in eutrophic, however non-polluted and more calciferous still waters with muddy substrate.
18/2, pp. 187–190.
Myriophyllum spicatum prefers fresh water, but is found in brackish water as well.
, „The micronucleus test“, Mutation Research, Vol.
For the sediment-free toxicity test, sterile plants are required.
31/1, pp. 9–15.
If the testing laboratory does not have regular cultures of Myriophyllum spicatum, sterile plant material may be obtained from another laboratory or (unsterile) plant material might be taken from the field or provided by a commercial supplier; if plants come from the field a taxonomic verification of the species should be envisaged.
61–118.
If collected from the field or provided by a commercial supplier, plants should be sterilised (1) and maintained in culture in the same medium as used for testing for a minimum of eight weeks prior to use.
29-80.
Field sites used for collecting starting cultures have to be free of obvious sources of contamination.
555–558.
Great care should be taken to ensure that the correct species is obtained when collecting Myriophyllum spicatum from the field, especially in regions where it can hybridise with other Myriophyllum species.
103–112.
If obtained from another laboratory they should be similarly maintained for a minimum of three weeks.
513–522.
The source of plant material and the species used for testing should always be reported.
245–249.
The quality and uniformity of the plants used for the test will have a significant influence on the outcome of the test and should therefore be selected with care.
CSGMT/JEMS.MMS – The Collaborative Study Group for the Micronucleus Test (1992), „Micronucleus test with mouse peripheral blood erythrocytes by acridine orange supravital staining: the summary report of the 5th collaborative study“, Mutation Research/Genetic Toxicology, Vol.
Young, rapidly growing plants without visible lesions or discoloration (chlorosis) should be used.
278/2-3, pp. 83–98.
Details about preparation of the test organism are given in Appendix 4.
CSGMT/JEMS.MMS – The Mammalian Mutagenesis Study Group of the Environmental Mutagen Society of Japan (1995), „Protocol recommended by the CSGMT/JEMS.MMS for the short-term mouse peripheral blood micronucleus test“.
Cultivation
The Collaborative Study Group for the Micronucleus Test (CSGMT) (CSGMT/JEMS.MMS, The Mammalian Mutagenesis Study Group of the Environmental Mutagen Society of Japan), Mutagenesis, Vol.
To reduce the frequency of culture maintenance (e.g. when no Myriophyllum tests are planned for a period), cultures can be held under reduced illumination and temperature (50 μE m–2 s–1, 20 ± 2 °C).
10/3, pp. 153–159.
Details of culturing are given in Appendix 3.
Mavournin (1994), „Bone marrow micronucleus assay: a review of the mouse stocks used and their published mean spontaneous micronucleus frequencies“, Environmental and Molecular Mutagenesis, Vol.
At least 14 to 21 days before testing, sufficient test organisms are transferred aseptically into fresh sterile medium and cultured for 14 to 21 days under the conditions of the test as a pre culture.
23/4, pp. 239–273.
Details for preparation of a pre-culture are given in Appendix 4.
Krishna, G., G. Urda, J. Paulissen (2000), „Historical vehicle and positive control micronucleus data in mice and rats“, Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Vol.
Only one nutrient medium is recommended for Myriophyllum spicatum in a sediment-free test system, as described in Appendix 2.
453/1, pp. 45–50.
A modification of the Andrews' medium is recommended for culturing and testing with Myriophyllum spicatum as described in (1).
Hayes, J. et al.
From five separately prepared nutrient stock solutions with addition of 3 % sucrose the modified Andrews' medium will be arranged.
(2009), „The rat bone marrow micronucleus test--study design and statistical power“, Mutagenesis, Vol.
Details about preparation of the medium are given in Appendix 2.
24/5, pp.
A tenfold concentrated, modified Andrews' medium is needed for obtaining the test solutions (by dilution as appropriate).
419–424.
The composition of this medium is given in Appendix 2.
Mammalian Mutagenicity Study Group, Environmental and Molecular Mutagenesis, Vol.
The addition of the nutrients will be achieved by using the tenfold concentrated, modified Andrews' medium.
(2011), „Improvement of in vivo genotoxicity assessment: combination of acute tests and integration into standard toxicity testing“, Mutation Research/Genetic Toxicology and Environmental Mutagenesis, Vol.
The stock solutions of the test chemical can be sterilised by autoclave at 121 °C for 20 minutes or by sterile filtration, provided that the sterilisation technique used does not denaturise the test chemical.
723/2, pp.
Test solutions can also be prepared in sterile demineralised water or medium, under sterile conditions.
108–120.
The thermo-stability and the adsorption on different surfaces should the taken into account in the selection of the sterilisation procedure of the stock solutions of the test chemical.
LeBaron, M.J. et al.
Because of that, it is recommended that the stock solutions be prepared under sterile conditions, i.e. using sterile material for dissolving the test chemical under sterile conditions (e.g. flame sterilisation, laminar-flow hoods, etc.) into sterile water.
(2013), „Influence of counting methodology on erythrocyte ratios in the mouse micronucleus test“, Environmental and Molecular Mutagenesis, Vol.
This technique of preparation of sterile stock solutions is valid for both substances and mixtures.
54/3, pp.
The highest tested concentration of the test chemical should normally not exceed its water solubility under the test conditions.
222–228.
For test chemicals of low water solubility it may be necessary to prepare a concentrated stock solution or dispersion of the chemical using an organic solvent or dispersant in order to facilitate the addition of accurate quantities of the test chemical to the test medium and aid in its dispersion and dissolution.
234–252.
Every effort should be made to avoid the use of such materials.
Asanami, S., K. Shimono (1997), „High body temperature induces micronuclei in mouse bone marrow“, Mutation Research/Genetic Toxicology and Environmental Mutagenesis, Vol.
There should be no phytotoxicity resulting from the use of auxiliary solvents or dispersants.
390/1-2, pp. 79–83.
For example, commonly used solvents which do not cause phytotoxicity at concentrations up to 100 μl/l, include acetone and dimethylformamide.
Asanami, S., K. Shimono, S. Kaneda (1998), „Transient hypothermia induces micronuclei in mice“, Mutation Research/Genetic Toxicology and Environmental Mutagenesis, Vol.
If a solvent or dispersant is used, its final concentration should be reported and kept to a minimum (≤ 100 μl/l), and all treatments and controls should contain the same concentration of solvent or dispersant.
413/1, pp. 7–14.
Further guidance on the use of dispersants is given in (5).
Spencer, P.J. et al.
Test and control groups
(2007), „Induction of micronuclei by phenol in the mouse bone marrow: I. Association with chemically induced hypothermia“, Toxicological Sciences, Vol.
Prior knowledge of the toxicity of the test chemical to Myriophyllum spicatum from a range-finding test will help in selecting suitable test concentrations.
97/1, pp.
In the definitive toxicity test, there should normally be five (like in the Lemna growth inhibition test, Chapter C.26 of this Annex) to seven test concentrations arranged in a geometric series; they should be chosen in order that the NOEC and EC50 values are bracketed by the concentration range (see below).
120–127.
Preferably the separation factor between test concentrations should not exceed 3,2; however, a larger value may be used where the concentration-response curve is flat.
Ishidate (1983), „An application of Acridine Orange fluorescent staining to the micronucleus test“, Mutation Research Letters, Vol.
Justification should be provided when fewer than five concentrations are used.
120/4, pp. 241–247.
At least five replicates should be used at each test concentration.
Langlois (1983), „A simple fluorescent staining procedure for micronuclei and RNA in erythrocytes using Hoechst 33258 and pyronin Y“, Mutation Research, Vol.
In setting the range of test concentrations (for range-finding and/or for the definitive toxicity test), the following should be considered:
120/4, pp. 269–275.
To determine an ECx, test concentrations should bracket the ECx value to ensure an appropriate level of confidence.
Staniforth (1989), „The automated bone marrow micronucleus test“, Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Vol.
For example, if estimating the EC50, the highest test concentration should be greater than the EC50 value.
213/1, pp. 91–104.
If the EC50 value lies outside of the range of test concentrations, associated confidence intervals will be large and a proper assessment of the statistical fit of the model may not be possible.
Galloway (1999), „Rapid method for improving slide quality in the bone marrow micronucleus assay; an adapted cellulose column procedure“, Mutation Research, Vol.
If the aim is to estimate the LOEC/NOEC, the lowest test concentration should be low enough so that growth is not significantly less than that of the control.
439/1, pp. 121–126.
In addition, the highest test concentration should be high enough so that growth is significantly lower than that in the control.
Adler (1990), „Application of antikinetochore antibody staining (CREST staining) to micronuclei in erythrocytes induced in vivo“, Mutagenesis, Vol.
If this is not the case, the test will have to be repeated using a different concentration range (unless the highest concentration is at the limit of solubility or the maximum required limit concentration, e.g. 100 mg/l).
5/4, pp. 411–415.