Council Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) (Text with EEA relevance)

• 4.For cognitive function testing in young adults (e.g. PND 60-70), a third set of 20 pups/sex/dose level is used (i.e. 1 male and 1 female per litter). Of these animals, 10 animals/sex/group (1 male or 1 female per litter) are killed at study termination and the brain is removed and weighed.

• 5.The remaining 20 animals/sex/group are reserved for possible additional tests.

Table 1

Pup No 27	Pup No 27	No of pups assigned to test	Examination/Test
m	f	No of pups assigned to test	Examination/Test
1	5	20 m + 20 f	Behavioural ontogeny
		10 m + 10 f	PND 22 brain weight/neuropathology/morphometry
		10 m + 10 f	PND 22 brain weight
2	6	20 m + 20 f	Detailed clinical observations
		20 m + 20 f	Motor activity
		20 m + 20 f	Sexual maturation
		20 m + 20 f	Motor and sensory function
		20 m + 20 f	Learning and memory (PND 25)
		10 m + 10 f	Young adult brain weight/neuropathology/morphometry ~ PND 70
3	7	20 m + 20 f	Learning and memory (young adults)
		10 m + 10 f	Young adult brain weight ~ PND 70
4	8	—	Reserve animals for replacements or additional tests

For this example, litters are culled to 4 males + 4 females; male pups are numbered 1 through 4, female pups 5 through 8.

Example 2

• 6.One set of 20 pups/sex/dose level (i.e. 1 male and 1 female per litter) is used for pre-weaning testing of behavioural ontogeny. Out of these animals, 10 pups/sex/dose level (1 male or 1 female per litter), are humanely killed at PND 11. The brains are removed, weighed and processed for histopathologic evaluation.

• 7.Another set of 20 animals/sex/dose level (1 male and1 female per litter) is used for post-weaning examinations (detailed clinical observations, motor activity, assessing age of sexual maturation and motor and sensory function). Of these animals, 10 animals/sex/dose level (i.e.1 male or 1 female per litter) are anesthetised and fixed via perfusion at study termination (approximately PND 70). After additional fixation in situ, the brain is removed, weighed and processed for neuropathological evaluation.

• 8.For cognitive function testing in adolescents and young adults, 10 pups/sex/dose level are used(i.e. 1 male or 1 female per litter). Different animals are used for testing for cognitive function tests at PND 23 and young adults. At termination, the 10 animals/sex/group tested as adults are killed, the brain is removed and weighed.

• 9.The remaining 20 animals/sex/group not selected for testing are killed and discarded at weaning.

Table 2

Pup No 28	Pup No 28	No of pups assigned to test	Examination/Test
m	f	No of pups assigned to test	Examination/Test
1	5	20 m + 20 f	Behavioural ontogeny
		10 m + 10 f	PND 11 brain weight/neuropathology/morphometry
2	6	20 m + 20 f	Detailed clinical observations
		20 m + 20 f	Motor activity
		20 m + 20 f	Sexual maturation
		20 m + 20 f	Motor and sensory function
		10 m + 10 f	Young adult brain weight/neuropathology/morphometry ~ PND 70
3	7	10 m + 10 f 29	Learning and memory (PND 23)
3	7	10 m + 10 f 29	Learning and memory (young adults)
			Young adult brain weight
4	8	—	Animals killed and discarded PND 21.

For this example, litters are culled to 4 males + 4 females; male pups are numbered 1 through 4, female pups 5 through 8.

Different pups are used for cognitive tests at PND 23 and in young adults (e.g. even/odd litters from total of 20).

Example 3

• 10.One set 20 pups/sex/dose level (i.e. 1 male and 1 female per litter) is used for brain weight and neuropathology assessment at PND 11. Out of these animals, 10 pups/sex/dose level (i.e. 1 male or 1 female per litter) are humanely killed at PND 11 and brains are removed, weighed and processed for histopathologic evaluation. In addition, brain weight data are collected using unfixed brains from the remaining 10 males and 10 females per dose level.

• 11.Another set of of 20 animals/sex/dose level (i.e. 1 male and 1 female per litter) are used for behavioural ontogeny (motor activity), post-weaning examinations (motor activity and assessing age of sexual maturation), and cognitive function testing in adolescents.

• 12.Another set of 20 animals/sex/dose level (i.e. 1 male and 1 female per litter) is used for motor and sensory function tests (auditory startle) and detailed clinical observations. Of these animals, 10 animals/sex/dose level (i.e. 1 male or 1 female per litter) are anesthetised and fixed via perfusion at study termination (approximately PND 70). After additional fixation in situ, the brain is removed, weighed and processed for neuropathological evaluation.

• 13.Another set of 20 pups/sex/dose level are usedfor cognitive function testing in young adults (i.e. 1 male and 1 female per litter). Of these, 10 animals/sex/group (i.e. 1 male or 1 female per litter) are killed at termination, the brain removed and weighed.

Table 3

Pup No 30	Pup No 30	No of pups assigned to test	Examination/Test
m	f	No of pups assigned to test	Examination/Test
1	5	10 m + 10 f	PND 11 brain weight/neuropathology/morphometry
		10 m + 10 f	PND 11 brain weight
2	6	20 m + 20 f	Behavioural ontogeny (motor activity)
		20 m + 20 f	Motor activity
		20 m + 20 f	Sexual maturation
		20 m + 20 f	Learning and memory (PND 27)
3	7	20 m + 20 f	Auditory startle (adolescents and young adults)
		20 m + 20 f	Detailed clinical observations
		10 m + 10 f	Young adult brain weight/neuropathology/morphometry ~ PND 70
4	8	20 m + 20 f	Learning and memory (young adults)
		10 m + 10 f	Young adult brain weight

For this example, litters are culled to 4 males + 4 females; male pups are numbered 1 through 4, female pups 5 through 8.

Appendix 2

Definitions

Chemical : A substance or a mixture

Test chemical : Any substance or mixture tested using this test method

B.54   UTEROTROPHIC BIOASSAY IN RODENTS: A SHORT-TERM SCREENING TEST FOR OESTROGENIC PROPERTIES

INTRODUCTION

• 1. This test method is equivalent to OECD Test Guideline (TG) 440 (2007). The OECD initiated a high-priority activity in 1998 to revise existing guidelines and to develop new guidelines for the screening and testing of potential endocrine disrupters (1). One element of the activity was to develop a test guideline for the rodent Uterotrophic Bioassay. The rodent Uterotrophic Bioassay then underwent an extensive validation programme including the compilation of a detailed background document (2)(3) and the conduct of extensive intra- and interlaboratory studies to show the relevance and reproducibility of the bioassay with a potent reference oestrogen, weak oestrogen receptor agonists, a strong oestrogen receptor antagonist, and a negative reference chemical (4)(5)(6)(7)(8)(9). This test method B.54 is the outcome of the experience gained during the validation test programme and the results obtained thereby with oestrogenic agonists.

• 1. The Uterotrophic Bioassay is a short-term screening test that originated in the 1930s (27)(28) and was first standardised for screening by an expert committee in 1962 (32)(35). It is based on the increase in uterine weight or uterotrophic response (for review, see 29). It evaluates the ability of a chemical to elicit biological activities consistent with agonists or antagonists of natural oestrogens (e.g. 17ß-estradiol), however, its use for antagonist detection is much less common than for agonists. The uterus responds to oestrogens in two ways. An initial response is an increase in weight due to water imbibition. This response is followed by a weight gain due to tissue growth (30). The uterus responses in rats and mice qualitatively are comparable.

• 1. This bioassay serves as an in vivo screening assay and its application should be seen in the context of the ‘OECD Conceptual Framework for the Testing and Assessment of Endocrine Disrupting Chemicals’ (Appendix 2). In this Conceptual Framework the Uterotrophic Bioassay is contained in Level 3 as an in vivo assay providing data about a single endocrine mechanism, i.e. oestrogenicity.

• 1. The Uterotrophic Bioassay is intended to be included in a battery of in vitro and in vivo tests to identify chemicals with potential to interact with the endocrine system, ultimately leading to risk assessments for human health or the environment. The OECD validation programme used both strong and weak oestrogen agonists to evaluate the performance of the assay to identify oestrogenic chemicals (4)(5)(6)(7)(8). Thereby the sensitivity of the test procedure for oestrogen agonists was well demonstrated besides a good intra- and interlaboratory reproducibility.

• 1. With regard to negative chemicals, only one ‘negative’ reference chemical already reported negative by uterotrophic assay as well as in vitro receptor binding and receptor assays was included in the validation programme, but additional test data, not related to the OECD validation programme, have been evaluated, giving further support to the specificity of the Uterotrophic Bioassay for the screening of oestrogen agonists (16).

INITIAL CONSIDERATIONS AND LIMITATIONS

• 1. Oestrogen agonists and antagonists act as ligands for oestrogen receptors a and b and may activate or inhibit, respectively, the transcriptional action of the receptors. This may have the potential to lead to adverse health hazards, including reproductive and developmental effects. Therefore, the need exists to rapidly assess and evaluate a chemical as a possible oestrogen agonist or antagonist. While informative, the affinity of a ligand for an oestrogen receptor or transcriptional activation of reporter genes in vitro is only one of several determinants of possible hazard. Other determinants can include metabolic activation and deactivation upon entering the body, distribution to target tissues, and clearance from the body, depending at least in part on the route of administration and the chemical being tested. This leads to the need to screen the possible activity of a chemical in vivo under relevant conditions, unless the chemical's characteristics regarding Absorption — Distribution — Metabolism — Elimination (ADME) already provide appropriate information. Uterine tissues respond with rapid and vigorous growth to stimulation by oestrogens, particularly in laboratory rodents, where the oestrous cycle lasts approximately 4 days. Rodent species, particularly the rat, are also widely used in toxicity studies for hazard characterisation. Therefore, the rodent uterus is an appropriate target organ for the in vivo screening of oestrogen agonists and antagonists.

• 1. This test method is based on those protocols employed in the OECD validation study which have been shown to be reliable and repeatable in intra- and interlaboratory studies (5)(7). Currently two methods, namely the ovariectomised adult female method (ovx-adult method) and the immature non-ovariectomised method (immature method) are available. It was shown in the OECD validation test programme that both methods have comparable sensitivity and reproducibility. However, the immature, as it has an intact hypothalamic-pituitary-gonadal (HPG) axis, is somewhat less specific but covers a larger scope of investigation than the ovariectomised animal because it can respond to chemicals that interact with the HPG axis rather than just the oestrogen receptor. The HGP axis of the rat is functional at about 15 days of age. Prior to that, puberty cannot be accelerated with treatments like GnRH. As the females begin to reach puberty, prior to vaginal opening, the female will have several silent cycles that do not result in vaginal opening or ovulation, but there are some hormonal fluctuations. If a chemical stimulates the HPG axis directly or indirectly, precocious puberty, early ovulation and accelerated vaginal opening result. Not only chemicals that act on the HPG axis do this but some diets with higher metabolisable energy levels than others will stimulate growth and accelerate vaginal opening without being oestrogenic. Such chemicals would not induce an uterotrophic response in OVX adult animals as their HPG axis does not work.

• 1. For animal welfare reasons preference should be given to the method using immature rats, avoiding surgical pre-treatment of the animals and avoiding also a possible non-use of those animals which indicate any evidence entering oestrous (see paragraph 30).

• 1. The uterotrophic response is not entirely of oestrogenic origin, i.e. chemicals other than agonists or antagonists of oestrogens may also provide a response. For example, relatively high doses of progesterone, testosterone, or various synthetic progestins may all lead to a stimulative response (30). Any response may be analysed histologically for keratinisation and cornification of the vagina (30). Irrespective of the possible origin of the response, a positive outcome of an Uterotrophic Bioassay should normally initiate actions for further clarification. Additional evidence of oestrogenicity could come from in vitro assays, such as the ER binding assays and transcriptional activation assays, or from other in vivo assays such as the female pubertal assay.

• 1. Taking into account that the Uterotrophic Bioassay serves as an in vivo screening assay, the validation approach taken served both animal welfare considerations and a tiered testing strategy. To this end, effort was directed at rigorously validating reproducibility and sensitivity for oestrogenicity — the main concern for many chemicals-, while little effort was directed at the antioestrogenicity component of the assay. Only one antioestrogen with strong activity was tested since the number of chemicals with a clear antioestrogenic profile (not obscured by some oestrogenic activity) is very limited. Thus this test method is dedicated to the oestrogenic protocol, while the protocol describing the antagonist mode of the assay is included in a Guidance Document (37). The reproducibility and sensitivity of the assay for chemicals with purely anti-oestrogenic activity will be more clearly defined later on, after the test procedure has been in routine use for some time and more chemicals with this modality of action are identified.

• 1. It is acknowledged that all animal based procedures will conform to local standards of animal care; the descriptions of care and treatment set forth below are minimal performance standards, and will be superseded by local regulations such as Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes (38). Further guidance of the humane treatment of animals is given by the OECD (25).

• 1. As with all assays using live animals, it is essential to ensure that the data are truly necessary prior to the start of the assay. For example, two conditions where the data may be required are:

• — high exposure potential (Level 1 of the Conceptual Framework, Appendix 2) or indications for oestrogenicity (Level 2) to investigate whether such effects may occur in vivo;

• — effects indicating oestrogenicity in Level 4 or 5 in vivo tests to substantiate that the effects were related to an oestrogenic mechanism that cannot be elucidated using an in vitro test.

• 1. Definitions used in this test method are given in Appendix 1.

PRINCIPLE OF THE TEST

• 1. The Uterotrophic Bioassay relies for its sensitivity on an animal test system in which the hypothalamic-pituitary-ovarian axis is not functional, leading to low endogenous levels of circulating oestrogen. This will ensure a low baseline uterine weight and a maximum range of response to administered oestrogens. Two oestrogen sensitive states in the female rodent meet this requirement:

• (i) immature females after weaning and prior to puberty; and

• (ii) young adult females after ovariectomy with adequate time for uterine tissues to regress.

• 1. The test chemical is administered daily by oral gavage or subcutaneous injection. Graduated test chemical doses are administered to a minimum of two treatment groups (see paragraph 33 for guidance) of experimental animals using one dose level per group and an administration period of three consecutive days for immature method and a minimum administration period of three consecutive days for ovx-adult method. The animals are necropsied approximately 24 hours after the last dose. For oestrogen agonists, the mean uterine weight of the treated animal groups relative to the vehicle group is assessed for a statistically significant increase. A statistically significant increase in the mean uterine weight of a test group indicates a positive response in this bioassay.

DESCRIPTION OF THE METHOD

Selection of animal species

• 1. Commonly used laboratory rodent strains may be used. As an example, Sprague-Dawley and Wistar strains of rats were used during the validation. Strains with uteri known or suspected to be less responsive should not be used. The laboratory should demonstrate the sensitivity of the strain used as described in paragraphs 26 and 27.

• 1. The rat and mouse have been routinely used in the Uterotrophic Bioassay since the 1930s. The OECD validation studies were only performed with rats based on an understanding that both species are expected to be equivalent and therefore one species should be enough for the world-wide validation in order to save resources and animals. The rat is the species of choice in most reproductive and developmental toxicity studies. Taking into consideration that a vast historical database exists for mice and thus to broaden the scope of the Uterotrophic Bioassay test method in rodents to the use of mice as test species, a limited follow-up validation study was carried out in mice (16). A bridging approach with a limited number of test chemicals, participating laboratories and without coded sample testing has been selected in keeping with the original intent to save resources and animals. This bridging validation study shows for the Uterotrophic Bioassay in young adult ovariectomised mice that, qualitatively and quantitatively, the data obtained in rats and mice correspond well with each other. Where the Uterotrophic Bioassay result may be preliminary to a long-term study, this allows animals from the same strain and source to be used in both studies. The bridging approach was limited to the OVX mice and the report does not provide a robust data set to validate the immature model, thus the immature model for mice is not considered under the scope of the current test method.

• 1. Thus, in some cases mice may be used instead of rats. A rationale should be given for this species, based on toxicological, pharmacokinetic, and/or other criteria. Modifications of the protocol may be necessary for mice. For example, the food consumption of mice on a body weight basis is higher than that of rats and therefore the phyto-oestrogen content in food should be lower for mice than for rats (9)(20)(22).

Housing and feeding conditions

• 1. All procedures should conform with local standards of laboratory animal care. These descriptions of care and treatment are minimum standards and will be superseded by local regulations such as Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes (38). The temperature in the experimental animal room should be 22 °C (with an approximate range ± 3 °C). The relative humidity should be a minimum of 30 % and preferably should not exceed a maximum 70 %, other than during room cleaning. The aim should be relative humidity of 50-60 %. Lighting should be artificial. The daily lighting sequence should be 12 hours light, 12 hours dark.

• 1. Laboratory diet and drinking water should be provided ad libitum. Young adult animals may be housed individually or be caged in groups of up to three animals. Due to the young age of the immature animals, social group housing is recommended.

• 1. High levels of phyto-oestrogens in laboratory diets have been known to increase uterine weights in rodents to a degree enough as to interfere with the Uterotrophic Bioassay (13)(14)(15). High levels of phyto-oestrogens and of metabolisable energy in laboratory diets may also result in early puberty, if immature animals are used. The presence of phyto-oestrogens results primarily from the inclusion of soy and alfalfa products in the laboratory diets and concentrations of phyto-oestrogens have been shown to vary from batch-to-batch of standard laboratory diets (23). Body weight is an important variable, as the quantity of food consumed is related to body weight. Therefore, the actual phyto-oestrogen dose consumed from the same diet may vary among species and by age (9). For immature female rats, food consumption on a body weight basis may be approximately double that of ovariectomised young adult females. For young adult mice, food consumption on a body weight basis may be approximately quadruple that of ovariectomised young adult female rats.

• 1. Uterotrophic Bioassay results (9)(17)(18)(19), however, show that limited quantities of dietary phyto-oestrogens are acceptable and do not reduce the sensitivity of the bioassay. As a guide, dietary levels of phyto-oestrogens should not exceed 350 μg of genistein equivalents/gram of laboratory diet for immature female Sprague Dawley and Wistar rats (6)(9). Such diets should also be appropriate when testing in young adult ovariectomised rats because food consumption on a body weight basis is less in young adult as compared to immature animals. If adult ovariectomised mice or more phyto-oestrogen-sensitive rats are to be used, proportional reduction in dietary phyto-oestrogen levels must be considered (20). In addition, the differences in available metabolic energy from different diets may lead to time shifts for the onset of puberty (21)(22).

• 1. Prior to the study, careful selection is required of a diet without an elevated level of phyto-oestrogens (for guidance see (6)(9)) or metabolisable energy, that can confound the results (15)(17)(19)(22)(36). Ensuring the proper performance of the test system used by the laboratory as specified in paragraphs 26 and 27 is an important check on both of these factors. As a safeguard consistent with good laboratory practice (GLP) representative sampling of each batch of diet administered during the study should be conducted for possible analysis of phyto-oestrogen content (e.g. in the case of high uterine control weight relative to historic controls or an inadequate response to the reference oestrogen, 17 alpha ethinyl estradiol). Aliquots should be analysed as part of the study or frozen at – 20 °C or in such a way as to prevent the sample from decomposing prior to analysis.

• 1. Some bedding materials may contain naturally occurring oestrogenic or antioestrogenic chemicals (e.g. corn cob is known to affects the cyclicity of rats and appears to be antioestrogenic). The selected bedding material should contain a minimum level of phyto-oestrogens.

Preparation of animals

• 1. Experimental animals without evidence of any disease or physical abnormalities are randomly assigned to the control and treatment groups. Cages should be arranged in such a way that possible effects due to cage placement are minimised. The animals should be identified uniquely. Preferably, immature animals should be caged with dams or foster dams until weaning during acclimatisation. The acclimatisation period prior to the start of the study should be about 5 days for young adult animals and for the immature animals delivered with dams or foster dams. If immature animals are obtained as weanlings without dams a shorter duration of the acclimatisation period may become necessary as dosing should start immediately after weaning (see paragraph 29).

PROCEDURE

Verification of Laboratory Proficiency

• 1. Two different options can be used to verify laboratory proficiency:

• — Periodic verification, relying on an initial baseline positive control study (see paragraph 27). At least every 6 months and each time there is a change that may influence the performance of the assay (e.g. a new formulation of diet, change in personnel performing dissections, change in animal strain or supplier, etc.), the responsiveness of the test system (animal model) should be verified using an appropriate dose (based on the baseline positive control study described in paragraph 27) of a reference oestrogen: 17a-ethinyl estradiol (CAS No 57-63-6) (EE).

• — Use of concurrent controls, by including a group administered with an appropriate dose of reference oestrogen in each assay.

If the system does not respond as expected, the experimental conditions should be examined and modified accordingly. It is recommended that the dose of reference oestrogen to be used in either approach be approximately the ED70 to 80.

• 1. Baseline Positive Control Study — Before a laboratory conducts a study under this test method for the first time, laboratory proficiency should be demonstrated by testing the responsiveness of the animal model, by establishing the dose response of a reference oestrogen: 17a-ethinyl estradiol (CAS No 57-63-6) (EE) with a minimum of four doses. The uterine weight response will be compared to established historical data (see reference (5)). If this baseline positive control study does not yield the anticipated results the experimental conditions should be examined and modified.

Number and condition of animals

• 1. Each treated and control group should include at least 6 animals (for both immature and ovx-adult method protocols).

Age of immature animals

• 1. For the Uterotrophic Bioassay with immature animals the day of birth must be specified. Dosing should begin early enough to ensure that, at the end of test chemical administration, the physiological rise of endogenous oestrogens associated with puberty has not yet taken place. On the other hand, there is evidence that very young animals may be less sensitive. For defining the optimal age each laboratory should take its own background data on maturation into consideration.

As a general guide, dosing in rats may begin immediately after early weaning on postnatal day 18 (with the day of birth being postnatal day 0). Dosing in rats preferably should be completed on postnatal day 21 but in any case prior to postnatal day 25, because, after this age, the hypothalamic-pituitary-ovarian axis becomes functional and endogenous oestrogen levels may begin to rise with a concomitant increase in baseline uterine weight means and an increase in the group standard deviations (2)(3)(10)(11)(12).

Procedure for ovariectomy

• 1. For the ovariectomised female rat and mouse (treatment and control groups), ovariectomy should occur between 6 and 8 weeks of age. For rats, a minimum of 14 days should elapse between ovariectomy and the first day of administration in order to allow the uterus to regress to a minimum, stable baseline. For mice, at least 7 days should elapse between ovariectomy and the first day of administration. As small amounts of ovarian tissue are sufficient to produce significant circulating levels of oestrogens (3), the animals should be tested prior to use by observing epithelial cells swabbed from the vagina on at least five consecutive days (e.g. days 10-14 after ovariectomy for rats). If the animals indicate any evidence entering oestrous, the animals should not be used. Further, at necropsy, the ovarian stubs should be examined for any evidence that ovarian tissue is present. If so, the animal should not be used in the calculations (3).

• 1. The ovariectomy procedure begins with the animal in ventral recumbency after the animal has been properly anesthetised. The incision opening the dorso-lateral abdominal wall should be approximately 1 cm lengthways at the mid-point between the costal inferior border and the iliac crest, and a few millimetres lateral to the lateral margin of the lumbar muscle. The ovary should be removed from the abdominal cavity onto an aseptic field. The ovary should be disconnected at the junction of the oviduct and the uterine body. After confirming that no massive bleeding is occurring, the abdominal wall should be closed by a suture and the skin closed by autoclips or appropriate suture. The ligation points are shown schematically in Figure 1. Appropriate post-operative analgesia should be used as recommended by a veterinarian experienced in rodent care.

Body weight

• 1. In the ovx-adult method, body weight and uterine weight are not correlated because uterine weight is affected by hormones like oestrogens but not by the growth factors that regulate body size. On the contrary, body weight is related to uterine weight in the immature model, while it is maturing (34). Thus, at the commencement of the study the weight variation of animals used, in the immature model, should be minimal and not exceed ± 20 % of the mean weight. This means that the litter size should be standardised by the breeder, to ensure that offspring of different mother animals will be fed approximately the same. Animals should be assigned to groups (both control and treatment) by randomised weight distribution, so that mean body weight of each group is not statistically different from any other group. Consideration should be given to avoid assignment of littermates to the same treatment group as far as practicable without increasing the number of litters to be used for the investigation.

Dosage

• 1. In order to establish whether a test chemical can have oestrogenic action in vivo, two dose groups and a control are normally sufficient and this design is therefore preferred for animal welfare reasons. If the purpose is either to obtain a dose-response curve or to extrapolate to lower doses, at least 3 dose groups are needed. If information beyond identification of oestrogenic activity (such as an estimate of potency) is required, a different dosing regimen should be considered. Except for treatment with the test chemical, animals in the control group should be handled in an identical manner to the test group subjects. If a vehicle is used in administering the test chemical, the control group should receive the same amount of vehicle used with the treated groups (or highest volume used with the test groups if different among groups).

• 1. The objective in the case of the Uterotrophic Bioassay is to select doses that ensure animal survival and that are without significant toxicity or distress to the animals after three consecutive days of chemical administration up to a maximum dose of 1 000 mg/kg/d. All dose levels should be proposed and selected taking into account any existing toxicity and (toxico-) kinetic data available for the test chemical or related materials. The highest dose level should first take into consideration the LD50 and/or acute toxicity information in order to avoid death, severe suffering or distress in the animals (24)(25)(26). The highest dose should represent the maximum tolerated dose (MTD); a study conducted at a dose level that induced a positive uterotrophic response would be accepted too. As a screen, large intervals (e.g. one half log units corresponding to a dose progression of 3,2 or even up to one log units) between dosages are generally acceptable. If there are no suitable data available, a range finding study may be performed to aid the determination of the doses to be used.

• 1. Alternatively, if the oestrogenic potency of an agonist can be estimated by in vitro (or in silico) data, these may be taken into consideration for dose selection. For example, the amount of the test chemical that would produce uterotrophic responses equivalent to the reference agonist (ethinyl estradiol) is estimated by its relative in vitro potencies to ethinyl estradiol. The highest test dose would be given by multiplying this equivalent dose by an appropriate factor e.g. 10 or 100.

Considerations for range finding

• 1. If necessary, a preliminary range finding study can be carried out with few animals. In this respect, OECD Guidance Document No 19(25) may be used defining clinical signs indicative of toxicity or distress to the animals. If feasible within this range finding study after three days of administration, the uteri may be excised and weighed approximately 24-hours after the last dose. These data could then be used to assist the main study design (select an acceptable maximum and lower doses and recommend the number of dose groups).

Administration of doses

• 1. The test chemical is administered by oral gavage or subcutaneous injection. Animal welfare considerations as well as toxicological aspects like the relevance to the human route of exposure to the chemical (e.g. oral gavage to model ingestion, subcutaneous injection to model inhalation or dermal adsorption), the physical/chemical properties of the test material and especially existing toxicological information and data on metabolism and kinetics (e.g. need to avoid first pass metabolism, better efficiency via a particular route) have to be taken into account when choosing the route of administration.

• 1. It is recommended that, wherever possible, the use of an aqueous solution/suspension be considered first. But as most oestrogen ligands or their metabolic precursors tend to be hydrophobic, the most common approach is to use a solution/suspension in oil (e.g. corn, peanut, sesame or olive oil). However, these oils have different caloric and fat content, thus the vehicle might affect total metabolisable energy (ME) intake, thereby potentially altering measured endpoints such as the uterine weight especially in the immature method (33). Thus, prior to the study, any vehicle to be used should be tested against controls without vehicles. Test chemicals can be dissolved in a minimal amount of 95 % ethanol or other appropriate solvents and diluted to final working concentrations in the test vehicle. The toxic characteristics of the solvent must be known, and should be tested in a separate solvent-only control group. If the test chemical is considered stable, gentle heating and vigorous mechanical action can be used to assist in dissolving the test chemical. The stability of the test chemical in the vehicle should be determined. If the test chemical is stable for the duration of the study, then one starting aliquot of the test chemical may be prepared, and the specified dosage dilutions prepared daily.

• 1. Dosage timing will depend of the model used (refer to paragraph 29 for the immature model and to paragraph 30 for ovx-adult model). Immature female rats are dosed with the test chemical daily for three consecutive days. A three-day treatment is also recommended for ovariectomised female rats but longer exposures are acceptable and may improve the detection of weakly active chemicals. With ovariectomised female mice, an application duration of 3 days should be sufficient without a significant advantage by an extension of up to seven days for strong oestrogen agonists, however, this relation was not demonstrated for weak oestrogens in the validation study (16) thus dosage should be extended up to 7 consecutive days in ovx-adult mice.The dose should be given at similar times each day. They should be adjusted as necessary to maintain a constant dose level in terms of animal body weight (e.g. mg of test chemical per kg of body weight per day). Regarding the test volume, its variability, on a body weight basis, should be minimised by adjusting the concentration of the dosing solution to ensure a constant volume on a body weight basis at all dose levels and for any route of administration.

• 1. When the test chemical is administered by gavage, this should be done in a single daily dose to the animals using a stomach tube or a suitable intubation cannula. The maximum volume of liquid that can be administered at one time depends on the size of the test animal. Local animal care guidelines should be followed, but the volume should not exceed 5 ml/kg body weight, except in the case of aqueous solutions where 10 ml/kg body weight may be used.

• 1. When the test chemical is administered by subcutaneous injection, this should be done in a single daily dose. Doses should be administered to the dorsoscapular or lumbar regions via sterile needle (e.g. 23- or 25-gauge) and a tuberculin syringe. Shaving the injection site is optional. Any losses, leakage at the injection site or incomplete dosing should be recorded. The total volume injected per rat per day should not exceed 5 ml/kg body weight, divided into 2 injection sites, except in the case of aqueous solutions where 10 ml/kg body weight may be used.

Observations

General and clinical observations

• 1. General clinical observations should be made at least once a day and more frequently when signs of toxicity are observed. Observations should be carried out preferably at the same time(s) each day and considering the period of anticipated peak effects after dosing. All animals are to be observed for mortality, morbidity and general clinical signs such as changes in behaviour, skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern).

Body weight and food consumption

• 1. All animals should be weighed daily to the nearest 0,1 g, starting just prior to initiation of treatment i.e. when the animals are allocated into groups. As an optional measurement, the amount of food consumed during the treatment period may be measured per cage by weighing the feeders. The food consumption results should be expressed in grams per rat per day.

Dissection and measurement of uterus weight

• 1. Twenty-four hours after the last treatment, the rats will be humanely killed. Ideally, the necropsy order will be randomised across groups to avoid progression directly up or down dose groups that could subtly affect the data. The bioassay objective is to measure both the wet and blotted uterus weights. The wet weight includes the uterus and the luminal fluid contents. The blotted weight is measured after the luminal contents of the uterus have been expressed and removed.

• 1. Before dissection the vagina will be examined for opening status in immature animals. The dissection procedure begins by opening the abdominal wall starting at the pubic symphysis. Then, uterine horn and ovaries, if present, are detached from the dorsal abdominal wall. The urinary bladder and ureters are removed from the ventral and lateral side of uterus and vagina. Fibrous adhesion between the rectum and the vagina is detached until the junction of vaginal orifice and perineal skin can be identified. The uterus and vagina are detached from the body by incising the vaginal wall just above the junction between perineal skin as shown in Figure 2. The uterus should be detached from the body wall by gently cutting the uterine mesentery at the point of its attachment along the full length of the dorsolateral aspect of each uterine horn. Once removed from the body, uterine handling should be sufficiently rapid to avoid desiccation of the tissues. Loss of weight due to desiccation becomes more important with small tissues such as the uterus (23). If ovaries are present, the ovaries are removed at the oviduct avoiding loss of luminal fluid from the uterine horn. If the animal has been ovariectomised, the stubs should be examined for the presence of any ovarian tissue. Excess fat and connective tissue should be trimmed away. The vagina is removed from the uterus just below the cervix so that the cervix remains with the uterine body as shown in Figure 2.

• 1. Each uterus should be transferred to a uniquely marked and weighed container (e.g. a petri-dish or plastic weight boat) with continuing care to avoid desiccation before weighing (e.g. filter paper slightly dampened with saline may be placed in the container). The uterus with luminal fluid will be weighed to the nearest 0,1 mg (wet uterine weight).

• 1. Each uterus will then be individually processed to remove the luminal fluid. Both uterine horns will be pierced or cut longitudinally. The uterus will be placed on lightly moistened filter paper (e.g. Whatman No 3) and gently pressed with a second piece of lightly moistened filter paper to completely remove the luminal fluid. The uterus without the luminal contents will be weighed to the nearest 0,1 mg (blotted uterine weight).

• 1. The uterus weight at termination can be used to ensure that the appropriate age in the immature intact rat was not exceeded, however, the historical data of the rat strain used by the laboratory are decisive in this respect (see paragraph 56 for interpretation of the results).

Optional investigations

• 1. After weighing, the uterus may be fixed in 10 % neutral buffered formalin to be examined histopathologically after Haematoxylin & Eosin (HE)-staining. The vagina may be investigated accordingly (see paragraph 9). In addition, morphometric measurement of endometrial epithelium may be done for quantitative comparison.

DATA AND REPORTING

Data

• 1. Study data should include:

• — the number of animals at the start of the assay,

• — the number and identity of animals found dead during the assay or killed for humane reasons and the date and time of any death or humane kill,

• — the number and identity of animals showing signs of toxicity, and a description of the signs of toxicity observed, including time of onset, duration, and severity of any toxic effects, and

• — the number and identity of animals showing any lesions and a description of the type of lesions.

• 1. Individual animal data should be recorded for the body weights, the wet uterine weight, and the blotted uterine weight. One-tailed statistical analyses for agonists should be used to determine whether the administration of a test chemical resulted in a statistically significant (p < 0,05) increase in the uterine weight. Appropriate statistical analyses should be carried out to test for treatment related changes in blotted and wet uterine weight. For example, the data may be evaluated by an analysis of covariance (ANCOVA) approach with body weight at necropsy as the co-variable. A variance-stabilising logarithmic transformation may be carried out on the uterine data prior to the data analysis. Dunnett and Hsu's test are appropriate for making pair wise comparisons of each dosed group to vehicle controls and to calculate the confidence intervals. Studentised residual plots can be used to detect possible outliers and to assess homogeneity of variances. These procedures were applied in the OECD validation programme using the PROC GLM in the Statistical Analysis System (SAS Institute, Cary, NC), version 8 (6)(7).

• 1. A final report shall include:

Testing facility:

• — Responsible personnel and their study responsibilities

• — Data from the Baseline Positive Control Test and periodic positive control data (see paragraphs 26 and 27)

Test chemical:

• — Characterisation of test chemicals

• — Physical nature and where relevant physicochemical properties

• — Method and frequency of preparation of dilutions

• — Any data generated on stability

• — Any analyses of dosing solutions

Vehicle:

• — Characterisation of test vehicle (nature, supplier and lot)

• — Justification of choice of vehicle (if other than water)

Test animals:

• — Species and strain and justification for their choice

• — Supplier and specific supplier facility

• — Age on supply with birth date

• — If immature animals, whether or not supplied with dam or foster dam and date of weaning

• — Details of animal acclimatisation procedure

• — Number of animals per cage

• — Detail and method of individual animal and group identification

Assay Conditions:

• — Details of randomisation process (i.e. method used)

• — Rationale for dose selection

• — Details of test chemical formulation, its achieved concentrations, stability and homogeneity

• — Details of test chemical administration and rationale for the choice of exposure route

• — Diet (name, type, supplier, content, and, if known, phyto-oestrogen levels)

• — Water source (e.g. tap water or filtered water) and supply (by tubing from a large container, in bottles, etc.)

• — Bedding (name, type, supplier, content)

• — Record of caging conditions, lighting interval, room temperature and humidity, room cleaning

• — Detailed description of necropsy and uterine weighing procedures

• — Description of statistical procedures

Results

For individual animals:

• — All daily individual body weights (from allocation into groups through necropsy) (to the nearest 0,1 g)

• — Age of each animal (in days counting day of birth as day 0) when administration of test chemical begins

• — Date and time of each dose administration

• — Calculated volume and dosage administered and observations of any dosage losses during or after administration

• — Daily record of status of animal, including relevant symptoms and observations

• — Suspected cause of death (if found during study in moribund state or dead)

• — Date and time of humane killing with time interval to last dosing

• — Wet uterine weight (to the nearest 0,1 mg) and any observations of luminal fluid losses during dissection and preparation for weighing

• — Blotted uterine weight (to the nearest 0,1 mg)

For each group of animals:

• — Mean daily body weights (to the nearest 0,1 g) and standard deviations (from allocation into groups through necropsy)

• — Mean wet uterine weights and mean blotted uterine weights (to the nearest 0,1 mg) and standard deviations

• — If measured, daily food consumption (calculated as grams of food consumed per animal)

• — The results of statistical analyses comparing both the wet and blotted uterine weights of treated groups relative to the same measures in the vehicle control groups.

• — The results of statistical analysis comparing the total body weight and the body weight gain of treated groups relative to the same measures in the vehicle control groups.

• 1. Summary of the important guidance facts of the test method

	Rat	Mice
Animals	Animals	Animals
Strain	Commonly used laboratory rodent strain	Commonly used laboratory rodent strain
Number of animals	A minimum of 6 animals per dose group	A minimum of 6 animals per dose group
Number of groups	A minimum of 2 test groups (see paragraph 33 for guidance) and a negative control group For guidance on positive control groups see paragraphs 26 and 27	A minimum of 2 test groups (see paragraph 33 for guidance) and a negative control group For guidance on positive control groups see paragraphs 26 and 27
Housing and feeding conditions	Housing and feeding conditions	Housing and feeding conditions
T° in animal room	22 °C ± 3 °C	22 °C ± 3 °C
Relative humidity	50-60 % and not below 30 % or above 70 %	50-60 % and not below 30 % or above 70 %
Daily lighting sequence	12 hours light, 12 hours dark	12 hours light, 12 hours dark
Diet and drinking water	Ad libitum	Ad libitum
Housing	Individually or in groups of up to three animals (social group housing is recommended for immature animals)	Individually or in groups of up to three animals (social group housing is recommended for immature animals)
Diet and bedding	Low level of phyto-oestrogens recommended in diet and bedding	Low level of phyto-oestrogens recommended in diet and bedding
Protocol	Protocol	Protocol
Method	Immature non-ovariectomised method (the preferred one). Ovariectomised adult female method	Ovariectomised adult female method
Age of dosing for immature animals	PND 18 at the earliest. Dosing should be completed prior to PND 25	Not relevant under the scope of the current test method.
Age of ovariectomy	Between 6 and 8 weeks of age.	Between 6 and 8 weeks of age.
Age of dosing for ovariectomised animals	A minimum of 14 days should elapse between ovariectomy and the 1st day of administration.	A minimum of 7 days should elapse between ovariectomy and the 1st day of administration.
Body weight	Body weight variation should be minimal and not exceed ± 20 % of the mean weight.	Body weight variation should be minimal and not exceed ± 20 % of the mean weight.
Dosing	Dosing	Dosing
Route of administration	Oral gavage or subcutaneous injection	Oral gavage or subcutaneous injection
Frequency of administration	Single daily dose	Single daily dose
Volume amount for gavage and injection	≤ 5 ml/kg body weight (or up to 10 ml/kg body weight in case of aqueous solutions) (in 2 injection sites for subcutaneous route)	≤ 5 ml/kg body weight (or up to 10 ml/kg body weight in case of aqueous solutions) (in 2 injection sites for subcutaneous route)
Duration of administration	3 consecutive days for immature model Minimum of 3 consecutive days for the OVX model	7 consecutive days for the OVX model
Time of necropsy	Approximately 24 hours after the last dose	Approximately 24 hours after the last dose
Results	Results	Results
Positive response	Statistically significant increase of the mean uterus weight (wet and/or blotted)	Statistically significant increase of the mean uterus weight (wet and/or blotted)
Reference oestrogen	17α-ethinyl estradiol	17α-ethinyl estradiol

GUIDANCE FOR THE INTERPRETATION AND ACCEPTANCE OF THE RESULTS

• 1. In general, a test for oestrogenicity should be considered positive if there is a statistically significant increase in uterine weight (p < 0,05) at least at the high dose level as compared to the solvent control group. A positive result is further supported by the demonstration of a biologically plausible relationship between the dose and the magnitude of the response, bearing in mind that overlapping oestrogenic and antioestrogenic activities of the test chemical may affect the shape of the dose-response curve.

• 1. Care must be taken in order not to exceed the maximum tolerated dose to allow a meaningful interpretation of the data. Reduction of body weight, clinical signs and other findings should be thoroughly assessed in this respect.

• 1. An important consideration for the acceptance of the data from the Uterotrophic Bioassay is the uterine weights of the vehicle control group. High control values may compromise the responsiveness of the bioassay and the ability to detect very weak oestrogen agonists. Literature reviews and the data generated during the validation of the Uterotrophic Bioassay suggest that instances of high control means do occur spontaneously, particularly in immature animals (2)(3)(6)(9). As the uterine weight of immature rats depends on many variables like strain or body weight, no definitive upper limit for the uterine weight can be given. As a guide, if blotted uterine weights in immature control rats are comprised between 40 and 45 mg, results should be considered as suspicious and uterine weights above 45 mg may lead to rerun the test. However, this needs to be considered on a case by case basis (3)(6)(8). When testing in adult rats incomplete ovariectomy will leave ovarian tissue that can produce endogenous oestrogen and retard the regression of the uterine weight.

• 1. Blotted vehicle control uterine weights less than 0,09 % of body weight for immature female rats and less than 0,04 % for ovariectomised young adult females appear to yield acceptable results (see Table 31 (2)). If the control uterine weights are greater than these numbers, various factors should be scrutinised including the age of the animals, proper ovariectomy, dietary phyto-oestrogens, and so on, and a negative assay result (no indication for oestrogenic activity) should be used with caution.

• 1. Historical data for vehicle control groups should be maintained in the laboratory. Historical data for responses to positive reference oestrogens, such as 17a-ethinyl estradiol, should also be maintained in the laboratory. Laboratories may also test the response to known weak oestrogen agonists. All these data can be compared to available data (2)(3)(4)(5)(6)(7)(8) to ensure that the laboratory's methods yield sufficient sensitivity.

• 1. The blotted uterine weights showed less variability in the course of the OECD validation study than the wet uterine weights (6)(7). However, a significant response in either measure would indicate that the test chemical is positive for oestrogenic activity.

• 1. The uterotrophic response is not entirely of oestrogenic origin, however, a positive result of the Uterotrophic Bioassay should generally be interpreted as evidence for oestrogenic potential in vivo, and should normally initiate actions for further clarification (see paragraph 9 and the ‘OECD Conceptual Framework for the Testing and Assessment of Endocrine Disrupting Chemicals’, Annex 2).

Figure 1

Schematic diagram showing the surgical removal of the ovaries

Image omitted; see official source

Text of image

Ovary

Oviduct

Cut here

Uterus

Incision

Mesometrium, vasculature and fat pad not shown

The procedure begins by opening dorso-lateral abdominal wall at the mid-point between the costal inferior border and the iliac crest, and a few millimetres lateral to the lateral margin of the lumbar muscle. Within the abdominal cavity, the ovaries should be located. On an aseptic field, the ovaries are then physically removed from the abdominal cavity, a ligature placed between the ovary and uterus to control bleeding, and the ovary detached by incision above the ligature at the junction of the oviduct and each uterine horn. After confirming that no significant bleeding persists, the abdominal wall should be closed by suture, and the skin closed, e.g. by autoclips or suture. The animals should be allowed to recover and the uterus weight to regress for a minimum of 14 days before use.

Figure 2

The removal and preparation of the uterine tissues for weight measurement.

Image omitted; see official source

Text of image

UTERINE WEIGHT

Disconnection line at necropsy

The procedure begins by opening the abdominal wall at the pubic symphysis. Then, each ovary, if present and uterine horn is detached from the dorsal abdominal wall. Urinary bladder and ureters are removed from the ventral and lateral side of uterus and vagina. Fibrous adhesion between the rectum and the vagina are detached until the junction of vaginal orifice and perineal skin can be identified. The uterus and vagina are detached from the body by incising the vaginal wall just above the junction between perineal skin as shown in the figure. The uterus should be detached from the body wall by gently cutting the uterine mesentery at the point of its attachment along the full length of the dorsolateral aspect of each uterine horn. After removal from the body, the excess fat and connective tissue is trimmed away. If ovaries are present, the ovaries are removed at the oviduct avoiding loss of luminal fluid from the uterine horn. If the animal has been ovarectomised, the stubs should be examined for the presence of any ovarian tissue. The vagina is removed from the uterus just below the cervix so that the cervix remains with the uterine body as shown in the figure. The uterus can then be weighed.

Appendix 1

DEFINITIONS:

Antioestrogenicity is the capability of a chemical to suppress the action of estradiol 17ß in a mammalian organism.

Chemical means a substance or a mixture.

Date of birth is postnatal day 0.

Dosage is a general term comprising of dose, its frequency and the duration of dosing.

Dose is the amount of test chemical administered. For the Uterotrophic Bioassay, the dose is expressed as weight of test chemical per unit body weight of test animal per day (e.g. mg/kg body weight/day).

Maximum Tolerable Dose (MTD) is the highest amount of a chemical that, when introduced into the body does not kill test animals (denoted by LD0) (IUPAC, 1993)

Oestrogenicity is the capability of a chemical to act like estradiol 17ß in a mammalian organism.

Postnatal day X is the Xth day of life after the day of birth.

Sensitivity is the proportion of all positive/active chemicals that are correctly classified by the test. It is a measure of accuracy for a test method that produces categorical results, and is an important consideration in assessing the relevance of a test method.

Specificity is the proportion of all negative/inactive chemicals that are correctly classified by the test. It is a measure of accuracy for a test method that produces categorical results and is an important consideration in assessing the relevance of a test method.

Test chemical means any substance or mixture tested using this test method.

Uterotrophic is a term used to describe a positive influence on the growth of uterine tissues.

Validation is a scientific process designed to characterise the operational requirements and limitations of a test method and to demonstrate its reliability and relevance for a particular purpose.

Appendix 2

Image omitted; see official source

Text of image

Note: Document prepared by the Secretariat of the Test Guidelines Programme based on the agreement reached at the 6th Meeting of the EDTA Task Force

OECD Conceptual Framework for the Testing and Assessment of Endocrine Disrupting Chemicals

Level 1

Sorting & prioritization based upon existing information

Level 2

In vitro assays providing mechanistic data

Level 3

In vivo assays providing data about single endocrine

Mechanisms and effects

Level 4

In vivo assays providing data about multiple endocrine

Mechanisms and effects

Level 5

In vivo assays providing data on effects from endocrine & other mechanisms

— physical & chemical properties, e.g., MW, reactivity, volatility, biodegradability,

— human & environmental exposure, e.g., production volume, release, use patterns

— hazard, e.g., available toxicological data

— ER, AR, TR receptor binding affinity

— Transcriptional activation

— Aromatase and steroidogenesis in vitro

— Aryl hydrocarbon receptor recognition/binding

— QSARs

— High Through Put Prescreens

— Thyroid function

— Fish hepatocyte VTG assay

— Others (as appropriate)

— Uterotrophic assay (estrogenic related)

— Hershberger assay (androgenic related)

— Non -receptor mediated hormone function

— Others (e.g. thyroid)

— enhanced OECD 407 (endpoints based on endocrine mechanisms)

— male and female pubertal assays

— adult intact male assay

— 1-generation assay (TG415 enhanced)1

— 2-generation assay (TG416 enhanced)1

— reproductive screening test (TG421 enhanced)1

— combined 28 day/reproduction screening test (TG 422 enhanced)1

1 Potential enhancements will be considered by VMG mamm

— Fish VTG (vitellogenin) assay (estrogenic related)

— Fish gonadal histopathology assay

— Frog metamorphosis assay

— Partial and full life cycle assays in fish, birds, amphibians & invertebrates (developmental and reproduction)

VMG mamm: Validation Management Group on Mammalian Testing and Assessment

NOTES TO THE FRAMEWORK:

• Note 1: Entering at all levels and exiting at all levels is possible and depends upon the nature of existing information needs for hazard and risk assessment purposes

• Note 2: In level 5, ecotoxicology should include endpoints that indicate mechanisms of adverse effects, and potential population damage

• Note 3: When a multimodal model covers several of the single endpoint assays, that model would replace the use of those single endpoint assays

• Note 4: The assessment of each chemical should be based on a case by case basis, taking into account all available information, bearing in mind the function of the framework levels.

• Note 5: The framework should not be considered as all inclusive at the present time. At levels 3, 4 and 5 it includes assays that are either available or for which validation is under way. With respect to the latter, these are provisionally included. Once developed and validated, they will be formally added to the framework.

• Note 6: Level 5 should not be considered as including definitive tests only. Tests included at that level are considered to contribute to general hazard and risk assessment.

LITERATURE

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B.55   HERSHBERGER BIOASSAY IN RATS: A SHORT-TERM SCREENING ASSAY FOR (ANTI)ANDROGENIC PROPERTIES

INTRODUCTION