. 

Nc~ro 

OFPT-~0)3-00 10 

P.O. Box 331, Millwood, VA 22646 (540) 83i-1602 

Task Force 

u~qi iiuumiuupup;~ii~p~,gi ; 

April 15, 2010 

Document Control Office (7407) 

Office of Pollution Prevention and Toxics 

Room 6428 

Environmental Protection Agency 

1200 Pennsylvania Ave, NW 

Washington, DC 20460 

Re: Testing Consent Order for Ethylene Dichloride; 

Request for Modification of Enforceable Consent 

Agreement (Docket No. OPPT-2003-0010) 

Dear Sirs : 

>> 

-, 

L4 

._., 

The HAP Task Force requests a modification to the Enforceable Consent 

Agreement for Ethylene Dichloride (the "ECA"), 68 Fed. Reg. 33125 (June 3, 2003). Six 

copies of this request are submitted. 

Last year, Table 1 to the ECA, which sets forth required testing, test standards, and 

reporting requirements, was amended to provide revised deadlines. 74 Fed. Reg. 36196 

(July 22, 2009). Since that time, the Task Force has discussed with EPA staff the 

possibility of further revising Table 1 so that the required oral subchronic neurotoxicity 

and reproductive toxicity studies could be conducted under a single combined protocol. 

This protocol would follow the OECD guideline 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., A tiered approach to life stages testing for agricultural chemical 

safety assessment, Crit. Rev. Toxicol. 36 :69-98 (2006) . If no final OECD guideline has 

been issued by the time 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, which is 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, EPA will want to review and approve a proposed protocol based on the 

CONTAINS NO CBI 

: 

. ~Y 

3~z~alL 
October 28, 2009 draft proposed guideline for the extended one-generation reproductive 

toxicity study. The Task Force will submit a proposed protocol by July 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 ECA appendix C.2 and C.6 12 

reporting extrapolation of dose-response 

(oral Tier II testing to inhalation) 

of a study reported by Daniel, et 

al ., (1994) 

OLCD Test Guideline / _ILSIHESI Aericultural Chemical 

Saf'etv Assessment 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., Brad v, 

~ 

A.M., 

Subchronic neurotoxici (oral) .md. . . 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~' 

k GA ., ppondix -F~'~ 

. Subchronic neurotoxicity route-to- 

, 

ECA appendix C.3 and C.6 -24- 

route extrapolation of dose-response (oral Tier II testing to inhalation) 

P " ,AU, r. . . :t, . r . .,I , 40 0`14 700. 440 _ 2S i43 

14~;A 

, , : 

l

: ., 

14,7 ` 

Reproductive toxicity route-to-route ECA appendix C.4 and C.6 ?S-- 

extrapolation of dose-response 

(oral data to inhalation, including 

Tier II testing and extant studies 

reported by Alumot, et al ., 

(1976), Rao, et al ., (1980), and 

Lane, et al ., (1982)) 

'Number of months after the date of publication of this Federal Register document, which announces that EPA has concluded the EPA 

Program Review, when the final report is due. In addition, every 6 months from the effective date of the Order until the end of the ECA 

testing program, interim reports describing the status of all testing to be performed under this ECA must be submitted by the Companies to 

EPA. 
I look forward to hearing from you. 

Sincerely yours, 

Peter E. Voytek; Ph.D'. 

Manager 

Enclosures 

cc: J. Schaeffer 

W. Caffey Norman, Esq. 
Draft version 28, October 2009 

OECD GUIDELINE FOR THE TESTING OF 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 of the 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 AND OBJECTIVES 

2. The main objective of the Extended One-Generation Reproductive Toxicity Study is to 

evaluate specific life stages not covered by other types of toxicity 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 from repeat-dose studies (including screening 

reproductive toxicity studies, e.g. TG 422), or short term endocrine 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 F 1 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 confirm information about the effects of a test 

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 from these tests, , should allow the 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 two weeks) and a two-week 

mating period. P males are further treated at least until weaning of the F, (i .e . a minimum of 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 from weaning to adulthood until postnatal day 

(PND) 70 or 90, depending on their cohort assignment . If a 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. A t 

weaning, selected offspring are assigned to specific subgroups (cohorts 1-3, see paragraphs 30 to 

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 if necessary revised in light of experience gained. 
Figure 1 : Scheme of the Extended One-Generation Reproductive Toxicity Study 

Extended One-Generation Protocol 

Dosing 

Pre-mating T Mating ~ Post-mating 

2 weeks I 2 weeks ~ 6 weeks 

------ - -----~--- ----

2 weeks ' 2 weeks Pregnancy Lactation 

------- - --- -~---- 

[

In-utero 

F' ! development Pre-weaning 

- ---- - 

Necropsy 

P animals 

1 

Post-weaninig 

Parental 

generation Cohort Designation Animals/Cohort 

Sexual Approximate age at 

Maturation necropsy (weeks) 

1 .A Reproductive 20 M +20 F Yes 13 

Target is 16 Reproductive 20 M +20 F Yes 14 or 20 if triggered 

20 litters 2A Neurotoxicity 10 M +10 F@ Yes g 

per group =B Neurotoxicity 10 M +10 F@ No 3 

3 Immunotoxicity 10 M +10 F@ Yes g 

Surplus Spares No 3 

4r one per litter and representative of 20 litters in total where possible 

DESCRIPTION OF THE METHOD/PREPARATIONS FOR THE TEST 

ANIMALS 

Selection of animal 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% of the 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 of the diet should be retained until finalisation of the report, 

in case the results necessitate a further analysis of diet 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 of copulation, 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 group from weaning 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 20 pregnant 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 of the P generation 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 show regular (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 P females 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 review of existing information is important for decisions on the route of administration, 

the choice of the vehicle, the selection of animal species, the selection of dosages 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, metabolism and 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, metabolism or induction of metabolism. 

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 (or relevant 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 TK data 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 of sample 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 of administration 

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 of an 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 of the 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 from previous studies, TK data from pregnant 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 of course, that human exposures 

are expected to be well below the point of saturation . In such cases, the highest dose level should 

be at or just 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 of the test substance . If dose levels are based on toxicity, 

the highest dose should be chosen with the aim to induce some systemic toxicity, but not death or 

severe suffering of the 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 allow for 

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 

of a 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 if a 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 of toxicity 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 from structurally- 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 of administration . 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 them at 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 nursing pups [5]. 

Dosing schedule and administration of doses 

24. Some information on oestrous cycles, male and female reproductive tract histopathology and 

testicular/epididymal sperm analysis may be available from previous repeat-dose toxicity studies 

of adequate duration. The duration of the pre-mating treatment in the Extended One-Generation 

Reproductive Toxicity Study therefore is aimed at the detection of effects 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. A 2-week, 

pre-mating treatment for both 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 allow the 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 sperm parameters are scheduled, the )? 

and F, males, will have been exposed for at least one entirespermatogenic process [7, 8, 9, 10 and 

supporting document] . 

25 . Pre-mating exposure scenarios for males could be adapted, if testicular toxicity (impairment 

of spermatogenesis) or effects on sperm integrity 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, may justify 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 of the 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 of liquid administered at one 

time should not exceed 1 mL/100 g body weight (0.4 mL/100 g body weight is the maximum for 

oil, e.g. corn oil) . A volume 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 TK data indicate a 

low placental 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 of parturition; omission of test substance administration on that day :is 

preferable to a disturbance of the birth process . 

Mating 

29. Each P female should be placed with a single, randomly selected, unrelated male from the 

same dose group (1 :1 pairing) until evidence of copulation 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 0 of pregnancy is defined as the day on which mating evidence is confirmed (a vaginal plug 

or sperm are found) . Animals should be separated as soon as possible after evidence of copulation 

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 of each litter should be adjusted by eliminating extra pups by 

random selection 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 of each sex per litter, partial adjustment (for example, 

six males and four females) is acceptable. Adjustments are not appropriate for litters of ten pups 

or less. 

Selection of pups for post-weaning studies (see Figure 1) 

31 . At weaning (around PND 21) pups from all 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 below the mean pup weight of the respective litter) 

should not be included as they are unlikely to be representative of the treatment group. 

On PND21, the selected F, pups are randomly assigned to one of three cohorts of animals as 

follows: 

Cohort 1 (1 A and 113) = Reproductive/developmental toxicity testing 

Cohort 2 (2A and 213) = Developmental neurotoxicity testing (PND21 or PND22 and 

adults 

Cohort 3 = Developmental immunotoxicity t esting (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 (PND 21 or PND 22). If there 

are insufficient numbers of animals, preference should be given to assign animals to Cohort 2A. 

Cohort 3 : 10 males and 10 females per group selected, one per litter (where possible) for a total 

of 20 pups per group . Additional pups may be required from the 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 P animals 

33 . A second mating is not normally recommended for the P animals 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 P and the selected F, animals a general clinical observation is made once a day. In the 

case of gavage 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 of difficult or prolonged parturition and all signs of toxicity are recorded . Twice 

daily, during the weekend once daily, all animals are observed for morbidity and mortality . 

35. In addition, a more detailed examination of all P and F, animals (after weaning) is conducted 

on a weekly basis and could conveniently be performed on an occasion when the animal is 

weighed, which would 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 of clonic 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. P animals are weighed on the first day of dosing 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 (PND 21) 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 from selection to respective cohort . 

Oestrous cycles 

38 . Preliminary information of test 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 1 A should also be monitored for a 

period of two weeks commencing around PND 75 . 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 of pairing, the date of insemination and the date. 

of parturition 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 (LD 0) 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 (PND 0 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 

PND 0 or at a later time should be examined for possible defects and cause of death. Live pups 

are counted and weighed individually on PND 0 or PND 1, and regularly thereafter, e.g ., at least 

on PND 4, 7, 14, and 21 . Clinical examinations, as applicable for the age of the 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) of each pup should be measured between PND 0 and PND 4 . 

Pup body weight should be collected on the day the AGD is measured and the AGD should 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 PND 12 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 from each 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 of day, and environmental distractions . Results of the 

neurotoxicity assays should be interpreted in relation to appropriate historical control reference 

ranges. Cohort 213 animals should be used for neuropathology assessment on PND 21 or PND 22. 

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 10 trials) should be determined, with 

test conditions optimized to produce intrasession habituation . These procedures should be 

consistent with OECD TG 426. 

47. At an appropriate time between PND 63 and PND 75, 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 from the least to the most interactive. A list 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 low as 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 of activity . 

48 . If existing information indicates the need for other functional testing (e.g., sensory, social., 

cognitive), these should be integrated without compromising the integrity of the 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 PND 56, plus or minus +-3 days of age, 10 male and 10 female cohort 3 animals from each 

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 IgM antibody 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 IgM antibody 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 from all 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 PFC response or serur,a 

for the ELISA assay. 

Assessment of potential reproductive toxicity (Triggered) 

50. Cohort 1B animals can be maintained on treatment beyond PND 90 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 PND 90, but not exceeding PND 120. 

Procedures should be similar to those for the P animals, although it may suffice to terminate the 

litters on PND 4 rather than follow them to weaning or beyond, depending on the concerns that 

triggered the extension of the study. The decision of whether 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 of triggers . 

TERMINAL OBSERVATIONS 

Clinical biochemistry / Haematology 

51 . Systemic effects should be monitored in P animals. Fasted blood samples from a defined site 

are taken from ten 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 TSH or 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 

serum should 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 from all 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 P animals 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 few days before the animals are mated for the second time. 

Unless existing data from repeated 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 from a defined 

site are taken from ten 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 of serum levels for thyroid hormones (T4 and 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 T4 and 

TSH analysis from weanlings subject to gross necropsy on PND 22 (F, pups not selected for 

cohorts) . 

Sperm parameters 

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 of suitable exposure duration 

show that sperm parameters 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 sperm and the analysis of functional and morphological 

sperm parameters may help to confirm or characterise the reproductive toxicity . Examination of 

sperm parameters should be performed in all cohort 1 A F, males. 

55. At termination, testis and epididymis weights are recorded for all P and F, (cohort l A) males. 

At least one testis and one epididymis are reserved for histopathological examination. The 

remaining epididymis is used for enumeration of cauda epididymis sperm reserves [17, 18]. In 

addition, sperm from the cauda epididymis (or vas deferens) is collected using methods that 

minimise damage for evaluation of sperm motility and morphology [ 19] . 

56. Sperm motility can either be evaluated immediately after sacrifice or recorded for later 

analysis. The percentage of progressively motile sperm could be determined either subjectively or 

objectively by computer-assisted motion analysis [20, 21, 22, 23, 24, 25] . For the evaluation of 

sperm morphology an epididymal (or vas deferens) sperm sample should be examined as fixed or 

wet preparations [26] and at least 200 spermatozoa 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 sperm heads may indicate defects in spermiation. 

57. If sperm samples are frozen, smears fixed and images for sperm motility 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/or cause of death and preserved. 

59. For adult P and F, females, a vaginal smear is examined on the day of necropsy to determine 

the stage of the oestrous cycle and allow correlation 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. 

Organ weight and Tissue Preservation - P and F, Adult Animals 

60. At the time of termination, body weights and wet weights of the organs listed below from all 

P animals and all F, adults, from relevant 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 and cervix), ovaries 

" Testes, epididymides (total and cauda for the samples used for sperm counts) 

" 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 1 A animals have all organs weighed and examined for histopathology . Histopathology 

will be performed on one half of the spleen, the other half being used for phenotypic analysis. 

63. For the investigation of pre- and postnatally induced immunotoxic effects, 10 male and 10 

female cohort IA animals from each 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 of the lymph nodes associated with and distant from the route of exposure (in 

addition to the weight of the adrenal glands and the spleen, already performed in all 

cohort 1 A animals) 

" splenic lymphocyte subpopulation analysis (CD4+ and CD8+ T lymphocytes, :B 

lymphocytes, and natural killer cells) using one half of the 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, if not 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 1B would be conducted if results from cohort IA are 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 2A animals and optional for cohort 2B animals, as provided in OECD TG 426 . 

Organ weight 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, from as 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 - P animals 

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 of all 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 1 A 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 of suspected reproductive or endocrine toxicants. Cohort 1 B 

should also undergo histological examination if results from cohort 1 A 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 2 animals 

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 PND 21 

or PND 22. 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 allow examination of olfactory 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 TG 426. 

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 appropriate judgment 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 from fewer 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 of tissue samples, from 

tissue fixation, through the dissection of tissue samples, tissue processing, and staining of slides 

should employ a counterbalanced design such that each batch contains representative samples 

from each 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 of animals 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 giving birth to a litter, and number of animals showing signs of toxicity. A description of 

the toxicity, including time of onset, 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 of analytical tools that may be 

appropriate for the data generated under this 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, TK data, including placental transfer and 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 from P, 

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 (if appropriate): 

" justification for choice of vehicle if other than water 

Test animals: 

" species/strain used 

" number, age and sex of animals 

" source, housing conditions, diet, nesting materials, etc . 

" individual weights of animals 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 of origin records for adult F, generation animals 

Test conditions : 

" rationale for dose level selection 

" details of test substance formulation/diet preparation, achieved concentrations 

" stability and homogeneity of the preparation in the vehicle or carrier (e.g. diet, drinking 

water), in the blood and/or milk under the conditions of use and storage between uses 

" details of the administration of the test substance 

" conversion from diet/drinking water test substance concentration (ppm) to the achieved 

dose (mg/kg body weight/day), if applicable 

" details of food and water quality (including diet composition, if available) 

" 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 if available, food efficiency (body weight gain per 

gram of 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 (if available) 

" body weight data for P animals 

" body weight data for the selected F, animals postweaning 

" time of death during the study or whether animals survived to termination 

" nature, severity and duration of clinical observations (whether reversible or not) 

" haematology, urinalysis and clinical chemistry data including TSH and T4 

" phenotypic analysis of spleen cells (T-, B-, NK-cells) 

" bone marrow cellularity 

" toxic response data 

" number of P and F, females with normal or abnormal oestrus cycle and cycle duration 

" time to mating (precoital interval, the number of days between pairing and mating) 

" toxic or other effects on reproduction, including numbers and percentages of animals that 

accomplished mating, pregnancy, parturition and lactation, of males inducing pregnancy, 

of females with signs of dystocia/prolonged or difficult parturition 
20 

" duration of pregnancy and, if available, parturition 

" numbers of implantations, litter size and percentage of male pups 

" number and percent of post-implantation loss, live births and stillbirths 

" litter weight and pup weight data (males, females and combined), the number of runts :if 

determined 

" number of pups with grossly visible abnormalities 

" toxic or other effects on offspring, postnatal growth, viability, etc. 

" data on physical landmarks in pups and other postnatal developmental data 

" data on sexual maturation of F, animals 

" data on functional observations in pups and adults, as applicable 

" body weight at sacrifice and absolute and relative organ weight data for the P and adult F, 

animals 

" necropsy findings 

" detailed description of all histopathological findings 

" total cauda epididymal sperm number, percent progressively motile sperm, percent 

morphologically normal sperm, and percent of sperm with each identified abnormality for 

P and F, males 

" numbers and maturational stages of follicles contained in the ovaries of P and F, females, 

where applicable 

" enumeration of corpora lutea in the ovaries of P females 

" statistical treatment of results, 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 of all test procedures used, and justification 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 of devices and the balancing of 

treatment groups in testing procedures; 

" a short justification explaining any decisions involving professional judgment . 

" a detailed description of all behavioural/functional, neuropathological and morphometric 

findings by sex, including both increases and decreases from controls ; 

" brain weight; 

" any diagnoses derived from neurological signs and lesions, including naturally-occurring 

diseases or conditions; 

" images of exemplar findings; 

" low-power images to assess homology of sections used for morphometry; 

" statistical treatment of results, including statistical models used to analyze the data, and 

the results, regardless of whether they were significant or not ; 

" list of study personnel, including professional training. 

" relationship of any other toxic effects to a conclusion about the neurotoxic potential of 

the test chemical, by sex and group; 

" impact of any toxicokinetic information on the conclusions; 

" similarities of effects to any known neurotoxicants ; 

" data supporting the reliability and sensitivity of the test method (i .e., positive and 

historical control data); 

" relationships, if any, between neuropathological and functional effects; 
21 

" 

NOAEL or benchmark dose for dams and offspring, by sex and group. 

discussion of the overall interpretation of the data based on the results, including a 

conclusion of whether or not the chemical caused developmental neurotoxicity and the 

NOAEL. 

Cohort 3 parameters 

" serum IgM antibody titres (sensitization to SRBC or KLH), or splenic IgM PFC units 

(sensitization to SRBC) 

" Performance of the method should be confirmed as part of the optimization process by 

laboratory setting up the assay for the first time, and periodically (e .g ., yearly) by all 

laboratories . 

Discussion of results 

Conclusions, including NOAEL values for parental 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 PND 90. 

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 organ weight 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 of dose-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 from other 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 

OECD TG 426 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 marrow cellularity, 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. OECD guidance document 43 should be consulted for aid in the interpretation of reproduction 

and neurotoxicity results [27]. 
23 

LITERATURE 

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Makris, S. L., Sheets, L. P., Speijers, G. J. A., Whitby, K. E. (2006) . A tiered approach to life 

stages testing for agricultural chemical safety assessment . Critical Reviews in Toxicology 36, 69-

98. 

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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 of an ILSI RSI expert working group . International Journal of 

Toxicology 24, 87-94. 

[5] Conolly R.B., Beck B.D., Goodman J .I . (1999). Stimulating research to improve the scientific 

basis of risk assessment. Toxicological Sciences 49, 1-4. 

[6] Takayama S., Akaike M., Kawashima K., Takahashi M., Kurokawa Y. (1995) . A 

Collaborative Study in Japan on Optimal Treatment Period and Parameters for Detection of Male 

Fertility Disorders Induced by Drugs in Rats. Journal of the American College of Toxicologists. 

14, 266-292. 

[7] Ulbrich, B., Palmer, A. K. (1995) . Detection of effects on male reproduction - a literature 

survey . Journal of the American College of Toxicologists. 14, 293-327. 

[8] Mangelsdorf, 1 ., Buschmann, J ., Orthen, B. (2003) . Some aspects relating to the evaluation of 

the effects of chemicals 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 of Toxicological Sciences 25, 1-21. . 

[10] Creasy, D. M. (2003). Evaluation of testicular toxicology: A synopsis and discussion of the 

recommendations proposed by the Society of Toxicologic Pathology. Birth Defects Research 

(Part B) 68, 408-415. 

[I 1 ] Goldman J .M., Murr A.S ., Buckalew A.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 of alterations 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 of Pubertal 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, Bynum J, 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 Sperm Production and Epididymal 

Sperm Reserves of Pubertal and Adult Rats. Journal of Reproduction 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 Sperm Motility. 

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 

of Andrology 13, 409-421 . 

[23] Slott V.L ., Suarez J.D., Perreault S .D. (1991) . Rat Sperm Motility 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 Sperm Motility Following Subchronic Epichlorhydrin Administration : Methodologic and 

Statistical Considerations. Journal of Andrology 10, 401-415. 

[26] Linder R.E ., Strader L.F ., Slott V.L ., Suarez J.D. (1992) . Endpoints of Spermatoxicity 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 of Andrology 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 ., Barlow N.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 of their 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 different mill 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 of clinical 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 of removal Temperature 

Involuntary Clonic & Tonic Ease of handling 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 of an 

integrated assessment of all data available within the study from the P generation and the F, 

generation in all the cohorts studied . Integrated assessment includes: 

" Comparison among effects, between P and 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 of the chemical tested, if available ; 

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 be judged 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 of absence of reproductive toxicity of 

the chemical tested, then normally the generation of an 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 of an 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 Trigger 3 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 Organ Weights Yes Yes No 

P, Reproductive Organ Histopathology Yes Yes No 

P, Andrology (sperm parameters) Yes No No 

P, Qualitative Ovarian Assessment Yes No No 

F, Endpoints 

F, Reproductive Organ Weights No Yes No 

F, Reproductive Organ Histopathology No Yes No 

F, Andrology (sperm parameters) No No No 

F, Qualitative Ovarian Assessment No No No 

*Criteria for establishing triggers taken from Cooper et al. 2006 and draft OECD Extended One-Generation 

Reproduction Toxicity Test Guideline (6-18-2008) ', 

'If biologically relevant, dose-related changes in estrous cycle length without overt toxicity in the dams. 

z In the absence of corresponding, treatment-related reproductive organ histopathology . i 

3 If significant, treatment-related decreases in litter size/pup survival are seen in the absence of severe maternal I 

toxicity or lethality. I 

4 Dose-related effects; in the absence of body weight-mediated changes in these parameters . 
p. 32 

29 

Table 2: List of potential offspring endpoints considered for triggering an FZ generation* 

Fl or P Endpoint 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 Trigger Z 

one eye, large head) 

IF, pup viability index (PNDO-4) Yes Yes Trigger 2 

IF, live birth index Yes Yes Trigger 2 

IF, pup bodyweight only Yes Yes Trigger 3 

*Criteria for establishing triggers taken from Cooper el al . 2006 and draft OECD Extended One-Generation 

Reproduction Toxicity Test Guideline (6-18-2008). 

1 Unless F, pup body weight decrease at a lower dose than the dose at which maternal body weight decreased 

z In the absence of severe maternal toxicity. , 

3 If the pup bodyweight decrease is significant and in absence of matemal body weight decrement.