natgillin/translations-raw huggingface dataset:
J.K. Eidem, H. Kleivdal, K. Kroll, N. Denslow, R. van Aerle, C. Tyler, G. Panter, T. Hutchinson og A. Goksoyr (2006), »Development and validation of a direct homologous quantitative sandwich ELISA for fathead minnow (Pimephales promelas) vitellogenin.
It has been reported that air humidity of less than 60 % RH can impede reproduction.
Aquatic Toxicology«, 78, s. 202-206.
Any suitable natural or synthetic water may be used.
Ankley (2006), »Evaluation of a commercial kit for measuring vitellogenin in the fathead minnow (Pimephales promelas)«, Ecotoxicology and Environmental Safety 64, s. 101-105.
Well water, dechlorinated tap water and artificial media (e.g. Elendt “M4” or “M7” medium, see below) are commonly used.
Comparison of vitellogenin in plasma and whole body homogenate from zebrafish (Danio rerio) and rainbow trout (Oncorhynchus mykiss)«, Nordisk Ministerråd, TemaNord 2001:597, s. 48-51.
If necessary, the culture water may be renewed by pouring or siphoning the used water from culture vessels carefully without destroying the tubes of larvae.
Porcher og A. Goksoyr (2004), »Development of quantitative vitellogenin-ELISAs for fish test species used in endocrine disruptor screening«, Analytical and Bioanalytical Chemistry 378, s. 621-633.
Feeding larvae
S. Orn, S. Yamani og L. Norrgren (2006), »Comparison of vitellogenin induction, sex ratio, and gonad morphology between zebrafish and Japanese medaka after exposure to 17 alpha-ethinylestradiol and 17 beta-trenbolone«, Archives of Environmental Contamination and Toxicology 51, s. 237-243.
Chironomus larvae should be fed with a fish flake food (Tetra Min®, Tetra Phyll® or other similar brand of proprietary fish food), at approximately 250 mg per vessel per day.
S. Scholz og N. Kluver (2009), »Effects of Endocrine Disrupters on Sexual, Gonadal Development in Fish, Sexual Development 3«, s. 136-151.
This can be given as a dry ground powder or as a suspension in water:
M. Fenske, G. Maack, C. Schafers og H. Segner (2005), »An environmentally relevant concentration of estrogen induces arrest of male gonad development in zebrafish, Danio rerio«, Environmental Toxicology and Chemistry 24, s. 1088-1098.
1,0 g of flake food is added to 20 ml of dilution water and blended to give a homogenous mix.
Nagahama (2004), »Two DM domain genes, DMY and DMRT1, involved in testicular differentiation and development in the medaka, Oryzias latipes«, Developmental Dynamics 231, s. 518-526.
This preparation may be fed at a rate of about 5 ml per vessel per day.
A. Shinomiya, H. Otake, K. Togashi, S. Hamaguchi og M. Sakaizumi (2004), »Field survey of sex-reversals in the medaka, Oryzias latipes: genotypic sexing of wild populations«, Zoological Science 21, s. 613-619.
(shake before use.)
Flick (2007), »Collapse of a fish population after exposure to a synthetic estrogen«, Proceedings of the National Academy of Sciences of the United States of America 104, s. 8897-8901.
Older larvae may receive more.
Kidd (2009), »Interspecies differences in biochemical, histopathological, and population responses in four wild fish species exposed to ethynylestradiol added to a whole lake«, Canadian Journal of Fisheries and Aquatic Sciences 66, s. 1920-1935.
Feeding is adjusted according to the water quality.
Tyler (2006), »Development of chronic tests for endocrine active chemicals — Part 1.
If the culture medium becomes “cloudy”, the feeding should be reduced.
An extended fish early-life stage test for oestrogenic active chemicals in the fathead minnow (Pimephales promelas)«, Aquatic Toxicology 77, s. 279-290.
Food additions should be carefully monitored.
Petersen, P. Jackson, K. Hylland, L. Norrgren og P. Bjerregaard (2006), »Detection of endocrine disrupters: Evaluation of a Fish Sexual Development Test (FSDT)«, Comparative Biochemistry and Physiology C-Toxicology & Pharmacology 144, s. 57-66.
Too little food will cause emigration of the larvae towards the water column, and too much food will cause increased microbial activity and reduced oxygen concentrations.
L. Andersen, K. Kinnberg, H. Holbech, B. Korsgaard og P. Bjerregaard (2004), »Evaluation of a 40 day assay for testing endocrine disrupters: Effects of an anti-estrogen and an aromatase inhibitor on sex ratio and vitellogenin concentrations in juvenile zebrafish (Danio rerio)«, Fish Physiology and Biochemistry 30, s. 257-266.
Both conditions can result in reduced growth rates.
J.E. Morthorst, H. Holbech og P. Bjerregaard (2010), »Trenbolone causes irreversible masculinization of zebrafish at environmentally relevant concentrations«, Aquatic Toxicology 98, s. 336-343.
Some green algae (e.g. Scenedesmus subspicatus, Chlorella vulgaris) cells may also be added when new culture vessels are set up.
Metcalf (2003), »Effects of the antiandrogens, vinclozolin and cyproterone acetate on gonadal development in the Japanese medaka (Oryzias latipes)«, Aquatic Toxicology 63, s. 391-403.
Feeding emerged adults
Tyler (2004), »Successful detection of (anti-) androgenic and aromatase inhibitors in pre-spawning adult fathead minnows (Pimephales promelas) using easily measured endpoints of sexual development«, Aquatic Toxicology 70, s. 11-21.
Some experimenters have suggested that a cotton wool pad soaked in a saturated sucrose solution may serve as a food for emerged adults.
Petersen og P. Bjerregaard (2007), »Effects of the fungicide prochloraz on the sexual development of zebrafish (Danio rerio)«, Comparative Biochemistry and Physiology C-Toxicology & Pharmacology 145, s. 165-170.
At 20 ± 2 °C adults will begin to emerge from the larval rearing vessels after approximately 13 - 15 days.
Kapitel C.14 i dette bilag, Væksttest på fiskeyngel.
Males are easily distinguished by having plumose antennae and thin body.
Kapitel C.4 i dette bilag, Bestemmelse af »let« bionedbrydelighed.
Egg masses
Kapitel C.37 i dette bilag, 21-dages assay med fisk:
Once adults are present within the breeding cage, all larval rearing vessels should be checked three times weekly for deposition of the gelatinous egg masses.
En korttidsscreeningstest for østrogene og androgene virkninger og aromatasehæmning.
If present, the egg masses should be carefully removed.
C. Schäfers, M. Teigeler, A. Wenzel, G. Maack, M. Fenske og H. Segner (2007), »Concentration- and time-dependent effects of the synthetic estrogen, 17 alpha-ethinylestradiol, on reproductive capabilities of the zebrafish, Danio rerio« Journal of Toxicology and Environmental Health-Part A, 70, 9-10 s. 768-779.
They should be transferred to a small dish containing a sample of the breeding water.
Forkortelser og definitioner
Egg masses are used to start a new culture vessel (e.g. 2 - 4 egg masses/vessel) or are used for toxicity tests.
Slutpunkt
First instar larvae should hatch after 2 - 3 days.
skaber effekt på populationsniveau
Set-up of new culture vessels
air saturation value = luftmætning
Once cultures are established it should be possible to set up a fresh larval culture vessel weekly or less frequently depending on testing requirements, removing the older vessels after adult midges have emerged.
Biomarkør
Using this system a regular supply of adults will be produced with a minimum of management.
skaber effekt på individuelt niveau
Preparation of test solutions “M4” and “M7”
et stof eller en blanding.
Elendt (1990) has described the “M4” medium.
days post hatch = dage efter udklækning
The “M7” medium is prepared as the “M4” medium except for the substances indicated in Table 1, for which concentrations are four times lower in “M7” than in “M4”.
Y-specifikt DM-domain-gen, der er nødvendig for udvikling af hanner hos japansk risfisk
The test solution should not be prepared according to Elendt and Bias (1990) for the concentrations of NaSiO3 · 5H2O, NaNO3, KH2PO4 and K2HPO4 given for the preparation of the stock solutions are not adequate.
Enzymkoblet immunadsorptionsteknik
Preparation of the “M7”-medium
Fiskevægt
Each stock solution (I) is prepared individually and a combined stock solution (II) is prepared from these stock solutions (I) (see Table 1).
Fiskens vådvægt (duppet tør)
Fifty ml from the combined stock solution (II) and the amounts of each macro nutrient stock solution which are given in Table 2 are made up to 1 litre of deionised water to prepare the “M7” medium.
Test af fisks kønsudvikling
A vitamin stock solution is prepared by adding three vitamins to deionised water as indicated in Table 3, and 0,1 ml of the combined vitamin stock solution are added to the final “M7” medium shortly before use.
Hypothalamus-hypofyse-gonadeaksen
The vitamin stock solution is stored frozen in small aliquots.
Interkønnet fisk
The medium is aerated and stabilised.
Fisk med mere end en oocyt i testes pr. 6 analyserede snit eller spermatogene celler i ovarierne (ja/nej)
Stock solutions of trace elements for medium M4 and M7
fiskens vådvægt pr. vandvolumen
Stock solutions (I)
Virkningsmekanisme
Amount (mg) made up to 1 litre of deionised water
revers transskriptasepolymerasekædereaktion
To prepare the combined stock solution (II):
Udifferentierede fisk
mix the following amounts (ml) of stock solutions (I) and make up to 1 litre of deionised water
fisk, hvis kønskirtler ikke viser synlige kønsceller
Final concentrations in test solutions (mg/l)
Testbetingelser for FSDT (ferskvandsarter)
MnCl2·4H2O [1]
Japansk risfisk (Oryzias latipes)
SrCl2·6H2O [1]
Trepigget hundestejle (Gasterostreus aculeatus)
MoO4·2H2O [1]
Gennemstrømning eller semistatisk
CuCl2·2H2O [1]
Fluorescerende pærer (bredspektret)
CaCl2·6H2O
Lysintensitet
EDTA·2H2O [1] [2]
10-20 μE/m2/s, 540-1080 lux eller 50-100 ft-c (rumniveauer i laboratoriet)
FeSO4·7H2O [1] [2]
12-16 t. lys, 8-12 t. mørke
Macro nutrient stock solutions for medium M4 and M7
Mindste kammerstørrelse
Amount made up to 1 litre of deionised water
Individuelle kamre skal indeholde et vandvolumen på minimum 7 l
Amount of macro nutrient stock solutions added to prepare medium M4 and M7
Testorganismernes alder ved eksponeringens start
(ml/l)
Nybefrugtede æg (tidligt blastulastadie)
Final concentrations in test solutions M4 and M7
Nyligt befrugtede æg
Vitamin stock solution for medium M4 and M7
Antal æg pr. behandling
All three vitamin solutions are combined to make a single vitamin stock solution.
Minimum 3 (plus passende kontroller)
Amount of vitamin stock solution added to prepare medium M4 and M7
Antal replikater pr. behandling
Thiamine hydrochloride
Minimum 4 (medmindre fordeling til kontrol ifølge kvadratrodsreglen)
Cyanocobalamin (B12)
Levende Artemia, frossen voksen saltsøkrebs, foder i flager osv. Det anbefales at fodre to gange dagligt.
Biotine
Specialyngelfoder, levende Artemia, frossen voksen saltsøkrebs, foder i flager osv. Det anbefales at fodre to gange dagligt.
These substances differ in M4 and M7, as indicated above.
Ingen, medmindre DO-koncentration falder under 60 % mætning
These solutions are prepared individually, then poured together and autoclaved immediately.
Ingen, medmindre DO-koncentration falder under 70 % mætning
Preparation of formulated sediment
Rent overflade-, brønd- eller rekonstitueret vand
SEDIMENT COMPOSITION
Varighed af eksponering for testkemikaliet
The composition of the formulated sediment should be as follows:
Klækningssucces, overlevelse, makromorfologi, VTG, histologisk undersøgelse af kønskirtler, genetisk køn Kønsfordeling
Sphagnum moss peat, as close to pH 5,5-6,0 as possible, no visible plant remains, finely ground (particle size ≤ 1 mm) and air dried
Klækningssucces, overlevelse makromorfologi, VTG, histologisk undersøgelse af kønskirtler, kønsfordeling
4 - 5
Testgodkendelseskriterier for samlede kontrolreplikater
> 50 % of the particles should be in the range of 50-200 μm
Klækningssucces > 80 %
CaCO3, pulverised, chemically pure
Overlevelse efter klækning ≥ 70 %
0,05 - 0,1
Vækst (fiskens vådvægt, duppet tørre) > 150 mg
Conductivity ≤ 10 μS/cm
Vækst (fiskens vådvægt, duppet tør) > 75 mg
PREPARATION
Vækst (fiskens vådvægt, duppet tør) > 120 mg
The suspension is conditioned for at least two days with gentle stirring at 20 ± 2 °C, to stabilise pH and establish a stable microbial component. pH is measured again and should be 6,0 ± 0,5.
Længde (standardlængde) > 20 mm
STORAGE
Kønsfordeling (% hanner eller hunner) 30-70 %
The dry constituents for preparation of the artificial sediment may be stored in a dry and cool place at room temperature.
Kemiske egenskaber for acceptabelt vand til fortynding
The formulated (wet) sediment should not be stored prior to its use in the test.
KONCENTRATION
It should be used immediately after the 7 days conditioning period that ends its preparation.
Partikler
OECD (1984), Earthworm, Acute Toxicity Test, Test Guideline No. 207, Guidelines for the Testing of Chemicals, OECD, Paris.
Fra testmetode C.14 / vejledning i testkoncentrationer
Meller, M., Egeler, P., Roembke, J., Schallnass, H., Nagel, R. and B. Streit (1998), Short-term toxicity of lindane, hexachlorobenzene and copper sulfate on tubificid sludgeworms (Oligochaeta) in artificial media, Ecotox.
Kolonne (antal koncentrationer mellem 100 og 10 eller mellem 10 og 1) [1]
Environ.
En række bestående af tre (eller flere) successive koncentrationer kan vælges fra en kolonne.
Safety, 39:
Midterpunkterne mellem koncentrationerne i kolonne (x) findes i kolonne (2x + 1).
10-20.
De anførte værdier kan repræsentere koncentrationer udtrykt i procent pr. volumen eller vægt (mg/l eller μg/l).
Chemical Characteristics of an Acceptable Dilution water
Værdierne kan multipliceres eller divideres med en hvilken som helst potens af 10, alt efter hvad der er aktuelt.
CONSTITUENT
Kolonne 1 kan eventuelt anvendes, hvis der har været betydelig tvivl om toksicitetsniveauet.
CONCENTRATIONS
Vejledning i homogenisering af hoved og hale fra yngel af zebrafisk, tykhovedet elritse, trepigget hundestejle og japansk risfisk
Hardness as CaCO3
Formålet med dette afsnit er at beskrive de procedurer, der går forud for en kvantitativ bestemmelse af VTG-koncentrationen.
However, it should be noted that if there is an interaction suspected between hardness ions and the test chemical, lower hardness water should be used (and thus, Elendt Medium M4 should not be used in this situation).
Der kan anvendes andre procedurer, som fører til en sammenlignelig kvantitativ bestemmelse af VTG.
Guidance for test performance
Det er valgfrit, om man vil bestemme VTG-koncentrationen i blodplasma eller lever i stedet for hoved-/halehomogenat.
Example of a breeding cage:
Hoved og hale skæres af fisken i overensstemmelse med testbeskrivelsen.
A gauze on the top and at least one side of the cage (mesh size ca.
Alle dissektionsinstrumenter og skærebrættet skylles og rengøres grundigt (f.eks. med 96 % ethanol) mellem håndtering af hver enkelt fisk for at forebygge »VTG-forurening« fra hunner eller inducerede hanner til ikkeinducerede hanner.
1 mm)
Når der er opnået en homogen masse, tilsættes en mængde iskold homogeniseringsbuffer på 4-10 gange vævsvægten (bemærk fortyndingen).
B aperture for placing the emerged adults inside the breeding cage and to remove the laid egg ropes from the crystallisation dishes (not shown in this graphic)
Prøverne lægges på is indtil centrifugering ved 4 °C ved 50000 g i 30 min.
C breeding cage size minimum 30 cm length, 30 cm height and 30 cm width
Anvend en pipette til at fordele portioner à 20-50 μl (bemærk mængden) supernatant i mindst to rør ved at dyppe spidsen af pipetten under fedtlaget på overfladen og omhyggeligt suge supernatanten op uden dele af hverken fedt eller pellets.
Example of a test vessel:
Anbringes på is under brug.
A pasteur pipette for air supply of the overlying water
12 ml Tris-HCl pH 7,4 + 120 μl proteaseinhibitorblanding (eller tilsvarende proteaseinhibitorblandinger).TRIS:
B glass lid to prevent emerged midges from escaping
TRIS-ULTRA PURE (ICN)Proteaseinhibitorblanding:
C water surface layer
Fra Sigma (til pattedyrvæv) produktnummer P 8340.
D test vessel (glass beaker minimum 600 ml)
Vejledning i kvantitativ bestemmelse af vitellogenin i hoved- og halehomogenat fra zebrafish (danio rerio) (ændret fra holbech et al., 2001).
E sediment layer
Der kan benyttes andre procedurer, hvor der anvendes homologe antistoffer og standarder.
Example of an exhauster for capturing adult midges (arrows indicate air flow direction):
Mikrotiterplader (certificeret Maxisorp F96, Nunc, Roskilde, Danmark), som tidligere er coatet med 5 μg/ml IgG-antistoffer mod zebrafisk-lipovitellin, optøs og vaskes tre gange med vaskebuffer (*).
A glass tube (inner diameter ca.
Vitellogeninstandard [1] af renset zebrafisk fortyndes serielt til 0,2, 0,5, 1, 2, 5, 10 og 20 ng/ml i fortyndingsbuffer (**), og prøverne fortyndes mindst 200 gange (for at forebygge matrixeffekt) i fortyndingsbuffer og påføres pladerne.
5 mm) connected to a self-priming pump B cork of vulcanised rubber, perforated with glass tube (A).
Der foretages en dobbelt assaykontrol.
On the inside, the opening of glass tube (A) is covered with some cotton and a gauze (mesh size ca.
150 μl tilsættes hver brønd.
1 mm) to prevent damaging the midges when they are sucked into the exhauster
Standarderne anvendes in duplo og prøverne in triplo.
C transparent container (plastic or glass, length ca.
Inkuberes natten over ved 4 °C på et rystebord.
15 cm) for captured midges
Pladerne vaskes fem gange med vaskebuffer (*).
D cork of vulcanised rubber, perforated with tube (E).
HRP bundet til en dextrankæde (f.eks. AMDEX A/S, Danmark) og konjugerede antistoffer fortyndes i vaskebuffer.
To release midges into the breeding cage, cork D is released from container C
Den faktiske fortynding afviger efter parti og alder.
E tube (plastic or glass, inner diameter ca.
150 μl tilsættes hver brønd, og pladerne inkuberes i en time ved stuetemperatur på et rystebord.
8 mm) to collect adult midges from vessel
Pladerne vaskes fem gange med vaskebuffer (*), og bunden af pladerne renses omhyggeligt med ethanol.
Schematic presentation of a life-cycle test:
150 μl TMB plus (***) tilføres hver brønd.
A 1st generation — test vessels containing a sediment-water system, eight replicates, 20 first instar larvae per vessel
Beskyt pladen mod lys med stanniol og hold øje med farveudviklingen på et rystebord.
B four test vessels for each breeding cage, A and B
Når standardkurven er fuldt udviklet, standses enzymaktiviteten ved at tilsætte 150 μl 0,2 M H2SO4 til hver brønd.
C breeding cages (A and B) for swarming, mating and oviposition
Vaskebuffer:
D crystallising dishes for deposition of egg ropes
PBS-stam (****)
E micro plates, one well for each egg rope
Juster pH til 7,3 og fyld op til 5 l med millipore H2O.
F 2nd generation — test vessels containing a sediment-water system, eight replicates, 20 first instar larvae per vessel
Opbevar ved 4 °C.
C.41.
Fortyndingsbuffer:
FISH SEXUAL DEVELOPMENT TEST
PBS-stam (****)
This test method is equivalent to OECD test guideline (TG) 234 (2011).
Juster pH til 7,3 og fyld op til 1 l med millipore H2O.
It is based on a decision from 1998 to develop new or update existing test methods for the screening and testing of potential endocrine disrupters.
TMB plus er et brugsklart substrat fremstillet af KemEnTec (Danmark).
The Fish Sexual Development Test (FSDT) was identified as a promising test method covering a sensitive fish life stage responsive to both oestrogen and androgen-like chemicals.
Det er lysfølsomt.
The test method went through an inter-laboratory validation exercise from 2006 to 2010, where Japanese medaka (Oryzias latipes), zebrafish (Danio rerio) and three spined stickleback (Gasterosteus aculeatus) were validated and fathead minnow (Pimephales promelas) was partially validated (41) (42) (43).
PBS stam
This protocol includes Japanese medaka, the three-spined stickleback and zebrafish.
HPO4.2H2O
The protocol is in principle an enhancement of OECD TG 210 Fish, Early Life Stage Toxicity Test (1), where the exposure is continued until the fish are sexually differentiated, i.e. about 60 days post-hatch (dph) for Japanese medaka, the three-spined stickleback and zebrafish (the exposure period can be shorter or longer for other species that are validated in the future), and endocrine-sensitive endpoints are added.
Battelle AP4.6.04 (1,18 mg/ml (AAA)), renset ifølge: N.D. Denslow, M.C. Chow, K.J.
The combination of the two core endocrine endpoints, vitellogenin (VTG) concentration and phenotypic sex ratio enable the test to indicate the mode of action of the test chemical.
Vejledning i fremstilling af vævssnit til kønsbestemmelse og stadieinddeling af kønskirtler
Due to the population-relevant change in phenotypic sex ratio, the FSDT can be used for hazard and risk assessment.
Formålet med dette afsnit er at beskrive de procedurer, der går forud for vurderingen af histologiske snit.
However, if the test is used for hazard or risk assessment, the stickleback should not be used because the validation data available so far showed that in this species the alterations of phenotypic sex ratio by the test chemicals were uncommon.
Der kan anvendes andre procedurer, som fører til en lignende kønsbestemmelse og stadieinddeling af kønskirtler.
The protocol is based on fish exposed via water to chemicals during the sex labile period in which the fish is expected to be most sensitive to the effects of endocrine disrupting chemicals that interfere with sexual development.
Med nogle få undtagelser er disse procedurer de samme for japansk risfisk (JMD) og zebrafisk (ZF).
Two core endpoints are measured as indicators of endocrine-associated developmental aberrations, the VTG concentrations and sex ratios (proportions of sex) determined via gonad histology.
Aflivning, obduktion og fiksering af væv
Gonadal histopathology (evaluation and staging of oocytes and spermatogenetic cells) is optional.
Mål:
Additionally, the genetic sex is determined whenever possible (e.g. in Japanese medaka and the three spined stickleback).
at sikre en human aflivning af fisk
The presence of a genetic sex marker is a considerable advantage as it increases the power of the sex ratio statistics and enables the detection of individual phenotypic sex reversal.
at opnå den nødvendige kropsvægt og de nødvendige mål
Other apical endpoints that should be measured include hatching rate, survival, length and body weight.
at vurdere sekundære kønskarakteristika
The test method might be adaptable to other species than those mentioned above provided that the other species undergo a validation equal to the one accomplished for Japanese medaka, the three-spined stickleback and zebrafish, that the control fish are sexually differentiated at the end of the test, that VTG levels are sufficiently high to detect significant chemical-related variations, and that sensitivity of the test system is established using endocrine active reference chemicals ((anti)-oestrogens, (anti)-androgens, aromatase inhibitors etc).
Fremgangsmåder:
VTG is normally produced by the liver of female oviparous vertebrates in response to circulating endogenous oestrogen (2).
Fisk aflives umiddelbart før obduktion.
The VTG synthesis is very limited, though detectable, in immature fish and adult male fish because they lack sufficient circulating oestrogen.
Medmindre der er mange prosektorer til rådighed, bør mange fisk derfor ikke aflives samtidig.
However, the liver is capable of synthesising and secreting VTG in response to exogenous oestrogen stimulation (3) (4) (5).
Ved hjælp af en lille ketsjer fjernes en fisk fra forsøgskammeret og transporteres til obduktionsområdet i transportbeholderen.
The measurement of VTG serves for the detection of chemicals with oestrogenic, anti-oestrogenic, androgenic modes of action and chemicals that interfere with steroidogenesis as for example aromatase inhibitors.
Fisken anbringes i aflivningsopløsningen.
The detection of oestrogenic chemicals is possible via the measurement of VTG induction in male fish, and it has been abundantly documented in the scientific peer-reviewed literature.
Fisken fjernes fra opløsningen, når vejrtrækningen er ophørt, og fisken ikke reagerer på eksterne stimuli.
VTG induction has also been demonstrated following exposure to aromatisable androgens (6) (7).