# . 

# Nc~ro OFPT-~0)3-0010 P.O . Box 331, Millwood, VA 22646 (540) 83i-1602 

# Task Force 

# u~qiiiuumiuupup;~ii~p~,gi; April 15, 2010 Document Control Office (7407) Office ofPollution Prevention andToxics Room6428 Environmental Protection Agency 1200 Pennsylvania Ave, NW Washington, DC 20460



## Re: Testing Consent Order for Ethylene Dichloride; Request for Modification ofEnforceable Consent Agreement (Docket No. OPPT-2003-0010) Dear Sirs: 

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The HAPTask Force requests a modification to the Enforceable Consent Agreement for Ethylene Dichloride (the "ECA"), 68 Fed. Reg. 33125 (June 3, 2003) . Six copies ofthis request are submitted. Last year, Table 1 to the ECA, which sets forth required testing, test standards, and reporting requirements, was amendedto provide revised deadlines. 74 Fed. Reg. 36196 (July 22, 2009) . Since that time, the Task Force has discussed with EPAstaffthe possibility offurther revising Table 1 so that the required oral subchronic neurotoxicity and reproductive toxicity studies could be conducted under a single combined protocol . This protocol wouldfollow the OECDguideline being developed for an extended onegeneration reproductive toxicity study, as proposed by the ILSI-HESI Agricultural Chemical Safety Assessment Task Group, see Cooper, R .L., Lamb, J.C., Barlow, S.1VL, Bentley, K

., Brady, A

.M ., Doerrer, N

.G ., Eisenbrandt, D

.L., Fenner-Crisp, P.A., Hines, R .N ., Irvine, L., Kimmel, C.A., Koeter, H ., Li, A .A., Makris, S.L., Sheets, L., Speijers, G .J.A. and Whitby, K

., Atiered approach to life stages testing for agricultural chemical safety assessment, Crit. Rev. Toxicol . 36:69-98 (2006) . Ifno final OECDguideline has been issued by thetime the study must be initiated to meet the agreed final report date, the Task Force would be content to conduct the study in accordance with the October 28, 2009 draft proposal, whichis enclosed. Both options are shown below as alternative test standards to avoid further delays in EPA's internal review . The deadlines for final reports are left open in Table 1 because I understand that, as an initial matter, EPAwill want to reviewand approve a proposed protocol based on the 

# CONTAINS NOCBI :

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### October 28, 2009 draft proposed guideline for the extended one-generation reproductive

toxicity study. TheTask Force will submit a proposed protocol byJuly 2010 . 

## As revised, Table 1 would read as follows (redline shown for your convenience) :

Table I. -Required Testing, Test Standards, and Reporting Requirements for EDC Deadline for final report' Testing segment Required testing Test standard (months) Tier II testing and/or extrapolation Subchronic toxicity route-to-route ECAappendix C.2 and C.6 12 reporting extrapolation ofdose-response (oral Tier II testing to inhalation) of a study reported by Daniel, et al ., (1994) OLCDTest Guideline / _ILSIHESI Aericultural Chemical Saf'etvAssessment Task Group Proposal for Extended OneGeneration Reproductive Toxicitv Studv (October 28, 2009 draft), see Cooper, R.1,., Lamb.J .C. Barlow S h1 Bentley. K., Bradv,

~A .M ., Subchronic neurotoxici md. (oral) .. . Doerrer, N.G . LiV ~enbrandt, reproductive toYicitr (oral) tcomhinedt D1 ., Fenner-Crisp, P.A., llines, - R .N.. Irvine, L., Kimmel, C .A., Kocter, H

., I,i, A .A., Makris. S.L.. Sheets. L., Speijers, G ..1 .A. I and Whitby, K. A tiered I approach to life stages testina ~I for agricultural chemical sal'etv ' assessment, Crit. Rev.'I'oxicol . li 36:69-98 (2006Z49-(.-klt- ;r_~,~=°-.r .3'~*am ,~;t~' kGA .,ppondix-F~'~

. Subchronic neurotoxicity route-to- , ECAappendix C

.3 andC .6 -24- routeextrapolation of dose-response (oral Tier II testing to inhalation)

P " ,AU, r. . . :t, . r. .,I , 40 0` 14

. 70044 0

_ 2S i43 14 ~;A , , : l: ., 14

, 7 ` Reproductive toxicity route-to-route ECAappendix C.4 and C.6 ?S-- extrapolation ofdose-response (oral data to inhalation, including Tier II testingand extant studies reported by Alumot,et al ., (1976), Rao, et al ., (1980), and Lane, et al ., (1982)) 'Numberofmonths after the date of publication ofthis Federal Register document,which announces that EPA has concludedthe EPA ProgramReview, when thefinal report is due. In addition, every 6months fromthe effective date of the Order until theend ofthe ECA testing program, interimreports describing the status of all testingto be performed under this ECAmust be submitted by the Companies to EPA .

### I look forwardto hearing fromyou. Sincerely yours, Peter E. Voytek; Ph.D'. Manager 

## Enclosures cc: J. Schaeffer W . Caffey Norman, Esq.

## Draft version 28, October 2009



# OECD GUIDELINE FORTHE TESTINGOF CHEMICALS Draft Proposal for an Extended One-Generation Reproductive Toxicity Study INTRODUCTION 1 . This Test Guideline (TG) is based on the International Life Science Institute (ILSI)-Health and Environmental Sciences Institute (HESI), Agricultural Chemical Safety Assessment (ACSA .) Technical Committee proposal for a life stage F, extended one generation reproductive study as published in Cooper et al ., 2006 [1] . Several improvements and clarifications have been made to the study design to provide flexibility and to stress the importance of starting with existing knowledge, while using in-life observations to guide and tailor the testing . This guideline provides a detailed description of the operational conduct of an Extended One-Generation Reproductive Toxicity Study and lists a set of endpoints that may be used to determine the need to produce an FZ generation. The TG describes three cohorts of F, animals: 

### " Cohort 1 : assesses reproductive/developmental endpoints; this cohort may be extended to include an Fz generation, depending on available data including observations during the in-life part ofthe study. " Cohort 2: assesses the potential impact of chemical exposure on the developing nervous system . " Cohort 3: assesses the potential impact of chemical exposure on the developing immune system . 

### INITIAL CONSIDERATIONS ANDOBJECTIVES 2 . The main objective of the Extended One-Generation Reproductive Toxicity Study is to evaluate specific life stages not covered by other types oftoxicity studies and test for effects that may occur as a result of pre- and postnatal chemical exposure. For reproductive endpoints, it is envisaged that, as a first step, available information fromrepeat-dose studies (including screening reproductive toxicity studies, e .g. TG 422), or short termendocrine disrupter screening assays, (e.g. Uterotrophic assay - TG 440 - and Hershberger assay - TG 441 ) is used to detect effects on reproductive organs for males and females. This would include spermatogenesis (testicular histopathology) for males and oestrous cycles, follicle counts/oocyte maturation and ovarian integrity (histopathology) for females. The Extended One-Generation Reproductive Toxicity Study then serves as a test for reproductive endpoints that require the interaction of males with females, females with conceptus, and females with offspring and the F1 generation until after sexual maturity (see supporting guidance ) 3. The TG is designed to provide an evaluation of the pre- and postnatal effects of chemicals on development as well as a thorough evaluation of systemic toxicity in pregnant and lactating females and young and adult offspring. Detailed examination of key developmental endpoints, such as offspring viability, neonatal health, developmental status at birth, and physical and functional development until adulthood, is expected to identify specific target organs in the

### offspring. In addition, the study will provide and/or confirminformation about the effects ofatest substance on the integrity and performance of the adult male and female reproductive systems. Specifically, but not exclusively, the following parameters are considered: gonadal function, the oestrous cycle, epididymal sperm maturation, mating behaviour, conception, pregnancy, parturition, and lactation. Furthermore, the information obtained from the developmental neurotoxicity and developmental immunotoxicity assessments will characterize potential effects in those systems. The data derived fromthese tests, , should allowthe determination of NcObserved Adverse Effect Levels (NOAELs), Lowest Observed Adverse Effect Levels (LOAELs,) and/or benchmark doses for the various endpoints and/or be used to characterize effects detected in previous repeat-dose studies and/or serve as a guide for subsequent testing. 4 . A schematic drawing of the protocol is presented in Figure 1 . The test substance is administered continuously in graduated doses to several groups of sexually mature males and females. This parental (P) generation is dosed for a defined pre-mating period (selected based on the available information for the test substance; but for a minimum of twoweeks) and a two-week mating period. P males are further treated at least until weaning of the F, (i .e. a minimumof 10 weeks of treatment) . They may be treated for longer if there is a need to clarify effects on reproduction. Treatment of the P females is continued during pregnancy and lactation until termination after the weaning of their litters (i .e. 8-10 weeks of treatment) . The F, offspring receive further treatment with the test substance fromweaning to adulthood until postnatal day (PND) 70 or 90, depending on their cohort assignment. Ifa second generation is triggered, the F, offspring will be maintained on treatment until weaning of the Fz, or until termination of the study. 5 . Clinical observations and pathology examinations are performed on all animals for signs of toxicity, with special emphasis on the integrity and performance of the male and female reproductive systems and the health, growth, development and function of the offspring. At weaning, selected offspring are assigned to specific subgroups (cohorts 1-3, see paragraphs 30to 32 and Figures I &2) for further investigations, including sexual maturation, reproductive organ integrity and function, neurological and behavioural endpoints, and immune functions. 

### 6. In conducting the study, the guiding principles and considerations outlined in the OECD Guidance Document n° 19 on the recognition, assessment, and use of clinical signs as humane endpoints for experimental animals used in safety evaluations [35] should be followed. 7. When a sufficient number of studies are available to ascertain the impact of this new stud;y design, the Test Guideline will be reviewed and ifnecessary revised in light ofexperience gained.Figure 1 : Scheme of the Extended One-Generation Reproductive Toxicity Study 

## Extended One-Generation Protocol

D osing Pre-mating T Mating ~ Post-mating 2weeks I 2weeks ~ 6weeks ------ - -----~--- ---- 2weeks ' 2weeks Pregnancy Lactation ------- - --- - ~ ---- [ In-utero F' ! development Pre-weaning - ---- - Necropsy Panimals 

# 1

Post-weaninig

Parental generation Cohort Desi gnation Animals/Cohort Sexual Approximate age at Maturation necro

p s y (weeks) 1 .A Reproductive 20 M +20 F Yes 13 Target is 16 Reproductive 20 M +20 F Yes 14 or 20 if triggered 20litters 2A Neurotoxicity 10 M +10F@ Yes g per group =B Neurotoxicity 10 M +10F@ No 3 3 Immunotoxicity 10 M +10F@ Yes g Surplus Spares No 3 4r one per litter and representative of 20 litters in total where possible



### DESCRIPTION OF THE METHOD/PREPARATIONS FORTHE TEST ANIMALS Selection ofanimal species and strain 8 . The choice of species for the reproductive toxicity test should be carefully considered in light of all available information. However, because of the extent of background data and the comparability to general toxicity tests, the rat is normally the preferred animal, and criteria and recommendations given in the protocol refer to this species. If another species is used., justification should be given and appropriate modifications to the protocol will be necessary. Strains with low

fecundity or a well-known high incidence of spontaneous developmental defects should not be used.

### Age, body weight and inclusion criteria 9. Healthy parental animals, which have not been subjected to previous experimental procedures, should be used. Both males and females should be studied and the females should be nulliparous and non-pregnant. The P animals should be sexually mature, of similar weight (within sex) at initiation of dosing, similar age (approximately 90 days) at mating, and representative of the species and strain under study. Animals should be acclimated for at least 5 days after arrival . The animals are randomly assigned to the control and treatment groups, in a manner which results in comparable mean body weight values among the groups (i .e. +/- 20%ofthe mean) . Housing and feeding conditions 

### 10 . The temperature in the experimental animal room should be 22 °C (+/- 3°) . Relative humidity should be between 30-70% with an ideal range of 50-60% . Artificial lighting should be set at 12 hours light, 12 hours dark. Conventional laboratory diets may be used with an unlimited supply of drinking water. Careful attention should be given to diet phytoestrogen content. Standardized, open-formula diets in which estrogenic substances have been reduced are recommended [2, 33] .The choice of diet may be influenced by the need to ensure a suitable admixture of a test substance when administered by this method. Content, homogeneity and stability of the test substance in the diets should be verified. The feed and drinking water should be regularly analysed for contaminants. Samples ofthe diet should be retained until finalisation of the report, in case the results necessitate a further analysis ofdiet ingredients. 11 . Animals should be caged in small groups of the same sex and treatment group. They may be housed individually to avoid possible injuries (e.g. males after the mating period) . Mating procedures should be carried out in suitable cages. After evidence ofcopulation, females that are presumed to be pregnant are housed separately in parturition or maternity cages where they are provided with appropriate and defined nesting materials. Litters are housed with their mothers until weaning. F, animals can be housed individually or in small groups of the same sex and treatment groupfromweaning to termination. Number and identification of animals 12. Normally, each test and control group should contain a sufficient number of mating pairs to yield at least 20pregnant females per dose group. The objective is to produce enough pregnancies to ensure a meaningful evaluation of the potential of the substance to affect fertility, pregnancy and maternal behaviour ofthe Pgeneration and growth and development of the F, offspring, from conception to maturity. Failure to achieve the desired number of pregnant animals does not necessarily invalidate the study and should be evaluated on a case-by-case basis, considering a possible causal relationship to the test substance. 13. Each P animal is assigned a unique identification number before dosing starts. If laboratory historical data suggest that a significant proportion of females may not showregular (4 or 5-day) oestrous cycles, then an assessment of oestrous cycles before start of treatment is advised. Alternatively, the group size may be increased to ensure that at least 20 females in each group would have regular (4 or 5-day) oestrous cycles at start of treatment. If Pfemales are monitored for oestrous cycles before treatment is started, the females should receive their identification before this procedure is initiated. All F, offspring are uniquely identified when neonates are first examined on postnatal day (PND) 0 or 1 . Records indicating the litter of origin should be maintained for all F, animals, and FZ animals where applicable, throughout the study.

### TEST SUBSTANCE Available information on the test substance 

### 14. The reviewof existing information is important for decisions on the route of administration, the choice of the vehicle, the selection ofanimal species, the selection ofdosages and potential modifications of the dosing schedule. Therefore, all the relevant available information on the test substance, i .e. physico-chemical, TK (including species-specific metabolism), toxicodynamic properties, structure-activity relationships (SARs), in vitro metabolic processes, results of previous toxicity studies and relevant information on structural analogues should be taken into consideration in planning the Extended One-Generation Reproductive Toxicity Study. Preliminary information on absorption, distribution, metabolismand elimination (ADME) and bioaccumulation may be derived from chemical structure, physico-chemical data, extent of plasma protein binding or TK studies, while results from toxicity studies give additional information, e.g

. on NOAEL, metabolismor induction ofmetabolism . Consideration of toxicokinetic data 15. Although not required, TK data from previously conducted dose range-finding or other studies are extremely useful in the planning of the study design, selection of dose levels and interpretation of results. Of particular utility are data which: 1) verify exposure of developing fetuses and pups to the test compound (orrelevant metabolites), 2) provide an estimate of internal dosimetry, and 3) evaluate for potential dose-dependent saturation of kinetic processes. Additional TK data, such as metabolite profiles, concentration-time courses, etc. should also be considered, if they are available. Supplemental TK data may also be collected during the main study, provided that it does not interfere with the collection and interpretation of the main study endpoints. As a general guide, the following TKdata set would be useful in planning the Extended OneGeneration Reproductive Toxicity Study: 

### " Late pregnancy (e.g., GD20) - maternal blood and foetal blood " Mid-lactation (PND 10) - maternal blood, pup blood and/or milk " Early post-weaning (e.g., PND28) - weanling blood samples Flexibility should be employed in determining the specific analytes (e.g. parent compound and/or metabolites) and sampling scheme. For example, the number and timing ofsample collection on a given sampling day will be dependent upon route of exposure and prior knowledge of T1C properties in non-pregnant animals. For dietary studies, sampling at a single consistent time on each of these days is sufficient, whereas gavage dosing may warrant additional sampling times to obtain a better estimate of the range of internal doses. However, it is not necessary to generate a full concentration time-course on any of the sampling days. If necessary, blood can be pooled by sex within litters for fetal and neonatal analyses. Route ofadministration 16. Selection of the route should take into consideration the route(s) most relevant for human exposure. Although the protocol is designed for administration of the test substance through the diet, it can be modified for administration by other routes (drinking water, gavage, inhalation, dermal), depending on the characteristics of the compound and the information required.

### 6 Choice of the vehicle 

### 17. Where necessary, the test substance is dissolved or suspended in a suitable vehicle. It is recommended that, where possible, the use ofan aqueous solution/suspension is considered first, followed by consideration of a solution/suspension in oil (e.g . corn oil). For vehicles other than water, the toxic characteristics of the vehicle should be known. Use of vehicles with potential intrinsic toxicity should be avoided (e.g., acetone, DMSO) . The stability of the test substance in the vehicle should be determined. Considerations should be given to the following characteristics if a vehicle or other additive is used to facilitate dosing: effects on the absorption, distribution, metabolism, or retention of the test substance; effects on the chemical properties of the test substance which may alter its toxic characteristics; and effects on the food or water consumption or the nutritional status ofthe animals. Dose Selection 18. Normally, the study should include at least three dose levels and a concurrent control . When selecting appropriate dose levels, the investigator should consider all available information ., including the dosing information fromprevious studies, TK data frompregnant or non-pregnan .t animals, the extent of lactational transfer, and estimates of human exposure. If TK data are available which indicate dose-dependent saturation of TK processes, care should be taken to avoid high dose levels which clearly exhibit saturation, provided ofcourse, that human exposures are expected to be well below the point ofsaturation. In such cases, the highest dose level should be at orjust slightly above the inflection point for transition to nonlinear TK behaviour. 

### 19. In the absence of relevant TK data, the dose levels should be based on toxic effects, unless limited by the physical/chemical nature ofthe test substance. Ifdose levels are based on toxicity, the highest dose should be chosen with the aimto induce some systemic toxicity, but not death or severe suffering ofthe animals. 20 . A descending sequence of dose levels should be selected in order to demonstrate any doserelated effect and to establish NOAELs or doses near the limit of detection that would allowfor derivation of a benchmark dose for the most sensitive endpoint(s) . To avoid large dose spacing between NOAELs and LOAELs, two- or four-fold intervals are frequently optimal . The addition ofa fourth test group is often preferable to using a very large interval (e.g., more than a factor of 10) between doses. 21 . Except for treatment with the test substance, animals in the control group are handled in an identical manner to the test group subjects. This group should be untreated or sham-treated or a vehicle-control group ifa vehicle is used in administering the test substance. If a vehicle is used, the control group should receive the vehicle in the highest volume used. Limit test 22. If there is no evidence oftoxicity at a dose of at least 1000 mg/kg body weight/day in repeatdose studies, or if toxicity would not be expected based upon data fromstructurally- and/or metabolically-related compounds, indicating similarity in the in vivo/in vitro metabolic properties, a study using several dose levels may not be necessary. In such cases, the Extended OneGeneration Reproductive Toxicity Study could be conducted using a control group and a single dose of at least 1000 mg/kg body weight/day. However, should evidence for reproductive or developmental toxicity be found at this limit dose, further studies at lower dose levels will be

### required to identify a NOAEL. These limit test considerations apply only when human exposure does not indicate the need for a higher dose level . PROCEDURES Exposure of offspring 23. Dietary exposure is the preferred method ofadministration. If gavage studies are performed, it should be noted that the pups will normally only receive test substance indirectly through the milk, until direct dosing commences for themat weaning . In diet or drinking water studies, the pups will additionally receive test substance directly when they commence eating for themselves during the last week of the lactation period. Modifications to the study design should be considered when excretion of the test substance in milk is poor and where there is lack of evidence for a continuous exposure of the offspring. In these cases, direct dosing of pups during the lactation period should be considered based on available TK information, offspring toxicity or changes in bio-markers [3, 4] . Careful consideration of benefits and disadvantages should be made prior to conducting direct dosing studies on nursingpups [5] . Dosing schedule and administration ofdoses 24. Some information on oestrous cycles, male and female reproductive tract histopathology and testicular/epididymal sperm analysis may be available fromprevious repeat-dose toxicity studies ofadequate duration. The duration of the pre-mating treatment in the Extended One-Generation Reproductive Toxicity Study therefore is aimed at the detection ofeffects on functional changes that may interfere with mating behaviour and fertilisation. The pre-mating treatment should be sufficiently long to achieve steady-state exposure conditions in P males and females. A2-week, pre-mating treatment forboth sexes is considered adequate in most cases. For females, this covers 3-4 complete oestrous cycles and should be sufficient to detect any adverse effects on cyclicity. For males, this is equivalent to the time required for epididymal transit of maturing spermatozoa and should allowthe detection of post-testicular effects on sperm(during the final stages of spermiation and epididymal sperm maturation) at mating. At the time of termination, when testicular and epididymal histopathology and analysis of spermparameters are scheduled, the )? and F, males, will have been exposed for at least one entirespermatogenic process [7, 8, 9, 10and supporting document] . 25. Pre-mating exposure scenarios for males could be adapted, if testicular toxicity (impairment of spermatogenesis) or effects on spermintegrity and function have been clearly identified in previous studies. Similarly, for females, known effects of the test substance on the oestrous cycle and thus sexual receptivity, mayjustify different pre-mating exposure scenarios. In special cases it may be acceptable that treatment of the P females is initiated only after a sperm-positive smear has been obtained (see supporting document) . 26. Once the pre-mating dosing period is established, the animals should be treated with the test substance continuously on a 7-days/week basis until necropsy. All animals should be dosed by the same method. Dosing should continue during the 2-week mating period and, for P females, throughout gestation and lactation up to the day of termination after weaning. Males should be treated in the same manner until termination at the time when the F, animals are weaned. For necropsy, priority should be given to females; they should be necropsied on the same/similar day of lactation. Necropsy of males can be spread over a larger number of days, depending on laboratory facilities . Unless already initiated during the lactation period, direct dosing of the

### selected F, males and females should begin at weaning and continue until scheduled necropsy, depending on cohort assigmnent (see Figure 2) . 27. For substances administered via the diet or drinking water, it is important to ensure that the quantities of the test substance involved do not interfere with normal nutrition or water balance. When the test substance is administered in the diet, either a constant dietary concentration (pprn) or a constant dose level in terms ofthe body weight of the animal may be employed; the option chosen should be specified. 28. When the test substance is administered by gavage, the volume ofliquid administered at one time should not exceed 1 mL/100 g body weight (0.4 mL/100g body weight is the maximumfor oil, e.g . corn oil) . Avolume of up to 2 mL/100 g body weight may be used for aqueous solutions in exceptional cases. Except for irritant or corrosive substances, which will normally reveal exacerbated effects with higher concentrations, variability in test volume should be minimised by adjusting the concentration to ensure a constant volume at all dose levels. The treatment should be given at similar times each day. The dose to each animal should normally be based on the most recent individual body weight determination and adjusted at least weekly in adult males and adult non-pregnant females, and every two days in pregnant females and F, animals when administered prior to weaning and during the 2 weeks following weaning. However, should TKdata indicate a lowplacental transfer of the test substance, the gavage dose during the last week of pregnancy may have to be adjusted to prevent administration of an excessively toxic dose to the dam . Females should not be treated by gavage, or any other route of treatment where the animal needs to be handled, on the day ofparturition; omission of test substance administration on that day :is preferable to a disturbance ofthe birth process. Mating 29. Each P female should be placed with a single, randomly selected, unrelated male fromthe same dose group (1 :1 pairing) until evidence ofcopulation is observed or 2 weeks have elapsed. If there are insufficient males, for example due to male death before pairing, then male(s) which have already mated may be paired(1 :1) with a second female(s) such that all females are paired. Day 0of pregnancy is defined as the day on which mating evidence is confirmed (a vaginal plug or spermare found) . Animals should be separated as soon as possible after evidence ofcopulation is observed. If mating has not occurred after 2 weeks, the animals should be separated without further opportunity for mating. Mating pairs should be clearly identified in the data. Litter size 

### 30 . On day 4 after birth, the size ofeach litter should be adjusted by eliminating extra pups by randomselection to yield, as nearly as possible, five males and five females per litter. Selective elimination of pups, i.e. based upon body weight, is not appropriate. Whenever the number of male or female pups prevents having five ofeach sex per litter, partial adjustment (for example, six males and four females) is acceptable. Adjustments are not appropriate for litters often pups or less. Selection of pups for post-weaning studies (see Figure 1) 31 . At weaning (around PND 21) pups fromall available litters up to 20 per dose group are selected for further examinations and maintained until sexual maturation (unless earlier testing is required) . Pups are selected randomly with the exception that obvious runts (animals with a body

### weight more than two standard deviations belowthe mean pup weight of the respective litter) should not be included as they are unlikely to be representative ofthe treatment group. On PND21, the selected F, pups are randomly assigned to one of three cohorts of animals as follows:Cohort 1 (1Aand 113) =Reproductive/developmental toxicity testing Cohort 2 (2A and 213) = Developmental neurotoxicity testing (PND21 or PND22 and adults Cohort 3 = Developmental immunotoxicity testing (PND56 +/- 3) 

### Cohort IA: One animal/sex/dose/litter (20/sex/dose) : priority selection for primary assessment of effects upon reproductive systems and of general toxicity. Cohort 113: One animal/sex/dose/litter (20/sex/dose) : priority selection for follow-up assessment of reproductive performance by mating F, animals, when triggered Cohort 2A: Total of 20 pups per group (10 males and 10 females per group; one male or one female per litter) are assigned for neurobehavioral testing followed by neurohistopathology assessment. Cohort 213: Total of 20 pups per group (lOmales and 10 females per group; one male or one female per litter) are assigned for Neurohistopathology at weaning (PND21 or PND22) . If there are insufficient numbers ofanimals, preference should be given to assign animals to Cohort 2A. Cohort 3: 10 males and 10 females per groupselected, one per litter (where possible) for a total of 20 pups per group . Additional pups may be required fromthe control group to act as positive control animals in the immune challenge assay. 32. Should there be an insufficient number of pups in a litter to serve all cohorts, the cohort 1 takes precedence as it can be extended to produce an Fz generation. Additional pups may be assigned to any of the cohorts in case of specific concern, i .e. if a chemical is suspected to be either a neurotoxicant, immunotoxicant or reproductive toxicant. These pups may be used for examinations at different timepoints, for the evaluation of supplementary endpoints, or to increase sample size in the cohort . Second mating of the Panimals 

### 33. A second mating is not normally recommended for the Panimals as it comes at the expense of losing important information on the number of implantation sites (and thus post-implantation and peri-natal loss data, indicators of a possible teratogenic potential) for the first litter. The need to verify or elucidate an effect in exposed females, would be served better by extending the study to include a mating of the F, generation. However, a second mating of the P males with untreated females is always an option to clarify equivocal findings or for further characterisation of effects on fertility observed in the first mating. IN-LIFE OBSERVATIONS Clinical Observations 34. For the Pand the selected F, animals a general clinical observation is made once a day. In the case ofgavage dosing, the timing of clinical observations should be prior to and post dosing (for

### 11) possible signs of toxicity associated with peak plasma concentration) . Pertinent behavioural changes, signs ofdifficult or prolonged parturition and all signs of toxicity are recorded. Twice daily, during the weekend once daily, all animals are observed formorbidity and mortality. 35. In addition, a more detailed examination of all Pand F, animals (after weaning) is conducted on a weekly basis and could conveniently be performed on an occasion when the animal is weighed, whichwould minimize handling stress. Observations should be carefully conducted and recorded using scoring systems that have been defined by the testing laboratory. Efforts should be made to ensure that variations in the test conditions are minimal . Signs noted should include, but not be limited to, changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g., lacrimation, piloerection, pupil size, unusual respiratory pattern) . Changes in gait, posture, response to handling, as well as the presence ofclonic or tonic movements, stereotypy (e.g. excessive grooming, repetitive circling) or bizarre behaviour (e.g. self-mutilation, walking backwards) should also be recorded. Body weight and food/water consumption 36. Panimals are weighed on the first day ofdosing and at least weekly thereafter. In addition, P females are weighed during lactation on the same days as the weighing of the pups in their litters. All F, animals are weighed individually at weaning (PND21) and at least weekly thereafter. Body weight is also recorded on the day when they attain puberty (completion of preputial separation or vaginal patency, between PND 30 and PND 50) . All animals are weighed at sacrifice. 37. During the study, food and water consumption (in the case of test substance administration in the drinking water) are recorded at least weekly on the same days as animal body weights (except during cohabitation) . The food consumption of each cage of F, animals is recorded weekly commencing fromselection to respective cohort . Oestrous cycles 38. Preliminary information oftest substance-related effects on the oestrous cycle may already be available from previous repeat-dose toxicity studies, and may be used in designing a test substance-specific protocol for the Extended One-Generation Reproductive Toxicity Study. Normally the assessment of oestrous cyclicity (by vaginal cytology) will start at the beginning of the treatment period and continue until confirmation of mating or the end of the 2-weeks mating period. If females have been screened for normal oestrous cycles before treatment, then it is useful to continue smearing as treatment starts, but if there is concern about non-specific effects at the start of treatment (such as an initial marked reduction in food consumption) then animals may be allowed to adapt to treatment for up to two weeks before the start of the 2-weeks smearing period leading into pairing. If the female treatment period is extended in this way (i .e. to a 4-weeks pre-mating treatment) then consideration should be made to purchasing animals younger and to extending the period of male treatment before pairing . When obtaining vaginal/cervical cells, care should be taken to avoid disturbance of mucosa and subsequently, the induction of pseudopregnancy [11, 12] . 39. Vaginal smears should be examined daily for all F, females in cohort IA, after the onset of vaginal patency, until the first cornified smears, in order to determine the time interval between these two events. Oestrous cycles for all F, females in cohort 1Ashould also be monitored for a period of two weeks commencing around PND75. In addition, should mating of the F, generation

### ll be necessary, the vaginal cytology in cohort 1B will be followed from the time of pairing until mating evidence is detected. Mating and pregnancy 

### 40 . In addition to the standard endpoints (body weight, food consumption, clinical observations including mortality/morbidity checks), the dates ofpairing, the date of insemination and the date . ofparturition are recorded and the precoital interval (pairing to insemination) and the duration of pregnancy (insemination to parturition) are calculated. The P females should be examined carefully at the time of expected parturition for any signs of dystocia. Any abnormalities in nesting behaviour or nursing performance should be recorded. 41 . The day on which parturition occurs is lactation day 0 (LD0) for the dam and postnatal day 0 (PND 0) for the offspring. Alternatively, all comparisons may be based on post-coital time to eliminate confounding of postnatal development data, by differences in the duration of pregnancy. This is especially important when the test substance exerts an influence on the duration of pregnancy . 

### Offspring parameters 42. Each litter should be examined as soon as possible after parturition (PND0 or 1) to establish the number and sex of pups, stillbirths, live births; and the presence of gross anomalies (externally visible abnormalities, including cleft palate; subcutaneous haemorrhages; abnormal skin colour or texture; presence of umbilical cord; lack of milk in stomach; presence of dried secretions) . In addition, the first clinical examination of the neonates should include a qualitative assessment of body temperature, state of activity and reaction to handling. Pups found dead on PND0 or at a later time should be examined for possible defects and cause of death. Live pups are counted and weighed individually on PND0 or PND 1, and regularly thereafter, e.g., at least on PND4, 7, 14, and 21 . Clinical examinations, as applicable for the age ofthe animals, should be repeated when the offspring are weighed, or more often if case specific findings have been made at birth. Signs noted could include, but may not be limited to, external abnormalities, changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity. Changes in gait, posture, response to handling as well as the presence of clonic or tonic movements, stereotypy or bizarre behaviour should also be recorded. 43. The anogenital distance (AGD) ofeach pup should be measured between PND0 and PND4. Pup body weight should be collected on the day the AGDis measured and the AGDshould be normalized to a measure of pup size, preferably the cube root of body weight [13] . The presence of nipples/arcolae in male pups should be checked on PND12 or 13. 44. All selected F, animals are evaluated daily for balano-preputial separation and vaginal patency, respectively commencing before the expected day for achievement of these endpoints to detect ifsexual maturation occurs early. Any abnormalities of genital organs, such as persistent vaginal thread, hypospadia or cleft penis, should be noted. Sexual maturity of F, females and males is compared to physical development by determining age and body weight at vaginal opening or balano-preputial separation, respectively [14] . Assessment of potential developmental neurotoxicity (cohort 2A and 2B) 45. Ten male and 10 female cohort 2A and 2B animals fromeach treatment group (1 male or 1 female per litter; all litters represented by at least 1 pup; randomly selected) should be used for neurotoxicity assessments. Cohort 2A animals should be subjected to auditory startle, functional

### 12 observational battery, motor activity, and neuropathology assessments. Efforts should be made to ensure that variations in all test conditions are minimal and are not systematically related to treatment. Among the variables that can affect behaviour are sound level (e.g., intermittent noise), temperature, humidity, lighting, odors, time ofday, and environmental distractions. Results of the neurotoxicity assays should be interpreted in relation to appropriate historical control reference ranges. Cohort 213animals should be used for neuropathology assessment on PND21 or PND22. 

### 46. Auditory startle test. An auditory startle test should be performed on PND 24 (tl day) using animals in cohort 2A. The day of testing should be counterbalanced across treated and control groups. Each session consists of 50 trials. In performing the auditory startle test, the mean response amplitude on each block of 10 trials (5 blocks of 10trials) should be determined, with test conditions optimized to produce intrasession habituation. These procedures should be consistent with OECDTG426. 47. At an appropriate time between PND63 and PND75, the cohort 2A F, animals are subjected to a functional observational battery and an automated test of motor activity. These procedures should be consistent with OECD TG 424 . The functional observational battery includes a thorough description of the subject's appearance, behavior, and functional integrity. This is assessed through observations in the home cage, after removal to a standard arena for observation (open field) where the animal is moving freely, and through manipulative tests. Testing should proceed fromthe least to the most interactive. Alist of measures is presented in Appendix A . All animals should be observed carefully by trained observers who are unaware of the animals' treatment status, using standardized procedures to minimize observer variability. Where possible, it is advisable that the same observer evaluates the animals in a given test. If this is not possible, some demonstration of inter-observer reliability is required. For each parameter in the behavioural testing battery, explicit operationally defined scales and scoring criteria are to be used. If possible, objective quantitative measures should be developed for observational endpoints which involve subjective ranking . For motor activity, each animal is tested individually. The test session should be long enough to demonstrate intra-session habituation for non-treated controls. Motor activity should be monitored by an automated activity recording apparatus which should be capable of detecting both increases and decreases in activity, (i .e., baseline activity as measured by the device should not be so lowas to preclude detection of decreases, nor so high as to preclude detection of increases in activity). Each device should be tested by standard procedures to ensure, to the extent possible, reliability of operation across devices and across days. To the extent possible, treatment groups should be balanced across devices. Treatment groups should be counter-balanced across test times to avoid confounding by circadian rhythms ofactivity. 

### 48. If existing information indicates the need for other functional testing (e.g., sensory, social ., cognitive), these should be integrated without compromising the integrity ofthe other evaluations conducted in the study. If this testing is performed in the same animals as used for standard auditory startle, FOB and motor activity testing, different tests should be scheduled to minimis~2 the risk of compromising the integrity of these tests. Supplemental procedures may be particularly useful when empirical observation, anticipated effects, or mechanistic/mode-of-actio:n indicate a specific type of neurotoxicity. Assessment of developmental immunotoxicity (cohort 3) 49. At PND56, plus or minus +-3 days ofage, 10 male and 10female cohort 3 animals fromeach treatment group (1 male or 1 female per litter; all litters represented by at least 1 pup; randomly selected) should be used to assess the primary IgMantibody response to a T cell dependent

### 13 antigen, such as Sheep Red Blood Cells (SRBC), or Keyhole Limpet Hemocyanin (KLH :) consistent with current immunotoxicity testing procedures [15, 16] . The response may be evaluated by counting specific plaque forming cells (PFC) in the spleen or by determining the titer of SRBC- or KLH-specific IgMantibody in the serum by ELISA, at the peak of the response. Responses typically peak four (PFC response) or five (ELISA) days after intravenous immunization. If the primary antibody response is assayed by counting plaque forming cells, it is permissible to evaluate subgroups of animals on separate days, provided that: subgroup immunization and sacrifice are timed so that PFCs are counted at the peak of the response; that subgroups contain an equal number of male and female offspring fromall dose groups, including controls; and that subgroups are evaluated at approximately the same postnatal age. Exposure to the test article will continue until the day before collecting spleens for the PFCresponse or serur,a for the ELISAassay. Assessment of potential reproductive toxicity (Triggered) 50 . Cohort 1B animals can be maintained on treatment beyond PND90 and bred to obtain a FZ generation if data from previous studies or early findings from this study raise concern for possible effects on reproduction or if equivocal effects on reproduction are obtained in the lP animals. Males and females of the same dose group should be cohabited (avoiding the pairing of siblings) for up to two weeks, beginning on or after PND90, but not exceeding PND 120 . Procedures should be similar to those for the Panimals, although it may suffice to terminate the litters on PND4 rather than followthemto weaning or beyond, depending on the concerns that triggered the extension of the study. The decision ofwhether a breeding of the F, generation is necessary should be based on a weight of the evidence approach that considers the nature and degree of the effects found in the P and F, generation, as well as other available pertinent information (e.g., mechanistic studies, subchronic toxicity studies) . See appendix B and the Guidance Document supporting this TG for a more detailed discussion oftriggers. 

### TERMINAL OBSERVATIONS Clinical biochemistry / Haematology 51 . Systemic effects should be monitored in P animals. Fasted blood samples froma defined site are taken fromten randomly selected P males and females per dose group at termination, stored under appropriate conditions and subjected to partial or full scale haematology, clinical biochemistry, assay of T4 and TSHor other examinations suggested by the known effect profile of the test substance (see supporting guidance) . The following haematological parameters should be examined: haematocrit, haemoglobin concentration, erythrocyte count, total and differential leukocyte count, platelet count and blood clotting time/potential . Investigations of plasma or serumshould include: glucose, total cholesterol, urea, creatinine, total protein, albumin, at least two enzymes indicative of hepatocellular effects (such as alanine aminotranferase, aspartate, aminotransferase, alkaline phosphatase, gamma glutamyl transpeptidase and sorbitol dehydrogenase)

. Measurements of additional enzymes and bile acids may provide useful information under certain circumstances. In addition, blood fromall animals may be taken and stored for possible analysis at a later time to help clarify equivocal effects or to generate internal exposure data. If a second mating of Panimals is not intended, the blood samples are obtained just prior to, or as part of, the procedure at scheduled sacrifice. In the case animals are retained, blood samples should be collected a fewdays before the animals are mated for the second time. Unless existing data fromrepeated dose studies indicate that the parameter is not affected by the test substance, urinalysis should be performed prior to terminationand the following parameters evaluated: appearance, volume, osmolality or specific gravity, pH, protein, glucose, blood and

### 114 blood cells, cell debris. Urine may also be collected to monitor excretion of test substance and/or metabolite(s) . 52. Systemic effects should be also monitored in F1 animals. Fasted blood samples froma defined site are taken fromten randomly selected cohort IA F, males and females per dose group alt termination, stored under appropriate conditions and subjected to standard clinical biochemistr},, including the assessment ofserumlevels for thyroid hormones (T4and TSH), haematology (total and differential leukocyte plus erythrocyte counts) and urinalysis assessments. 53. The surplus pups at PND 4 are subject to gross necropsy and consideration given to measuring serum

thyroid hormone (T4) concentrations. If necessary, neonatal (PND 4) blood can be pooled by litters for biochemical/thyroid hormone analyses. Blood is also collected for T4and TSH analysis fromweanlings subject to gross necropsy on PND 22 (F, pups not selected for cohorts) . 

### Spermparameters 54. Sperm

parameters may have already been examined as part of systemic toxicity studies. Such data may be helpful in the planning of the Extended One-Generation Reproductive Toxicity Study as well as in the interpretation of the results. If existing data ofsuitable exposure duration showthat spermparameters are not affected by the test substance and if the mating of the P generation does not produce evidence for impaired sperm function, the analysis of sperm parameters is optional for P males. However, in the case that clear or equivocal effects on reproduction (e.g. reductions in sperm-positive or fertile matings, decreased litter size, histopathologic evidence of impaired spermatogenesis from previous repeat-dose studies) are observed, a quantitation of epididymal spermand the analysis of functional and morphological spermparameters may help to confirmor characterise the reproductive toxicity. Examination of sperm parameters should be performed in all cohort 1AF, males. 55. At termination, testis and epididymis weights are recorded for all Pand F, (cohort lA) males. At least one testis and one epididymis are reserved for histopathological examination. The remaining epididymis is used for enumeration of cauda epididymis spermreserves [17, 18] . In addition, spermfromthe cauda epididymis (or vas deferens) is collected using methods that minimise damage for evaluation ofsperm motility and morphology [19] . 56. Spermmotility can either be evaluated immediately after sacrifice or recorded for later analysis. The percentage of progressively motile spermcould be determined either subjectively or objectively by computer-assisted motion analysis [20, 21, 22, 23, 24, 25] . For the evaluation of spermmorphology an epididymal (or vas deferens) spermsample should be examined as fixed or wet preparations [26] and at least 200spermatozoa per sample classified as either normal (both head and midpiece/tail appear normal) or abnormal . Examples of morphologic sperni abnormalities would include fusion, isolated heads, and misshapen heads and/or tails [27] . Misshapen or large spermheads may indicate defects in spermiation . 57. If spermsamples are frozen, smears fixed and images for spermmotility analysis recorded at the time of necropsy [28], subsequent analysis may be restricted to control and high-dose males_ However, if treatment-related effects are observed, the lower dose groups should also be evaluated.

### 1:5 Gross necropsy 58. At the time of termination or premature death all P and F, animals are necropsied and examined macroscopically for any structural abnormalities or pathological changes. Special attention should be paid to the organs of the reproductive system . Pups that are humanely killed in a moribund condition and dead pups should be recorded and, when not macerated, examined for possible defects and/orcause ofdeath and preserved. 

### 59. For adult P and F, females, a vaginal smear is examined on the day ofnecropsy to determine the stage ofthe oestrous cycle and allowcorrelation with histopathology in reproductive organs. The uteri of all P females (and F, females, if applicable) are examined for the presence and number of implantation sites, in a manner which does not compromise histopathological evaluation. Organweight and Tissue Preservation - Pand F, Adult Animals 60 . At the time of termination, body weights and wet weights of the organs listed below fromall Panimals and all F, adults, fromrelevant cohorts (as outlined below), are determined as soon as possible after dissection to avoid drying. These organs should then be preserved in an appropriate fixative. Unless specified otherwise, paired organs can be weighed individually or combined, consistent with the typical practice of the performing laboratory. 

### " Uterus (with oviducts andcervix), ovaries " Testes, epididymides (total and cauda for the samples used for spermcounts) " Prostate (dorsolateral and ventral part combined) . Care should be exercised when trimming the prostate complex to avoid puncture of the fluid filled seminal vesicles. In the event of a treatment-related effect on total prostate weight, the dorsolateral and ventral segments should be carefully dissected after fixation, and weighed separately. " Seminal vesicles with coagulating glands and their fluids (as one unit) " Brain, liver, kidneys, heart, spleen, thymus, pituitary, thyroid (post-fixation), adrenal glands and known target organs or tissues. 61 . In addition to the organs listed above, samples of peripheral nerve, muscle, spinal cord, eye plus optic nerve, gastrointestinal tract, urinary bladder, lung, trachea (with thyroid and parathyroid attached), bone marrow, vas deferens (males), and mammary gland (males and females) and vagina should be preserved in an appropriate fixative. 

### 62. Cohort 1Aanimals have all organs weighed and examined for histopathology. Histopathology will be performed on one halfofthe spleen, the other halfbeing used for phenotypic analysis. 63. For the investigation of pre- and postnatally induced immunotoxic effects, 10 male and 10 female cohort IA animals fromeach treatment group (1 male or 1 female per litter; all litters represented by at least 1 pup; randomly selected) will be subject to the following at termination: weighing ofthe lymph nodes associated with and distant fromthe route ofexposure (in addition to the weight of the adrenal glands and the spleen, already performed in all cohort 1A animals) " splenic lymphocyte subpopulation analysis (CD4+ and CD8+ T lymphocytes, :B lymphocytes, and natural killer cells) using one half ofthe spleen, 0 histopathology on the collected lymph nodes and bone marrow

### 16 Analysis of splenic lymphocyte subpopulations in nonimmunized (cohort IA) animals will determine if exposure is related to a shift in the immunological steady state distribution of "helper" (CD4+) or cytotoxic (CD8+) thymus-derived lymphocytes or natural killer (NK) cells (rapid responses to neoplastic cells and pathogens) . 64. Cohort 1B animals, ifnot required to produce the FZ generation, have the following organs weighed and corresponding tissues processed to the block stage: Vagina (not weighed) - Uterus with cervix 

### - Ovaries, - Testes (at least one) - Epididymides - Seminal vesicles and coagulating glands - Prostate - Pituitary 

### - Identified target organs Histopathology in cohort 1Bwould be conducted ifresults fromcohort IAare equivocal . 65. Cohorts 2A and 213: Developmental Neurotoxicity Testing (PND21 or PND 22 and adult offspring) . Cohort 2A animals are terminated after behavioural testing, with brain weight recorded and full neurohistopathology for purposes of neurotoxicity assessment . Cohort 2:B animals are terminated on PND 21 or PND 22, with brain weight recorded and microscopic examination of the brain for purposes of neurotoxicity assessment. Perfusion fixation is required for cohort 2Aanimals and optional for cohort 2Banimals, as provided in OECDTG426. Organweight and Tissue Preservation - F, weanlings 66. The pups not selected for cohorts, including runts, are terminated after weaning, on PND 22, unless the results indicate the need for further in-life investigations. Terminated pups are subjected to gross necropsy on reproductive organs, as described in paragraph 64. For up to 10 pups per sex per group, fromas many litters as possible, brain, spleen, and thymus should be weighed and retained in an appropriate fixative. . In addition, mammary tissues for these male and female pups should be preserved for possible further histopathological analysis driven by findings in related parameters within this study or existing information. Gross abnormalities and target tissues should be saved for possible histological examination. Histopathology - Panimals 67. Full histopathology of the organs listed in Paragraph 64 is performed for all high dose and control P animals. Organs demonstrating treatment-related changes should also be examined in all animals at the lower dose groups to aid in determining a NOAEL. Additionally, reproductive organs ofall animals suspected of reduced fertility, e.g., those that failed to mate, conceive, sire, or deliver healthy offspring, or for which oestrus cyclicity or sperm number, motility, or morphology were affected, and all gross lesions should be subjected to histopathological evaluation.

### 17 Histopathology - F1 animals Cohort 1 animals 68. Full histopathology of the organs listed in Paragraph 64 is performed for all high dose arid control adult cohort 1A animals. All litters should be represented by at least 1 pup per sex. Organs and tissues demonstrating treatment-related changes and all gross lesions should also be examined in all animals in the lower dose groups to aid in determining a NOAEL. 69. All cohort 1 B animals have their reproductive and endocrine tissues processed to the block stage. As described in paragraph 30, cohort 113 reproductive and endocrine organs should be examined for histopathology in cases ofsuspected reproductive or endocrine toxicants. Cohort 1 B should also undergo histological examination if results fromcohort 1A are equivocal . 70 . Ovaries of adult females should contain primordial and growing follicles as well as corpora lutea, therefore, a histopathological examination should be aimed at detecting a quantitative evaluation of primordial and small growing follicles, as well as corpora lutea, in F, females; the number of animals, ovarian section selection, and section sample size should be statistically appropriate for the evaluation procedure used. Follicular enumeration may first be conducted on control and high dose animals, and in the event of an adverse effect in the latter, lower doses should be examined. Examination should include enumeration of the number of primordial follicles, which can be combined with small growing follicles, for comparison of treated and control ovaries. Corpora lutea assessment should be conducted in parallel with oestrous cyclicity testing so that the stage of the cycle can be taken into account in the assessment. Oviduct, utenis and vagina are examined for appropriate organ-typic development. 

### 71 . Detailed testicular histopathology examinations are conducted on the F, males in order to identify treatment-related effects on testis differentiation and development and on spermatogenesis. When possible, sections of the rete testis should be examined. Caput, corpus, and cauda of the epididymis and the vas deferens are examined for appropriate organ-typlic development as well as for the parameters required for the P males. Cohort 2animals 72. Neurohistopathology is performed for 10 high dose and control cohort 2A animals per sex following completion of neurobehavioral testing (not to exceed PND 90 +/- 3 days) . Brain histopathology is performed for 10 high dose and control cohort 2B animals per sex on PND21 or PND22. Organs or tissues demonstrating treatment-related changes should also be examined for all animals at the lower dose groups to aid in determining a NOAEL . For cohort 2A and 2:B animals, multiple sections are examined from the brain to allowexamination ofolfactory bulbs, cerebral cortex, hippocampus, basal ganglia, thalamus, hypothalamus, mid-brain (thecum, tegmentum, and cerebral peduncles), brain-stem and cerebellum . For cohort 2A only, the eyes (retina and optic nerve) and samples of peripheral nerve, muscle and spinal cord are examined. All neurohistological procedures should be consistent with OECD TG426 . 73. Morphometric (quantitative) evaluations should be performed on representative areas of the brain (homologous sections carefully selected based on reliable microscopic landmarks) and may include linear and/or areal measurements of specific brain regions. The neuropathologist should exercise appropriatejudgment as to whether sections prepared for measurement are homologous with others in the sample set and therefore suitable for inclusion, since linear measurements in particular may change over a relatively short distance [31] . Non-homologous sections should not

### 18 be used. While the objective is to sample all animals reserved for this purpose (10/sex/dose level), smaller numbers may still be adequate. However, samples fromfewer than 6 animals/sex/dose level would generally not be considered sufficient for the purposes of this test guideline. Stereology may be used to identify treatment-related effects on parameters such as volume or cell number for specific neuroanatomic regions. All aspects of the preparation oftissue samples, from tissue fixation, through the dissection oftissue samples, tissue processing, and staining of slides should employ a counterbalanced design such that each batch contains representative samples fromeach dose group. When morphometric or stereological analyses are to be used, then brain tissue should be embedded in appropriate media at all dose levels at the same time in order to avoid shrinkage artifacts known to be associated with prolonged storage in fixative. 

### REPORTING Data 74. Data are reported individually and summarised in tabular form . Where appropriate, for each test group and each generation, the following should be reported: number ofanimals at the start of the test, number of animals found dead during the test or killed for humane reasons, time of any death or humane kill, number of fertile animals, number of pregnant females, number of females givingbirth to a litter, and number ofanimals showing signs oftoxicity. Adescription of the toxicity, including time ofonset, duration, and severity should also be reported. 75. Numerical results should be evaluated by an appropriate, and accepted statistical method. The statistical methods should be selected as part of the study design and should appropriately address non-normal data (e.g., count data), censored data (e.g., limited observation time), nonindependence (e.g., litter effects and repeated measures), and unequal variances. Generalized linear mixed models and dose-response models cover a broad class ofanalytical tools that may be appropriate for the data generated underthis TG . The report should include sufficient information on the method of analysis and the computer program employed, so that an independent reviewer/statistician can evaluate/re-evaluate the analysis. Evaluation of results 

### 76. The findings should be evaluated in terms of the observed effects including necropsy and microscopic findings. The evaluation includes the relationship, or lack thereof, between the dose and the presence, incidence, and severity of abnormalities, including gross lesions. Target organs, fertility, clinical abnormalities, reproductive and litter performance, body weight changes, mortality and any other toxic and developmental effects should also be assessed. Special attention should be given to sex specific changes. The physico-chemical properties of the test substance:, and when available, TKdata, including placental transferand milk excretion, should be taken into consideration when evaluating the test results. Test Report 77. The test report should include the following information obtained in the present study fromP, F, animals and FZ animals (where relevant) : Test substance: " All relevant available information on the substance, toxicokinetic and toxicodynamir, properties of the test substance " identification data

### 19 " purity Vehicle (ifappropriate) : " justification for choice ofvehicle ifotherthan water Test animals: 

### " species/strain used " number, age and sex ofanimals " source, housing conditions, diet, nesting materials, etc. " individual weights ofanimals at the start of the test 

### " vaginal smear data for P females before initiation of treatment (if data are collected at that time) " P generation pairing records indicating male and female partner of a mating and mating success " litter oforigin records for adult F, generation animals 

### Test conditions: " rationale for dose level selection " details oftest substance formulation/diet preparation, achieved concentrations " stability and homogeneity ofthe preparation in the vehicle or carrier (e.g. diet, drinking water), in the blood and/or milk under the conditions ofuse and storage between uses " details ofthe administration ofthe test substance " conversion fromdiet/drinking water test substance concentration (ppm) to the achieved dose (mg/kg body weight/day), ifapplicable " details offood and waterquality (including diet composition, ifavailable) " a detailed description of the randomization procedures to select pups for culling and to assign pups to test groups 

### " environmental conditions Results (summary and individual data by sex and dose) : " food consumption, water consumption ifavailable, food efficiency (body weight gain per gramof food consumed, except for the period of cohabitation and during lactation), and test material consumption (for dietary/drinking water administration) for P and F, animals 

### " absorption data (ifavailable) " body weight data for Panimals " body weight data for the selected F, animals postweaning " time ofdeath during the study or whether animals survived to termination " nature, severity and duration ofclinical observations (whether reversible or not) " haematology, urinalysis and clinical chemistry data including TSHand T4 " phenotypic analysis ofspleen cells (T-, B-, NK-cells) " bone marrow

cellularity " toxic response data " number of Pand F, females with normal or abnormal oestrus cycle and cycle duration " time to mating (precoital interval, the number ofdays between pairing and mating) " toxic or other effects on reproduction, including numbers and percentages ofanimals that accomplished mating, pregnancy, parturition and lactation, of males inducing pregnancy, offemales with signs ofdystocia/prolonged or difficult parturition

### 20 " duration ofpregnancy and, ifavailable, parturition " numbers ofimplantations, litter size and percentage ofmale pups " number and percent ofpost-implantation loss, live births and stillbirths " litter weight and pup weight data (males, females and combined), the number ofrunts :if determined " number ofpups with grossly visible abnormalities " toxic orother effects on offspring, postnatal growth, viability, etc. " data on physical landmarks in pups and other postnatal developmental data " data on sexual maturation ofF, animals " data on functional observations in pups and adults, as applicable " body weight at sacrifice and absolute and relative organ weight data for the Pand adult F, animals " necropsy findings 

### " detailed description ofall histopathological findings " total cauda epididymal sperm number, percent progressively motile sperm, percent morphologically normal sperm, and percent ofsperm with each identified abnormality for Pand F, males 

### " numbers and maturational stages offollicles contained in the ovaries of Pand F, females, where applicable " enumeration ofcorpora lutea in the ovaries of Pfemales " statistical treatment ofresults, where appropriate Cohort 2 parameters: 

### " a detailed description of the procedures used to standardize observations and procedures as well as operational definitions for scoring observations; " a list ofall test procedures used, andjustification for their use; " details of the behavioural/functional, neuropathological and morphometric procedures used, including information and details on automated devices; " procedures for calibrating and ensuring the equivalence ofdevices and the balancing of treatment groups in testing procedures; " a shortjustification explaining any decisions involving professionaljudgment . 

### " a detailed description of all behavioural/functional, neuropathological and morphometric findings by sex, including both increases and decreases fromcontrols; " brain weight; " any diagnoses derived fromneurological signs and lesions, including naturally-occurring diseases or conditions; " images ofexemplarfindings; " low-power images to assess homology ofsections used for morphometry; 

### " statistical treatment ofresults, including statistical models used to analyze the data, and the results, regardless ofwhether they were significant ornot; " list ofstudy personnel, including professional training. " relationship ofany other toxic effects to a conclusion about the neurotoxic potential of the test chemical, by sex and group; " impact ofany toxicokinetic information on the conclusions; " similarities ofeffects to any known neurotoxicants; " data supporting the reliability and sensitivity of the test method (i .e., positive and historical control data) ; " relationships, ifany, between neuropathological and functional effects;

### 21 " NOAEL or benchmark dose for dams and offspring, by sex and group. discussion ofthe overall interpretation of the data based on the results, including a conclusion ofwhether or not the chemical caused developmental neurotoxicity and the NOAEL. 

### Cohort 3 parameters " serumIgMantibody titres (sensitization to SRBC or KLH), or splenic IgM PFC units (sensitization to SRBC) 

### " Performance ofthe method should be confirmed as part ofthe optimization process by laboratory setting up the assay for the first time, and periodically (e.g., yearly) by all laboratories. Discussion ofresults Conclusions, including NOAEL values forparental and offspring effects All information not obtained during the study, but useful for the interpretation of the results, should also be provided. INTERPRETATION OF RESULTS 78. An Extended One-Generation Reproductive Toxicity Study will provide information on the effects of repeated exposure to a substance during all phases of the reproductive cycle, as necessary. In particular, the study provides information on the reproductive system, and on development, growth, survival, and functional endpoints of offspring up to PND90 . 79. Interpretation of the results of the study should take into account all available information on the substance including physico-chemical, TK and toxicodynamic properties, available relevant information on structural analogues, and results of previously-conducted toxicity studies with the test substance (e.g., acute toxicity, toxicity after repeated application, mechanistic studies and studies assessing if there are substantial qualitative and quantitative species differences in in vivo/in vitro metabolic properties) . Gross necropsy and organweight results should be assessed in context with observations made in other repeat-dose studies, when feasible. Decreases in offspring growth might be considered in relationship to an influence of the test substance on milk composition [32] . 80 . Cohort 2(Developmental Neurotoxicity) Neurobehavioral and neuropathology results should be interpreted in the context of all findings, using a weight-of-evidence approach with expert judgment . Patterns of behavioural or morphological findings, if present, as well as evidence ofdose-response should be discussed. The evaluation of developmental neurotoxicity, including human epidemiological studies or case reports, and experimental animal studies (e.g., toxicokinetic data, structure-activity information, data fromother toxicity studies) should be included in this characterization. Evaluation of data should include a discussion of both the biological and statistical significance. The evaluation should include the relationship, if any, between observed neuropathological and behavioural alterations. For guidance on the interpretation of developmental neurotoxicity results, refer to OECDTG426 and Tyl et al ., 2008 [34] .

### 22 81 . Cohort 3 (Developmental Immunotoxicity) Suppression or enhancement of immune function as assessed by TDAR (T-cell dependent antibody response) should be evaluated in the context of all observations made. Significance of the outcome of TDAR may be supported by other effects on immunologically related indicators (bone marrowcellularity, weight and histopathology of lymphoid tissues, lymphocyte subset distribution)

. Effects established by TDAR may be less meaningful in case of other toxicities observed at lower exposure concentrations 82. OECDguidance document 43 should be consulted for aid in the interpretation ofreproduction and neurotoxicity results [27] .

### 23

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.B. Ahlmark, J . Locklear, T. Spahr, G.F. Leviness, M .F. Goelz, J .K. Haseman, R .R. Newbold, and D .B. Forsythe (1999) . Phytoestrogen content of purified open and closed formula laboratory animal diets. Lab. Anim . Sci . 49, 530- 536

. [3] Zoetis, T., Walls, l . (2003) . Principles and Practices for Direct Dosing of Pre-Weaning Mammals in Toxicity Testing and Research. ILSI Press, Washington, DC . [4] Moser V

.C., Walls I ., Zoetis T. (2005) . Direct dosing of preweaning rodents in toxicity testin, ; and research: Deliberations ofan ILSI RSI expert working group. International Journal of Toxicology 24, 87-94 . 

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., Takahashi M

., Kurokawa Y

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., Palmer, A

. K . (1995) . Detection ofeffects on male reproduction - a literature survey. Journal ofthe American College ofToxicologists. 14, 293-327. [8] Mangelsdorf, 1., Buschmann, J ., Orthen, B . (2003) . Some aspects relating to the evaluation of the effects ofchemicals on male fertility. Regulatory Toxicology and Pharmacology 37, 356-369 . [9] Sakai, T. et al . (2000) . Collaborative work to evaluate toxicity on male reproductive organs by repeated dose studies in rats - overview of the studies. Journal ofToxicological Sciences 25, 1-21. . [10] Creasy, D

. M . (2003) . Evaluation of testicular toxicology: Asynopsis and discussion of the recommendations proposed by the Society of Toxicologic Pathology. Birth Defects Research (Part B) 68, 408-415

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., Murr A.S., BuckalewA.R., Ferrell J .M ., Cooper R

.L. (2007) . The rodent estrous cycle: Characterization of vaginal cytology and its utility in toxicological studies. Birth Defects Research Part B-Developmental and Reproductive Toxicology 80(2), 84-97. [12] Sadleir, R

.M .F.S. (1979) . Cycles and Seasons, In: Reproduction in Mammals: I . Germ Cells and Fertilization, C

.R. Auston and R

.V. Short (eds.), Cambridge, New York. [13] Gallavan R

.H. Jr, Holson J .F., Stump D .G., Knapp J.F., Reynolds V .L. (1999) . Interpreting the toxicologic significance ofalterations in anogenital distance: potential for confounding effects of progeny body weights. Reproductive Toxicology 13: 383-390

### 24 [14] Korenbrot C

.C., Huhtaniemi I .T., Weiner R .I . (1977) . Preputial Separation as an External Sign ofPubertal development in the male rat. Biological Reproduction 17, 298-303. 

### [15] Ladics GS. 2007. Use of SRBC antibody responses for immunotoxicity testing. Methods 41,9-19 [16] Gore ER, Gower J, Kurali E, Sui JL, BynumJ, Ennulat D, Herzyk DJ . 2004. Primary antibody response to keyhole limpet hemocyanin in rat as a model for immunotoxicity evaluation. Toxicology 197, 23-35. [l7] Gray L.E., Ostby J., Ferrell J., Rehnberg G ., Linder R

., Cooper R

., Goldman J., Slott V ., Laskey J . (1989) . A Dose-Response Analysis of Methoxychlor-Induced Alterations of Reproductive Development and Function in the Rat. Fundamental and Applied Toxicology 

### 12,92-108. [18] Robb G.W

., Amann R.P., Killian G .J. (1978) . Daily SpermProduction and Epididymal SpermReserves ofPubertal and Adult Rats. Journal ofReproduction and Fertility 54, 103-107. [19] Klinefelter G

.R., Gray L.E. Jr, Suarez J .D. (1991) . The Method of Sperm Collection Significantly Influences Sperm Motion Parameters Following Ethane Dimethanesulfonate Administration in the Rat. Reproductive Toxicology 5, 39-44 . [20] Seed J., Chapin R .E., Clegg E.D., Dostal L.A., Foote R .H., Hurtt M

.E., Klinefelter G

.R., Makris S.L., Perreault S.D., Schrader S., Seyler D ., Sprando R

., Treinen K

.A., Veeramachaneru D .N., Wise L.D. (1996) . Methods for Assessing Sperm Motility, Morphology, and Counts in the Rat, Rabbit, and Dog: a Consensus Report. Reproductive Toxicology 10, 237- 244 . [21] Chapin R

.E., Filler R

.S., Gulati D

., Heindel J .J., Katz D .F., Mebus C

.A., Obasaju F., Perreault S.D., Russell S.R., Schrader S. (1992) . Methods for Assessing Rat SpermMotility. Reproductive Toxicology 6, 267-273. [22] Klinefelter G.R., Roberts N .L., Suarez J .D. (1992) . Direct Effects of Ethane Dimethanesulphonate on Epididymal Function in Adult Rats: an In Vitro Demonstration. Journal ofAndrology 13, 409-421 . [23] Slott V

.L., Suarez J .D., Perreault S.D. (1991) . Rat SpermMotility Analysis: Methodologic Considerations. Reproductive Toxicology 5, 449-458 . [24] Slott, V

.L., Perreault, S.D., (1993) . Computer-Assisted Sperm Analysis of Rodent Epididymal Sperm

Motility Using the Hamilton-Thorn Motility Analyzer. In: Methods in Toxicology, Part A

., Academic, Orlando, Florida. pp. 319-333. 

### [25] Toth G

.P., Stober J .A., Read E.J., Zenick H., Smith M .K . (1989) . The Automated Analysis of Rat SpermMotility Following Subchronic Epichlorhydrin Administration: Methodologic and Statistical Considerations. Journal ofAndrology 10, 401-415. [26] Linder R

.E., Strader L.F., Slott V.L., Suarez J .D . (1992) . Endpoints ofSpermatoxicity in the Rat After Short Duration Exposures to Fourteen Reproductive Toxicants. Reproductive Toxicology 6, 491-505 .

### 2 :5 [27] OECD Guidance Document n° 43 on Mammalian Reproductive Toxicity Testing and Assessment 

### http://www

.olis.oecd.orp,/olis/2008doc.nsf/LinkTo/NT0000358A/$FILE/JT03249193 .pdf [28] Working, P.K., Hurtt, M . (1987) . Computerized Videomicrographic Analysis of Rat Sperna Motility. Journal ofAndrology 8, 330-337. 

### [29] Russell, L.D. et al ., (1990)

. Histological and Histopathological Evaluation of the Testis, Cache River Press, Clearwater, Florida. [30] Lanning L.L., Creasy D .M ., Chapin R

.E., Mann P

.C., BarlowN

.J., Regan K

.S., Goodman D .G. (2002) Recommended Approaches for the Evaluation of Testicular and Epididyma .l Toxicity. Toxicologic Pathology 30, 507-520 . [31] Bolin, B

., R . Garman, K

. Jensen, G

. Krinke, B

. Stuart, and an ad Hoc Working Group of the STP Scientific and Regulatory Policy Committee (2006) . A `Best Practices' Approach to Neuropathologic Assessment in Developmental Neurotoxicity Testing - for Today. Toxicological Pathology, 34, 296-313. [32] Stutz, N

., Bongiovanni, B., Rassetto, M ., Ferri, A

., Evangelista de Duffard, A . M ., and Duffard, R

. (2006) . Detection of 2,4-dichlorophenoxyacetic acid in rat milk of dams exposed during lactation and milk analysis oftheir major components. Food Chemicals Toxicology 44, 8- 16. [33] Thigpen JE, Setchell KDR, Haseman JK, Saunders HE, Caviness GF, Kissling GE, Grant MG, Forsythe DB

. (2007) . Variations in phytoestrogen content between differentmill dates of the same diet produces significant differences in the time of vaginal opening in CD-1 mice and F344 rats but not in CD Sprague Dawley rats. Environmental health perspectives 115(12), 1717-1726 . 

### [34] Tyl, R.W

., Crofton, K., Moretto, A., Moser, V., Sheets, L.P. and Sobotka, T.J. Identification and interpretation of developmental neurotoxicity effects: A report from the ILS1 Research Foundation/Risk Science Institute expert working group on neurodevelopmental endpoints. Neurotoxicology and Teratology 30 : 349-381, 2008. [35] OECD (2000), Guidance Document on the recognition, assessment, and use ofclinical signs as humane endpoints for experimental animals used in safety evaluations, Series on Testing and assessment No 19, ENV/JM/MONO(2000)7, OECD, Paris.

### Appendix A Measures and Observations Included in the Functional Observational Battery (cohort 2) 

### Home Cage &Open Field Manipulative PhysioloQic Posture Ease ofremoval Temperature Involuntary Clonic &Tonic Ease ofhandling Body weight Palpebral Closure Muscle Tone Pupil response Piloerection Approach Response Pupil size Salivation Touch Response Lacrimation Auditory Response Vocalizations Tail Pinch Response Rearing Righting Response Gait Abnormalities Landing Foot Splay Arousal Forelimb Grip Strength Stereotypy Hindlimb Grip Strength Bizarre Behavior Stains Respiratory Abnormalities

### 27 Appendix B Procedure for the decision on the internal triggering for producing a second generation (mating of the F, offspring to produce Fz litters) in the Extended One-Generation Reproductive Toxicity Study . 

### 1 . The decision on triggering an FZ generation within the study is made on the basis ofan integrated assessment ofall data available within the study fromthe Pgeneration and the F, generation in all the cohorts studied. Integrated assessment includes:

" Comparison among effects, between Pand F, generations, and between cohorts; " Consideration of relationships among different end point parameters tested; 

### " Dose-response analyses and comparison thereof between end points; and " Data on kinetics ofthe chemical tested, ifavailable; 2 . Special attention should be given to specific end point parameters that are relevant for the decision on triggering which are given in tables 1 and 2. The trigger parameters listed in the table if affected either alone or in combination need not automatically lead to the need for an Fz.These end point parameters should bejudged in the context of all findings within the study to arrive at an overall conclusion on the need for an Fz generation. 3. If the integrated data analysis shows a clear picture ofabsence of reproductive toxicity of the chemical tested, then normally the generation ofan F2 is not necessary. If the integrated data analysis shows an uncertainty about the reproductive hazard of the compound tested, within the integrated assessment of the findings in the study, then an Fz generation may be deemed necessary, if the parameters studied in the Fz are deemed likely to be able to clarify the uncertainty. 4 . Statistical significance without biological relevance should not in isolation trigger an Fz generation. 5 . Reproductive effects should be weighed against adult paternal and maternal effects observed to assess the specificity of the effect and its possible causation by primary adult toxicit:,~. Equivocal reproductive effects observed only in the presence of severe parental toxicity may not lead to generation ofan Fz.

### p. 31 

### 28

Table 1 : List of potential reproductive endpoints considered for triggering an FZ generation* Reproductive Endpoint Endpoint available i~ Endpoint Decision time to determine FZ. ~ evaluated in F2. Fertility Endpoints P, Estrous Cycle Evaluation Yes No Trigger' P, Fertility (# implantations, pregnancy Yes No Trigger' rate, gestational interval) F, Litter parameters (litter size, litter Yes No Trigger3

weight, sex ratio) F, Developmental landmarks (AGD, Yes No Trigger' nipple retention, puberty onset, PPS, VO) F, Estrous Cycle Evaluation Yes No Trigger Developmental Endpoints P, Reproductive OrganWeights Yes Yes No P, Reproductive Organ Histopathology Yes Yes No P, Andrology (spermparameters) Yes No No P, Qualitative OvarianAssessment Yes No No F, Endpoints F, Reproductive Organ Weights No Yes No F, Reproductive Organ Histopathology No Yes No F, Andrology (spermparameters) No No No F, Qualitative Ovarian Assessment No No No *Criteria for establishing triggers taken fromCooper etal . 2006and draft OECDExtended One-Generation Reproduction Toxicity Test Guideline (6-18-2008) ', 'Ifbiologically relevant, dose-related changes in estrous cycle length without overt toxicity in the dams. zIn the absence ofcorresponding, treatment-related reproductive organ histopathology. i 3Ifsignificant, treatment-related decreases in litter size/pup survival are seen in the absence ofsevere maternal I toxicity or lethality. I 4Dose-related effects; in the absence of body weight-mediated changes in these parameters.

### p. 32 

### 29

Table 2: List of potential offspring endpoints considered fortriggering an FZ generation* Fl or PEndpoint Will the endpoint be Endpoint available in time to evaluated in F~ Z. Decision determine FZ? Offspring Endpoints jMaternal (P) bodyweight same dose Yes Yes Not a Trigger' as IF, pup bodyweight 1 lactation index (PND4-21) Yes Yes Trigger F, pup mortality Yes Yes Trigger' F, pup malformations (e.g. ., hypospadia, cryptocordysm, Yes Yes TriggerZ one eye, large head) IF, pup viability index (PNDO-4) Yes Yes Trigger2 IF, live birth index Yes Yes Trigger2 IF, pup bodyweight only Yes Yes Trigger3 *Criteria for establishing triggers taken fromCooper el al . 2006and draftOECDExtended One-Generation Reproduction Toxicity Test Guideline (6-18-2008) . 1Unless F, pup body weight decrease at a lower dose than the dose at which maternal body weight decreased z In the absence ofsevere maternal toxicity. , 3 If the pup bodyweight decrease is significant and in absence of matemal body weight decrement.